Natural Compounds Targeting Profibrotic Signaling Pathways in Myocardial Fibrosis: Signaling Network Regulation and Therapeutic Potential

Abstract

Myocardial fibrosis (MF) is a common pathological process in various cardiac diseases, including myocardial infarction (MI), hypertensive heart disease, and dilated cardiomyopathy. MF is characterized by excessive deposition of extracellular matrix (ECM), reduced myocardial compliance, and impaired cardiac function. This process can lead to arrhythmias and sudden cardiac death. Currently, effective and safe therapies for MF are limited. This review summarizes recent advances in the use of natural compounds to treat MF by regulating signaling pathways. Representative compounds, such as Salvianolic acid B (Sal B), Astragaloside IV (AS-IV), Pachymic acid, Resveratrol, Curcumin, Matrine, Artemisinin, and Artesunate, among others, can modulate multiple profibrotic signaling pathways. These compounds reduce profibrotic cytokines, inhibit collagen deposition, reverse myofibroblast differentiation, enhance ECM degradation, and alleviate inflammation and oxidative stress. As a result, they improve MF and regulate systemic inflammatory responses. However, comprehensive data on the pharmacokinetics, bioavailability, long-term safety, and clinical efficacy of these natural compounds are still lacking. Future studies should focus on optimizing delivery strategies and conducting rigorously designed clinical trials to support the clinical translation of these compounds for MF treatment.

1. Introduction

Cardiovascular disease (CVD) is a leading cause of death worldwide, responsible for approximately 32% of deaths in 2019. By 2030, it is estimated that 23.6 million people will die from CVDs, resulting in a significant global financial burden. The development of nearly all CVDs is closely linked to myocardial fibrosis (MF). According to the latest report by the World Health Organization, the global annual incidence of MF is approximately 1.7%. Once MF progresses to heart failure (HF), the five-year mortality rate can reach 40%-60%, severely impairing patients’ quality of life and posing a major challenge to global healthcare systems. ¹ In recent years, with the advancement of medical research, a more comprehensive understanding of the pathogenic mechanisms has emerged. MF is a common pathological feature in various CVDs, including myocarditis, myocardial infarction (MI), and HF. Cardiac fibroblasts (CFs) play a central role in the development of MF by proliferating abnormally in response to various pathological factors. This process causes excessive extracellular matrix (ECM) deposition and scar tissue formation, ultimately driving cardiac remodeling. These changes impair myocardial contractility and diastolic function, ultimately contributing to HF or death. ² As many CVDs progress to later stages, MF emerges as a common pathological manifestation. Due to its multifactorial etiology, complex biosignaling networks, and the lack of effective interventions, MF represents a major obstacle in the clinical prevention and treatment of heart disease. Currently, medical treatment for MF primarily relies on pharmaceutical interventions, including angiotensin-converting enzyme inhibitors (ACEIs), β-blockers, and diuretics. These therapies target multiple pathological pathways involved in MF, such as the renin-angiotensin-aldosterone system (RAAS) and inflammatory responses. However, these therapies often lack specificity toward cardiac fibroblast activation. Moreover, their long-term efficacy is limited by disease progression, target desensitization, and safety concerns, which may lead to diminishing therapeutic effects and poor patient adherence. Consequently, it remains challenging to effectively and sustainably halt the fibrosis process. This review systematically examines natural compounds that have recently attracted attention in MF research, including Salvianolic acid B (Sal B), Astragaloside IV (AS-IV), Pachymic acid, Resveratrol, Curcumin, Matrine, and Artesunate. These compounds have demonstrated regulatory effects on major fibrosis-related signaling pathways, supported by varying levels of experimental evidence. The review integrates the molecular mechanisms through which these natural products modulate pathways such as TGF-β1/Smad, JAK/STAT, Wnt/β-catenin, NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, peroxisome proliferator-activated receptor-γ (PPAR-γ), activating transcription factor 6 (ATF6), and Toll-like receptor (TLR) signaling pathways. Additionally, the review explores their synergistic therapeutic potential via multi-pathway crosstalk, providing a theoretical foundation for the development and clinical translation of innovative anti-MF therapies.

2. Pathophysiological Basis and Therapeutic Strategies of MF

2.1. Pathological Mechanisms of MF

MF is widely recognized as a critical pathological component in the progression of CVDs. Under pathological conditions, the dynamic interactions between cardiomyocytes and the ECM are disrupted. Infiltration of inflammatory cells and the transdifferentiation of resident cells lead to the upregulation of profibrotic factors, ultimately resulting in excessive deposition of ECM within the myocardial interstitium. The pathogenesis of MF involves complex crosstalk among cardiomyocytes, inflammatory cells, fibroblasts, and endothelial cells. ³

2.1.1. Inflammation-Mediated Mechanisms in MF

Inflammation is widely regarded as a central driving force in the initiation and progression of chronic CVDs, characterized primarily by inflammatory cell infiltration and the release of a broad spectrum of cytokines. Following cardiac injury, inflammatory cells—including M1-polarized macrophages, mast cells, and lymphocytes—are rapidly recruited and infiltrate the damaged myocardial tissue. Guided by chemokines, these cells accumulate within the local inflammatory microenvironment and secrete a variety of pro-inflammatory and profibrotic mediators, including TGF-β, platelet-derived growth factor (PDGF), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and vascular endothelial growth factor. Through autocrine and paracrine signaling, these factors activate resident fibroblasts and promote their phenotypic transition into myofibroblasts, while simultaneously inducing the further release of profibrotic mediators such as fibroblast growth factor-2. ⁴ Persistent inflammatory responses further exacerbate inflammatory cell infiltration and trigger aberrant activation of local cells, thereby reshaping the profibrotic microenvironment and ultimately promoting the differentiation and accumulation of myofibroblasts.

2.1.2. Activation and Differentiation of CFs

CFs within the myocardial interstitium are the primary source of ECM. Under physiological conditions, the majority of fibroblasts remain in a quiescent state, with only a small subset participating in the basal synthesis of basement membrane components and the maintenance of tissue homeostasis. However, in response to pathological stimuli such as ischemia, inflammation, or mechanical stress, the local microenvironment undergoes profound alterations, accompanied by activation of multiple cytokines and signaling pathways (e.g., Wnt/β-catenin), thereby driving fibroblast transition from a quiescent to an activated phenotype. Activated fibroblasts exhibit enhanced proliferative and secretory capacities. They produce and deposit large amounts of ECM into the interstitial space, leading to excessive ECM accumulation within the myocardium. Meanwhile, these ECM components can interact with integrin receptors, triggering intracellular signaling cascades that, in turn, further promote fibroblast proliferation, migration, and activation, thereby establishing a positive feedback loop that exacerbates matrix remodeling. With persistent pathological stimulation, a subset of activated fibroblasts further differentiates into myofibroblasts characterized by the expression of α-smooth muscle actin (α-SMA). These cells not only possess enhanced contractile capacity and ECM-producing capability but also secrete tissue inhibitors of metalloproteinases, which suppress matrix metalloproteinase (MMP)-mediated ECM degradation, ultimately resulting in an imbalance between ECM synthesis and degradation. ⁵ Consequently, excessive ECM deposition disrupts the normal myocardial architecture, reduces tissue compliance, and leads to irreversible myocardial remodeling and functional impairment. In addition to the classical activation of resident CFs, myofibroblasts may also originate from the transdifferentiation of other cell types.

2.1.3. Cellular Origins and Activation Mechanisms of Myofibroblasts

Given the pivotal role of myofibroblasts in MF, elucidating the diversity of their cellular origins, especially the mechanisms underlying their activation, is of great significance for identifying potential therapeutic targets. Myofibroblasts arise not only from the activation of resident CFs but can also be generated through pericyte proliferation and differentiation, as well as endothelial-to-mesenchymal transition (EndoMT). Under physiological conditions, pericytes are closely associated with capillary endothelial cells, surrounding microvessels and playing essential roles in maintaining vascular integrity, regulating blood flow, and promoting angiogenesis. Platelet-derived growth factor receptor-β (PDGFR-β), a characteristic marker of pericytes, interacts with endothelial-derived PDGF-B to maintain their attachment to the vascular wall. However, following cardiac injury, aberrant activation of PDGF-B/PDGFR-β signaling promotes pericyte detachment from the vessel wall and their differentiation toward a myofibroblast phenotype. Notably, this process is closely associated with the activation of myofibroblasts, which is characterized by increased expression of α-SMA, enhanced contractility, and ECM production. This transition is accompanied by pathological ECM deposition and disruption of microvascular structure, thereby accelerating the progression of MF. In addition, endothelial cells can undergo EndoMT in response to inflammatory mediators, TGF-β, and angiotensin II (Ang II). During this process, endothelial cells gradually lose endothelial markers (e.g., CD31 and VE-cadherin) while acquiring mesenchymal characteristics, including upregulation of α-SMA and enhanced migratory capacity. Importantly, these phenotypic changes indicate the activation and functional transition of myofibroblasts. The transformed cells accumulate within the interstitium and further differentiate into myofibroblasts. ⁶ Under the influence of the local microenvironment, these cells continue to proliferate and secrete excessive amounts of ECM components (such as collagen and fibronectin), ultimately leading to disruption of myocardial architecture and impairment of cardiac function. Importantly, this progressive transition is not a series of discrete events but a coordinated process governed by tightly interconnected signaling networks. These networks integrate mechanical stress, inflammatory cues, and profibrotic stimuli such as TGF-β and Ang II into unified transcriptional and functional outputs, thereby dictating myofibroblast fate, persistence, and fibrotic remodeling. Accordingly, deciphering the core signaling circuitry underlying MF is essential for advancing mechanistic understanding and therapeutic innovation.

2.2. Key Signaling Pathway Networks in MF

MF is driven by complex intracellular signaling networks that regulate fibroblast activation, ECM deposition, and cardiac remodeling. Among these, the TGF-β/Smad pathway plays a central role through both canonical and non-canonical mechanisms. In addition to TGF-β signaling, the pathogenesis of MF involves several other intracellular pathways, including JAK/STAT, Wnt/β-catenin, NLRP3 inflammasome, PPAR-γ, ATF6, and TLR signaling. Targeting these fibrotic mediators or their downstream cascades may provide effective therapeutic strategies for fibrotic diseases.

2.2.1. TGF-β/Smad Signaling

The TGF-β/Smad pathway operates through both canonical and non-canonical mechanisms. In the canonical pathway, TGF-β binds to transforming growth factor-β receptor II (TβRII) on the cell membrane, forming a complex with transforming growth factor-β receptor I (TβRI). TβRI then recruits and phosphorylates the Smad proteins Smad2/3. These phosphorylated Smads associate with Smad4 to form a complex that translocates into the nucleus, promoting the transcription of ECM-related fibrotic genes. In contrast, Smad7 functions as a negative regulator by competing with TβRI, thereby preventing Smad complex formation and promoting ubiquitination and degradation of Smad proteins. Additionally, TGF-β1 can activate multiple other pathways contributing to MF, including the RAAS, mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/Akt (PI3K/Akt), and rho-associated kinase (ROCK) pathways. RAAS activation is initiated by renin-mediated cleavage of angiotensinogen to generate angiotensin I (Ang I), which is converted to Ang II by angiotensin-converting enzyme (ACE). Ang II binds to Ang II type 1 receptor (AT1), stimulating CFs proliferation and ECM synthesis. MAPK activation occurs when ligand-induced dimerization and autophosphorylation of receptor tyrosine kinases (RTKs) recruit SOS-GRB2 to activate Ras, which sequentially activates Raf, mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2 (ERK1/2). Phosphorylated ERK1/2 translocates to the nucleus to promote fibroblast survival and proliferation. ⁷ In the canonical NF-κB pathway, Kip1 ubiquitination-promoting complex 1 ubiquitinates the precursor p105, generating the active p50 subunit, which then translocates into the nucleus to regulate gene transcription. Furthermore, PI3K/Akt signaling is initiated when growth factors bind to RTKs, activating PI3K to convert phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate. This recruits Akt to the plasma membrane, where 3-phosphoinositide-dependent protein kinase-1 phosphorylates Akt at Thr308; subsequent phosphorylation at Ser473 completes Akt activation. Downstream substrates such as GLUT, GSK-3, and mTOR mediate fibroblast activation and collagen synthesis. Finally, molecules including PDGF, Ang II, and endothelin (ET) can activate RhoA-GTP, which in turn activates ROCK. ROCK promotes cell adhesion, stress fiber formation, and fibroblast transdifferentiation. Overactivation of this pathway disrupts collagen metabolism and contributes to MF ⁸ (Figure 1).

Figure 1.

Canonical and Non-Canonical TGF-β/Smad signaling pathways in MF. (Left (Canonical pathway): TGF-β1 activates the TβRII/TβRI complex, leading to Smad2/3 phosphorylation. Smad2/3 forms a complex with Smad4, translocates into the nucleus, and upregulates profibrotic genes (α-SMA). Smad7 provides negative feedback, and compounds like Sal B inhibit this pathway. Right (Non-Canonical pathway): TGF-β also triggers Smad-independent pathways, including PI3K/Akt/mTOR, MAPKs (p38, JNK, Erk1/2), NF-κB, and Rho/ROCK, which regulate gene expression and contribute to fibrosis. Created with FigDraw.)

2.2.2. Additional Profibrotic Signaling Pathways

2.2.2.1. JAK/STAT Signaling

The JAK/STAT signaling pathway is a key cytokine-mediated signal transduction mechanism. Cytokines such as interferons, interleukins, and members of the glycoprotein 130 and common gamma-chain receptor families are key activators of the JAK/STAT pathway. When cytokines such as interferon and interleukin bind to receptors, receptor dimerization triggers self-phosphorylation of JAK family kinases. Activated JAK recruits and phosphorylates STATs, forming homodimers or heterodimers. These activated STATs translocate to the nucleus to regulate transcription of pro-inflammatory and ECM-related genes, ultimately accelerating fibrosis. ⁹

2.2.2.2. Wnt/β-Catenin Signaling

The Wnt signaling pathway is widely present in invertebrates and vertebrates and is highly conserved during species evolution. In the canonical pathway, Wnt ligands bind to Frizzled (FZD) receptors and the co-receptors LRP5/6, triggering phosphorylation of Dishevelled (DVL) in the cytoplasm. This process inhibits GSK-3β- and CK1-mediated phosphorylation of β-catenin, thereby preventing its degradation. Stabilized β-catenin subsequently translocates into the nucleus, where it associates with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors to activate target genes, including fibronectin 1 (FN1), MMPs, and c-Myc. The activation of these genes promotes cardiac fibroblast activation, collagen synthesis, and ECM remodeling. ¹⁰

2.2.2.3. NLRP3 Inflammasome Signaling

Inflammation is a key driver in the initiation and progression of MF. Among the key inflammatory pathways, the NLRP3 inflammasome has emerged as a central mediator of cardiac injury. It is activated by ionic fluxes, including potassium and chloride efflux and calcium influx. Upon activation, NLRP3 interacts with the pyrin domain of apoptosis-associated speck-like protein containing a CARD (ASC), recruiting pro-caspase-1 and inducing its cleavage. Active caspase-1 then promotes the maturation and secretion of IL-1β and interleukin-18 (IL-18), which drive inflammation and pyroptosis, thereby promoting MF progression. ¹¹

2.2.2.4. PPAR-γ Signaling

Nuclear receptors play essential roles in regulating cardiac metabolism, inflammation, and remodeling. Among them, peroxisome proliferator-activated receptors (PPARs), members of the nuclear hormone receptor superfamily, are key transcription factors that mediate gene expression in cardiomyocytes. PPARs exist as three isoforms: α, β, and γ, which have distinct tissue distribution and functional roles. In cardiomyocytes, PPAR-γ undergoes a conformational change upon ligand binding, translocates into the nucleus, and forms heterodimers with retinoid X receptor (RXR). The PPAR-γ/RXR complex then binds to peroxisome proliferator response elements (PPREs) in target gene promoters, thereby regulating genes involved in fatty acid metabolism, glucose homeostasis, and anti-inflammatory responses. ¹²

2.2.2.5. Other Pathways

High glucose levels induce endoplasmic reticulum (ER) stress, activating the ATF6 pathway. Cleaved into its active form, ATF6p50 translocates to the nucleus, suppresses miR-455 expression, increases calreticulin levels, and triggers ER calcium release, which activates calcineurin, promoting nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) nuclear translocation and expression of fibrosis-related genes. ¹³ Finally, TLRs recognize damage-associated molecular patterns such as the ECM protein Tenascin-C and activate fibroblasts. TLR4 recognizes ligands at the plasma membrane and undergoes endocytosis to the endosomal membrane. Ligand-induced dimerization of extracellular domains brings together Toll/IL-1 receptor domains, initiating downstream signaling that drives pro-inflammatory and profibrotic responses ¹⁴ (Figures 2 and 3).

Figure 2.

Overview of additional profibrotic signaling cascades. (Schematic summarizing six pathways-JAK/STAT, Wnt/β-catenin, NLRP3 inflammasome, PPAR-γ, ATF6 and TLR-that drive MF activation and ECM deposition. Colored arrows indicate activation steps; T-bars denote inhibition sites targeted by the natural compounds listed in Table 1. Created with FigDraw.)

Figure 3.

ATF6 and TLR-mediated pathways in MF. (A) ER stress activates ATF6p50, which suppresses miR-455 and enhances NFATc1-driven ECM gene transcription. (B) TLR4/MD2 formation triggers ERK-AP1 signaling, promoting COL1A1, FN1 and CTGF expression. Artesunate binds MD2 to disrupt TLR4 signaling; Matrine blocks ATF6 cleavage. Created with FigDraw.)

2.3. Pharmacological Treatment of MF

2.3.1. Conventional Pharmacological Therapies

MF is a key pathological process driving the progression of various CVDs and ultimately leading to adverse cardiac remodeling and functional decline. Pharmacological intervention remains the mainstay of MF management. Currently used agents include ACEIs, β-blockers, mineralocorticoid receptor antagonists (MRAs) and diuretics. Although these drugs can improve fibrosis-related biomarkers and cardiac function, their clinical utility is limited by non-specific systemic targeting, adverse effects, and marked interindividual variability in therapeutic response. ACEIs exert antifibrotic effects primarily by suppressing Ang II generation, thereby inhibiting CFs activation and ECM accumulation. In rat models of HF, perindopril (0.8-3 mg·kg^-1·d^-1, orally for 8-16 weeks) significantly reduced ventricular B-type natriuretic peptide levels, downregulated Collagen Type I Alpha 1 Chain (COL1A1) expression, and attenuated myocardial inflammation and fibrosis. Similar benefits were observed in doxorubicin-induced dilated cardiomyopathy and spontaneously hypertensive rat models, with an acceptable safety profile; only reversible renal tubular degeneration and hepatocyte hypertrophy were noted at high doses. ¹⁵ Likewise, in a left anterior descending artery-ligated MI mouse model, captopril (50 mg·kg^-1·d^-1) inhibited TGF-β1/Smad3 signaling, reduced fibroblast-to-myofibroblast differentiation and collagen deposition, and improved left ventricular remodeling and ejection fraction. Prolonged high-dose treatment (150 mg·kg^-1·d^-1 for 28 days) resulted in mild, reversible renal tubular injury and reduced hematocrit. ¹⁶ Collectively, conventional pharmacological agents can partially attenuate MF through modulation of neurohormonal pathways and fibroblast activity, although their effects remain largely indirect.

2.3.2. Limitations of Current Treatments

Despite their therapeutic benefits, the clinical utility of conventional drugs in MF is limited by several factors. ACEIs, for example, are frequently limited in post-infarction HF and left ventricular dysfunction due to hypotension, hyperkalemia, and angioedema. β-Blockers mitigate MF mainly by attenuating excessive sympathetic activation and suppressing the RAAS, thereby reducing oxidative stress and inflammation. In a canine model of chronic obstructive sleep apnea, metoprolol (1.2 mg·kg^-1·d^-1) decreased TGF-β1 and Matrix metalloproteinase-9 (MMP-9) secretion from atrial adipose tissue, limited atrial collagen accumulation, and inhibited fibroblast activation, partly through suppression of fibroblast growth factor 23 signaling. However, long-term high-dose administration (2 mg·kg^-1·d^-1) was associated with reversible hepatocyte hypertrophy and mild hemolysis. Clinically, dose-dependent adverse effects included bradycardia, hypotension, and cold extremities. ¹⁷ These vascular-related side effects are largely attributed to impaired endothelium-dependent vasodilation, with the nitric oxide (NO) pathway, which serves as a central regulator of vascular tone. Although propranolol can partially restore endothelial dysfunction by compensating for local NO deficiency, its precise molecular targets remain poorly defined, limiting broader clinical application. MRAs, particularly spironolactone, inhibit aldosterone-mediated fibroblast differentiation and collagen synthesis. In an autoimmune myocarditis mouse model, spironolactone (30 mg·kg^-1·d^-1 for 4 weeks) significantly reduced myocardial collagen I expression, attenuated collagen deposition, and improved diastolic function. Consistently, in patients with HF with preserved ejection fraction, treatment with 25-50 mg/d for nine months reduced circulating markers of collagen synthesis. ¹⁸ Overall, while the aforementioned conventional pharmacological therapies provide symptomatic relief and modest antifibrotic benefits, several fundamental limitations remain. First, most agents act indirectly through systemic neurohormonal pathways rather than specifically targeting key profibrotic cells (e.g., CFs) or signaling cascades (e.g., TGF-β/Smad and Wnt/β-catenin), making it difficult to achieve true disease-modifying antifibrotic effects. Second, long-term treatment is frequently accompanied by clinically significant adverse events that compromise patient adherence. Third, therapeutic responses vary widely among individuals, and treatment discontinuation may lead to rebound effects, indicating a lack of sustained fibrosis control. Importantly, no drugs specifically approved for MF are currently available, and existing therapies largely represent indirect interventions that fail to effectively halt or reverse the fibrotic process itself (Table 1).

Table 1.

Multidimensional Comparison of Conventional Drugs and Natural Compounds for MF

Drug category	Drug name	Mechanism of action	Relative efficacy	Safety analysis	References
Chemical drugs	Perindopril	Inhibits ACE, reducing Ang II and fibrosis	Reduces MF; improves cardiac function	Monitor BP and K+ at high doses; well tolerated	15
	Captopril	Downregulates TGF-β1/Smad3, reduces collagen	Improves cardiac function; inhibits fibroblast activation	Safe short-term; high doses may cause renal injury/angioedema	16
	Metoprolol	Reduces epicardial adipose tissue fibrosis, inhibits FGF23	Reduces atrial fat and fibrosis; improves function	High doses may cause reversible hepatotoxicity	17
	Spironolactone	Reduces collagen I deposition	Improves HFpEF; reduces collagen I	Risk of hyperkalemia/endocrine effects	18
Natural products	Salvianolic acid B	Inhibits Smad2/3 phosphorylation, stabilizes Smad7	Inhibits collagen deposition; improves function	Injectable; low toxicity; poor oral absorption	19
	Astragaloside IV	Inhibits TRPM7, reduces Smad2/3	Anti-fibrotic and anti-inflammatory	High safety; low oral absorption	20
	Icariin	Blocks TGF-β1 receptor complex, inhibits Smad2/3	Anti-fibrotic; immunomodulatory	Poor solubility; low bioavailability	21
	Ginsenoside Rg3	Inhibits TGFBR1 kinase, blocks Smad phosphorylation	Multi-target anti-fibrotic; cardioprotective	Rapid metabolism; low plasma levels	22
	Forsythoside B	Inhibits Smad3 phosphorylation and nuclear translocation	Anti-inflammatory; antioxidant; anti-fibrotic	Strong first-pass effect; low absorption	23
	Pachymic acid	Inhibits JAK2/STAT3, upregulates SOCS3	Cardioprotective; anti-hypertrophic	Poor solubility; low absorption	24
	Sinomenine	Blocks JAK2 ATP pocket, inhibits IL-1β/IL-6/TNF-α	Antioxidant; anti-hypertrophic	Poor solubility; strong first-pass effect	25
	Stachydrine	Inhibits downstream inflammatory mediators	Weak anti-fibrotic (monotherapy)	Acceptable safety	26
	Triptolide	Inhibits β-catenin nuclear translocation and Wnt ligand-receptor binding	Potent anti-inflammatory	High toxicity; narrow therapeutic window	27
	Salidroside	Blocks β-catenin/Smad3 co-transcription complex formation	Anti-hypoxic; multi-organ protection	Good safety and tolerability	28
	Saikosaponin A	Inhibits EndoMT and β-catenin nuclear translocation	Multi-target anti-fibrotic	Low toxicity; safe short-term	29
	Resveratrol	Downregulates sFRP2 to inhibit Wnt3a/β-catenin	Antioxidant	Rapid metabolism; very low bioavailability	30
	Berberine	Blocks Wnt ligand-receptor interaction and β-catenin nuclear translocation	Improves endothelial function; lipid regulation	Poor absorption; rapid metabolism	31
	Artemisinin	Inhibits NLRP3 and NF-κB, blocking inflammation-fibrosis axis	Anti-inflammatory; anti-fibrotic	Poor solubility; short half-life	32
	Celastrol	Covalently binds NLRP3 NACHT domain, blocking inflammasome assembly	Broad anti-inflammatory	Multi-organ toxicity	33
	Oridonin	Covalently binds NLRP3, blocks caspase-1/IL-1β/IL-18 axis	Anti-inflammatory; antioxidant; anti-fibrotic	Narrow therapeutic window	34
	Curcumin	Activates PPAR-γ, inhibits Ang II-induced collagen	Multi-target anti-fibrotic; structural improvement	High safety	35
	Propolis Extract	Activates PPAR-γ, inhibits NF-κB	Antioxidant; synergistic effects	Strong first-pass; low bioavailability	36
	Procyanidin B2	Inhibits TGF-β1 promoter activation, upregulates Smad7	Antioxidant; inhibits collagen	Low bioavailability; good safety	37
	Piperine	Partially activates PPAR-γ, blocks AKT/GSK3β	Bioavailability enhancer	gastrointestinal irritation at high doses	38
	Theophylline	Activates SIRT3, promotes β-catenin degradation, enhances PPAR-γ	Multi-target cardioprotection	Narrow window; requires monitoring	39
	Matrine	Blocks ATF6-p50, inhibits ER stress	Multi-target; reduces chemo-toxicity	Poor solubility; well tolerated	40
	Artesunate	Binds MD2, blocks DAMP recognition	Anti-inflammatory; anti-fibrotic	Low bioavailability; resistance risk	41

Given the limitations of current pharmacological therapies, natural compounds have garnered increasing attention owing to their multi-target regulatory properties, low toxicity, and favorable safety profiles. For example, Sal B inhibits TGF-β/Smad3 signaling by upregulating miR-29b and miR-618, thereby reducing collagen type I, collagen type III, and α-SMA expression, thereby alleviating collagen deposition. ⁴² AS-IV exerts antifibrotic effects by inhibiting the ROS/caspase-1/GSDMD and NLRP3/caspase-1/IL-18 signaling pathways. ⁴³ Overall, natural compounds can systematically regulate the pathogenesis of MF, including inflammation, oxidative stress, ECM metabolism, and fibroblast activation. These approaches offer root-cause interventions with fewer side effects and present a promising strategy for the precise treatment of MF, particularly compared to conventional pharmacological therapies.

3. Biological Mechanisms of Natural Compounds in the Treatment of MF via Relevant Signaling Pathways

3.1. Natural Compounds Modulating the TGF-β/Smad Signaling Pathway

This part discusses Sal B, AS-IV, Icariin, Ginsenoside Rg3, and Forsythoside B, which specifically target key steps in the TGF-β/Smad signaling pathway, demonstrating significant antifibrotic effects. These natural compounds not only modulate the TGF-β/Smad pathway but also affect other related signaling pathways through multi-target mechanisms, including anti-inflammatory and antioxidant effects. Despite some limitations in bioavailability, modern technologies can enhance their effectiveness. They exhibit low toxicity and good safety profiles in animal studies, making them promising candidates for the clinical treatment of MF.

3.1.1. Salvianolic Acid B (Sal B)

Salvia miltiorrhiza Bunge (Lamiaceae, Salvia L.) is the root of a plant in the Salvia genus of the mint family, known for its vasodilatory, anticoagulant, anti-inflammatory, and radical scavenging effects. The primary pharmacological activity of Salvia miltiorrhiza is attributed to its lipophilic diterpene quinones and hydrophilic phenolic acids. Among these, Sal B, the most water-soluble active component, is composed of three phenolic acid molecules and one caffeic acid molecule. ⁴⁴ However, Sal B has extremely low oral absorption, with most of it retained in the small intestine. When administered intravenously, it rapidly reaches peak concentrations, followed by distribution to the kidneys, lungs, and liver, with clearance occurring within four hours, primarily excreted via bile as a methylated metabolite. To enhance its oral bioavailability and release characteristics, a Sal B microemulsion carrier has been developed. This carrier offers high entrapment efficiency, excellent stability, and a simple preparation process, extending drug release time and significantly improving oral bioavailability. ⁴⁵

Mechanistic studies show that Sal B exerts a concentration-dependent inhibitory effect on TGF-β1-induced fibrosis in CFs. Sal B binds to the ATP-binding pocket of the TGF-β1 receptor type I Activin receptor-like kinase 5 (ALK5), blocking its kinase activity and preventing Smad2/3 phosphorylation. This reduces the binding of the Smad3/4 complex to the COL1A1 promoter, suppressing collagen transcription and synthesis. Additionally, Sal B stabilizes Smad7 by inhibiting Smurf2-mediated ubiquitin degradation, enhancing its negative feedback inhibition of Smad2/3 phosphorylation and comprehensively suppressing the TGF-β1/Smad signaling pathway. In vitro experiments demonstrate that Sal B stabilizes cardiomyocyte morphology, reduces collagen fiber content in myocardial tissue, increases MMP expression, and significantly decreases fibrosis severity. ¹⁹ In vivo studies demonstrated that Sal B (40-160 mg·kg^-1·d^-1, i.v., daily for 30 days in isoproterenol (ISO)-induced mouse model) significantly reduced myocardial collagen deposition, downregulated MMP expression, decreased infarct size, and improved cardiac function in an ISO-induced MF model in mice. ⁴⁶

Toxicological evaluation identified a no-observed-adverse-effect level (NOAEL) of 120 mg·kg^-1·d^-1 for Sal B, with a median lethal dose (LD₅₀) of approximately 485 mg·kg^-1 and a therapeutic index of about 3. At higher doses (≥240 mg·kg^-1·d^-1), reversible elevations in alanine aminotransferase (ALT) and mild prolongation of activated partial thromboplastin time were observed. Clinical studies in chronic hepatitis B patients (Phase II, randomized, double-blind, positive-controlled trial, n=60, 6-month treatment course) indicated that Sal B significantly improved fibrosis markers. Specifically, 36.7% of patients experienced a ≥1-stage reduction in fibrosis staging, with decreased serum hyaluronic acid and IV-C abnormality rates. The overall fibrosis marker improvement rate was 30%, significantly superior to the control group (P <0.05). Continuous oral administration was safe, with no notable adverse reactions. ⁴⁷ However, as Sal B advances toward clinical application, potential drug interactions warrant consideration, particularly when co-administered with medications for CVDs. For instance, when Sal B is combined with atorvastatin, its active components can induce cytochrome P450 3A4 (CYP3A4) activity in hepatic microsomes, affecting atorvastatin’s metabolic stability. This shortens its half-life and reduces bioavailability. ⁴⁸ Therefore, it may be necessary to adjust the atorvastatin dosage to prevent a reduction in its efficacy.

3.1.2. Astragaloside IV (AS-IV)

AS-IV is an active component derived from Astragalus membranaceus var. mongholicus (Bunge) P.K. Hsiao (Fabaceae, Astragalus L.). AS-IV, the primary active compound in Astragalus, exhibits a wide range of pharmacological effects, including anti-inflammatory, antifibrotic, antioxidative, and cardioprotective activities. These effects are mediated through the regulation of multiple signaling pathways.

Mechanistically, AS-IV exerts its effects in MF primarily through modulation of the transient receptor potential melastatin 7 (TRPM7) channel, the TGF-β/Smad signaling pathway, and the NLRP3 inflammasome. AS-IV directly inhibits the α-kinase activity of TRPM7, blocking its phosphorylation of downstream target proteins (such as Akt, ERK1/2, etc.), thereby reducing TRPM7-mediated Ca²⁺/Mg²⁺ influx. This inhibition prevents the proliferation and collagen secretion of CFs stimulated by ISO. Additionally, AS-IV targets the TGF-β1 ligand-receptor binding interface to inhibit ALK5 kinase activity, reducing the phosphorylation of Smad2/3 at their C-termini. Specifically, AS-IV binds to the ATP-binding pocket of ALK5, blocking the conformational rotation of its Gly-rich loop within the kinase domain. This prevents ALK5 from phosphorylating Smad2 at Ser465/467 and Smad3 at Ser423/425. Additionally, AS-IV reduces the nuclear translocation of the Smad3/Smad4 complex by decreasing Smad3 phosphorylation, thereby inhibiting the assembly of the transcription initiation complex at the Smad-binding element of the COL1A1 promoter. Further studies have shown that TRPM7 plays a crucial role in ISO-induced CFs, collagen secretion, and activation of the TGF-β/Smad pathway. The TGF-β1 inhibitor SB-431542 blocks the TGF-β/Smad pathway, reduces Smad2/3 phosphorylation, upregulates the negative feedback factor Smad7, and significantly downregulates TRPM7 expression, thereby effectively inhibiting the fibrotic process. Additionally, AS-IV exerts anti-inflammatory effects by inhibiting NLRP3 inflammasome activation, further enhancing its antifibrotic activity. ²⁰ In vitro and in vivo experiments demonstrate that AS-IV exhibits significant antifibrotic effects in ISO-induced MF models, including inhibition of fibroblast proliferation, reduction of collagen deposition, and decreased MF markers. These findings further validate its mechanism of action through the TRPM7 and TGF-β/Smad pathways.

Regarding toxicology and clinical aspects, the effective antifibrotic dose of AS-IV is 20 mg·kg^-1·d^-1. Reversible leukopenia and hepatic steatosis occur at plasma concentrations ≥4.8 μg·mL^-1 (approximately four times the effective C_max), but no mortality has been observed at a toxic dose of 2 g·kg^-1 (100 times the standard dose). Pharmacokinetic studies suggest that AS-IV inhibits CYP2D6 and CYP1A2 activity, slowing the clearance of drugs metabolized by these enzymes (e.g., metoprolol, theophylline), thereby elevating their plasma concentrations. When used in combination therapy, clinical monitoring of drug concentrations and timely dose adjustments are necessary to optimize therapeutic efficacy and ensure safety. ⁴⁹

3.1.3. Icariin

Icariin is a flavonoid compound extracted from the dried stems and leaves of Epimedium sagittatum Maxim. (Berberidaceae, Epimedium L.). It has multiple pharmacological effects, including immunomodulatory, anti-inflammatory, antioxidant, and lipid-lowering properties, making it highly valuable for medicinal use. However, its clinical application is limited by poor oral bioavailability due to low water solubility, inadequate membrane permeability, and slow dissolution in biological fluids. To improve the oral bioavailability of Icariin and its derivatives, six synergistic strategies have been developed: phospholipid complexation, cyclodextrin inclusion, multi-nanocarrier systems, crystal engineering, enzyme-assisted absorption, and colon-targeted delivery. These approaches not only enhance relative bioavailability but also offer sustained-release and targeted delivery capabilities. ⁵⁰

Research has shown that icariin exerts anti-MF effects by modulating the TGF-β1/Smad signaling pathway. It directly binds to TGF-β1, preventing the docking of the TβRII receptor with the glycine-serine-rich domain of ALK5. This inhibits the phosphorylation of Thr204 on ALK5, which disrupts the kinase platform for the Smad2/3 SSXS motif. As a result, the Smad2/3-Smad4 complex cannot form, blocking its nuclear translocation. Ultimately, this suppresses COL1A1/FN1 transcription and inhibits the TGF-β/Smad pathway. In MI models, TGF-β1 is overexpressed in ischemic myocardial tissue. Icariin can directly downregulate TGF-β1 and its downstream Smad2/3 signaling, thereby inhibiting IL4-induced M2 macrophage activation and reducing collagen deposition. ²¹

Toxicological studies indicate that the single-dose oral LD₅₀ of icariin in rats ranges from 3.2 to 3.8 g·kg^-1(approximately 160 times the effective dose). Repeated dosing over 90 days caused reversible elevations in liver enzymes aspartate aminotransferase (AST). At doses ≥800 mg·kg^-1·d^-1, testicular atrophy and renal tubular vacuolar degeneration were observed. Long-term use requires regular monitoring of ALT, AST, and serum testosterone levels. ⁵¹ A phase I randomized, double-blind, placebo-controlled trial (n=30) provided the first systematic evaluation of the safety and pharmacokinetics of Epimedium Extract in humans. The results showed that a single oral dose of 370 mg was well tolerated, with a half-life of 12-17 hours. Plasma concentrations remained consistently above the effective threshold required to inhibit TGF-β1/Smad3 signaling and collagen deposition, establishing a safety dose and pharmacokinetic basis for subsequent long-term efficacy studies in MF. ⁵² Regarding drug interactions, icariin, when combined with statins or anti-inflammatory drugs, can alleviate myocardial and vascular fibrosis. In experimental models, it has shown synergistic potential with various cardiovascular drugs, enhancing efficacy while reducing toxicity. However, clinical pharmacokinetic evidence remains limited, and further validation randomized controlled trial (RCT) and systematic drug interaction studies is needed to guide the safety and dose optimization of combination therapies.

3.1.4. Ginsenoside Rg3

Ginsenoside Rg3, derived from the root of Panax ginseng C.A. Mey. (Araliaceae, Panax L.), is one of the primary active components of ginseng. It exhibits a wide range of biological activities such as antibacterial, antitumor, anti-inflammatory, antioxidant, and anti-apoptotic effects. However, its clinical application is limited by rapid biotransformation into secondary metabolites such as Ginsenoside Rh2, mediated by gastric acid, gut microbiota, and P-glycoprotein (P-gp) transporters, resulting in low plasma concentrations and poor bioavailability. To overcome these limitations, nanocarrier-based delivery systems have been employed in both preclinical and clinical studies, significantly increasing the maximum plasma concentration (C_max) and area under the concentration-time curve (AUC) of Ginsenoside Rg3 and its metabolites, thereby establishing a strong foundation for clinical translation. ⁵³

In vivo investigations demonstrate that knockout of the transforming growth factor-β receptor type 1 (TGFBR1) gene alleviates Ginsenoside Rg3-mediated suppression of phosphorylated Smad2/3 and collagen synthesis, contributing to protection against maladaptive ventricular remodeling. In vitro experiments further demonstrated that it interacts with the ATP-binding pocket of TGFBR1, inhibiting its kinase activity. This prevents TGF-β1-induced autophosphorylation of TGFBR1 and phosphorylation of downstream Smad2 at Ser465/467 and Smad3 at Ser423/425. Conversely, overexpression of TGFBR1 via adenoviral vectors partially reverses Ginsenoside Rg3’s inhibitory effects on fibroblast proliferation, collagen synthesis, and Smad2/3 activation. Consequently, Ginsenoside Rg3 inhibits TGFBR1 kinase activity by binding to its ATP pocket, preventing Smad2/3 phosphorylation and nuclear translocation. This reduces Smad3/4 binding to the collagen gene promoters such as COL1A1. Simultaneously, Ginsenoside Rg3 upregulates nuclear Smad7, which recruits SMAD-specific E3 ubiquitin protein ligase 2 (Smurf2) to mediate the ubiquitination and degradation of TGFBR1. This initiates a “kinase inactivation-receptor degradation” cascade that suppresses collagen transcription and MF. ²²

Studies indicate that the effective oral dose of Ginsenoside Rg3 in the CAL HF mouse model ranges from 7.5 to 30 mg·kg^-1·d^-1. At 30 mg·kg^-1·d^-1, Ginsenoside Rg3 significantly increases aminoacylase 1, inhibited TGF-β1/Smad3 signaling, and reduced collagen deposition. In contrast, the high-dose group (≥600 mg·kg^-1·d^-1 in dogs) exhibited reversible ALT elevation and mild tubular vacuolation, suggesting a broad safety margin. ⁵⁴ However, the lack of in vivo pharmacokinetic and pharmacodynamic models for combined drug therapy in cardiovascular conditions makes it difficult to predict dose-plasma concentration relationships in humans. Additionally, the long-term risks of hepatic and renal toxicity, as well as arrhythmia, remain unknown, limiting its broader clinical adoption.

3.1.5. Forsythoside B

Forsythoside B is derived from the dried fruit of Forsythia suspensa (Thunb.) Vahl (Oleaceae, Forsythia Vahl), which contains the highest concentration of this compound. As a key bioactive component of Forsythia suspensa, Forsythoside B exhibits a range of pharmacological activities, including anti-inflammatory, antioxidant, antibacterial, antiviral, anticancer, and antiallergic effects. ⁵⁵ Research has shown that Forsythoside B significantly inhibits ISO-induced collagen synthesis, reduces serum levels of the fibrosis marker TGF-β, and simultaneously downregulates the expression of the collagen-associated protein α-SMA. As a key profibrotic cytokine, TGF-β promotes CFs proliferation and transformation.

Mechanistically, Forsythoside B, via its α,β-unsaturated bond, binds to the ATP-binding pocket of ALK5 (TβRI), inhibiting phosphorylation of Smad3 at C-terminal Ser423/425 and preventing Smad3/Smad4 nuclear translocation. This reduces the enrichment of Smad-binding elements in the COL1A1 promoter region, thereby blocking the TGF-β1/Smad signaling pathway at both receptor and transcriptional levels. Consequently, collagen expression is reduced, leading to a reduction in MF. Further studies have shown that Forsythoside B can attenuate MF and improve cardiac function by inhibiting Smad3 phosphorylation, nuclear translocation, as well as reducing Smad4 protein expression. ²³ This weakens the transcriptional regulation of downstream fibrotic genes, such as collagen III and α-SMA, by the Smad3/Smad4 complex, thereby decreasing excessive ECM deposition.

Both in vitro and in vivo studies confirm that Forsythoside B possesses various biological effects, including anti-inflammatory, antioxidant, and cardioprotective properties. However, high doses or long-term use may result in liver damage and anaphylactoid reactions, limiting its clinical application. Research on drug interactions involving Forsythoside B is still limited, and its potential risks and safety in combination therapy require further systematic investigation.

3.2. Natural Compounds Modulating the JAK/STAT Signaling Pathway

Pachymic acid, sinomenine (SIN), and stachydrine (STA) have been selected for modulating the JAK/STAT signaling pathway due to their distinct mechanisms in combating MF. Pachymic acid inhibits JAK2 phosphorylation, upregulates the negative feedback factor Suppressor of cytokine signaling 3 (SOCS3), and alleviates both inflammation and MF. SIN binds to the ATP binding pocket of JAK2, inhibiting STAT3 activation and reducing the expression of inflammatory factors, thus mitigating fibrosis progression; STA mimics the ATP binding pocket, interfering with JAK2 kinase activity, inhibiting STAT3 phosphorylation, and reducing collagen synthesis. These natural compounds not only exhibit significant advantages in regulating the JAK/STAT pathway but also show good cardiovascular protective effects with low toxicity and favorable pharmacokinetic properties.

3.2.1. Pachymic Acid

Pachymic acid is derived from Poria cocos (also known as Wolfiporia extensa), a saprophytic fungus that grows on various pine trees. As one of the key bioactive components of Poria cocos, pachymic acid exhibits a range of biological activities, including anti-inflammatory, antioxidant, antiviral, and antibacterial effects. Additionally, it demonstrates cardioprotective properties by inhibiting neuronal apoptosis and alleviating MF. These effects suggest that pachymic acid holds promising therapeutic potential for CVDs, including ischemia-reperfusion injury and HF. Pharmacokinetic studies indicate that pachymic acid exhibits poor water solubility and extremely low oral bioavailability, which limits its clinical application. To improve its solubility and oral absorption, several synergistic strategies can be employed, including solid dispersion, micronization, and structural modification by introducing hydrophilic groups. ⁵⁶

Research indicates that cardiomyocyte apoptosis, MF, HF, and ischemia-reperfusion-induced cardiac dysfunction following acute MI are closely associated with the JAK2/STAT3 signaling pathway. Pachymic acid binds to the Glu955-Lys859 region of JAK2, blocking autophosphorylation at Tyr1007/1008, inhibiting STAT3 phosphorylation at Tyr705, and preventing its nuclear translocation. It downregulates the transcriptional levels of genes such as cellular FOS proto-oncogene (c-Fos), cyclin D1, MMP-2, and COL1A1, thereby reducing collagen synthesis and secretion. Simultaneously, it releases the inhibitory effect of STAT3 on the cytokine-regulated SOCS3, allowing SOCS3 to further inhibit JAK2 activity through negative feedback, thus establishing a regulatory loop. Pro-inflammatory cytokines (such as IL-6, TNF-α) induce the activation of SOCS3, which negatively regulates cytokine signaling by inhibiting the JAK/STAT pathway and plays a crucial role in immune and inflammatory regulation. In a rat model of MI, pachymic acid significantly upregulates the expression of p-JAK2/JAK2, p-STAT3/STAT3, and SOCS3 proteins in myocardial tissue. When JAK2/STAT3/SOCS3 pathway activators are administered, the expression of these proteins increases further, reducing the anti-inflammatory effects of pachymic acid. Therefore, pachymic acid exerts cardioprotective effects by inhibiting the JAK2/STAT3/SOCS3 signaling pathway, suppressing inflammatory responses, reducing MF, and ultimately improving cardiac function in AMI rats. ²⁴

In vitro and in vivo studies suggest that the effective concentration of pachymic acid in the HL-1 cell hypoxia/reoxygenation model ranges from 20 to 60 μg·mL^-1. In animal models, the minimum effective dose of 10 mg·kg^-1·d^-1 (administered via intraperitoneal injection for 3 consecutive days) significantly reduces the MI area. Toxicological evaluation reveals a single-dose oral LD₅₀ of 1.8 g·kg^-1, which is 180 times the effective dose. At doses ≥160 mg·kg^-1·d^-1, only mild, reversible opacity and vacuolation in the liver and kidneys were observed, with no significant toxicity in the heart, nervous system, or hematopoietic system. ⁵⁷ Moreover, Pachymic acid’s multi-modal mechanisms, including anti-inflammatory, antioxidant, and calcium overload inhibitory actions are particularly effective in conditions such as myocardial hypertrophy, ischemia-reperfusion injury, and hypertension. Nonetheless, combined pharmacotherapy involving Pachymic acid may pose risks of adverse or toxic reactions, necessitating careful evaluation of potential drug interactions and safety profiles during co-administration.

3.2.2. Sinomenine (SIN)

SIN is derived from Sinomenium acutum (Thunb.) Rehder and E.H. Wilson (Menispermaceae, Sinomenium Diels). As a significant natural bioactive compound, SIN exhibits potent antioxidant effects by scavenging oxygen free radicals and improving pathological conditions such as MF, cardiac hypertrophy, and atherosclerosis through the modulation of myocardial cell physiology. ⁵⁸ However, SIN’s poor solubility in both water and lipids limits its dissolution in gastrointestinal fluids and impairs mucosal penetration. Additionally, it undergoes significant first-pass metabolism via CYP3A enzymes in the intestinal wall and liver, substantially reducing its systemic bioavailability.

The JAK/STAT signaling pathway plays a critical role in regulating immune responses, cell proliferation, inflammation, gene transcription, and fibrotic and oxidative stress responses. JAK2, a widely expressed non-receptor tyrosine kinase, can be activated by various cytokines. Its phosphorylation recruits STAT3, which binds to JAK2’s phospho-tyrosine residues, transmitting extracellular signals intracellularly and regulating gene expression, including Suppressor of Cytokine Signaling 1 (SOCS1). In MF models, the expression levels of p-JAK2, p-STAT3, and SOCS1 proteins are elevated compared to controls. However, following SIN treatment, the expression levels of these proteins were significantly reduced. This mechanism involves the indolequinoline moiety of SIN forming hydrogen bonds with Val863 and Glu864 in the JAK2 ATP pocket, thereby blocking the transfer of ATP γ-phosphate to Tyr1007/1008 and significantly reducing JAK2’s self-phosphorylation efficiency. The inactivated JAK2 is unable to recruit STAT3, resulting in reduced phosphorylation of STAT3 at Tyr705 and inhibiting its nuclear translocation. As a result, the nuclear STAT3 levels decrease, and its binding to the gamma-activated sequence (GAS) element of the SOCS1 promoter is reduced by approximately fourfold. Simultaneously, JAK2-STAT3-mediated IKKβ activation is hindered, which downregulates NF-κB-p65-Ser536 phosphorylation. This ultimately inhibits the transcription of IL-1β, IL-6, and TNF-α, disrupting the JAK2-STAT3-NF-κB profibrotic signaling axis. This suggests that SIN mitigates myocardial injury by suppressing JAK2 and STAT3 phosphorylation, reducing SOCS1 expression, decreasing the release of inflammatory cytokines, enhancing antioxidant capacity, and alleviating inflammation and oxidative stress-induced damage to cardiac tissue. This ultimately reverses MF and improves cardiac function. ²⁵

Both in vitro and in vivo experiments using the ISO-induced mouse model of myocardial hypertrophy and fibrosis demonstrated that oral administration of 120 mg·kg^-1·d^-1 of SIN for 28 days significantly inhibited hypertrophy and collagen deposition. Its LD₅₀ in mice was 1.38 g·kg^-1, approximately 11.5 times the minimum effective dose. The 90-day NOAEL in dogs was 60 mg·kg^-1·d^-1, providing a cardiac safety margin of ≥200-fold. High-dose toxicity primarily affected the liver and gastrointestinal tract, resulting in reversible damage that resolved within 2 weeks after discontinuing treatment. ⁵⁹ Clinical studies suggest that SIN, as a natural compound ingredient with multiple pharmacological activities, when combined with other drugs, can significantly enhance therapeutic effects while reducing side effects. The combination of SIN with statins such as simvastatin or lovastatin has been shown to inhibit vascular endothelial inflammation, modulate immune cell function, and suppress vascular smooth muscle cell proliferation, thereby delaying the progression of atherosclerosis. Notably, short-term co-administration of SIN with CYP3A4 inhibitors like simvastatin or lovastatin may competitively inhibit CYP3A4, leading to elevated plasma concentrations of SIN and an increased risk of toxicity. ⁶⁰ Consequently, careful dose adjustment, treatment duration regulation, and plasma drug concentration monitoring are essential to ensure safety during combination therapy.

3.2.3. Stachydrine (STA)

STA, derived from Leonurus japonicus Houtt. (Lamiaceae, Leonurus L.), is a natural bioactive compound with notable cardiovascular protective effects. It exerts pharmacological actions through multiple signaling pathways, including cardiomyocyte protection, inhibition of myocardial hypertrophy and fibrosis progression, and alleviation of HF. STA holds promising potential for preventing and treating CVDs. STA is rapidly absorbed after oral administration. In pathological models characterized by blood deficiency, cold coagulation, blood stasis, or renal Yang deficiency, its half-life and mean residence time are prolonged, while clearance (CL) is reduced. These findings suggest that certain pathological conditions may delay drug elimination and enhance bioavailability. ⁶¹

In terms of molecular mechanisms, the JAK/STAT signaling pathway plays a critical role in stress-induced myocardial hypertrophy and ischemia/reperfusion-induced cardiac dysfunction. The JAK family includes JAK1, JAK2, JAK3, and TYK2, with JAK1 and JAK2 widely distributed across various tissues, while JAK3 is primarily expressed in leukocytes. STA, as a direct substrate of JAK, can transmit signals directly to the cell nucleus to regulate specific gene expression. STA binds to the Gly993-Leu1010 region of JAK2 kinase through an “ATP-pocket mimic” mechanism, forming dual hydrogen bonds with Leu1010 and Glu1007. This interaction locks the kinase in a “DFG-out” inactive conformation, preventing STAT3 from approaching the catalytic center and reducing phosphorylation efficiency at the Tyr705 site. Unphosphorylated STAT3 loses its ability to bind importin α3, significantly reducing its nuclear translocation. Additionally, the DNA-binding domain of STAT3 shows a sharp decrease in affinity for the GAS, halting collagen transcription and inhibiting the abnormal activation of the myocardial JAK2-STAT3 pathway. Based on these mechanisms, STA exerts dual protective effects in both platelets and cardiac tissue. It suppresses proinflammatory cytokines (e.g., IL-1β, IL-6, TNF-α), reducing downstream inflammatory markers (IL-1β, IL-10β, TNF-α) and oxidative stress markers (MDA, ROS). ²⁶ Simultaneously, it enhances superoxide dismutase activity, improves interactions between platelets and neutrophils, and reduces thrombosis formation while inhibiting platelet-mediated thromboinflammation. Moreover, it downregulates the expression of STAT3 target genes (IL-1β, IL-10β, TNF-α), decreases the collagen I/III ratio, reduces the Masson’s fibrosis area, and lowers the expression of ANP/β-MHC, ultimately attenuating MF and providing comprehensive cardiovascular protection.

In vivo experiments using the transverse aortic constriction (TAC) mouse model demonstrated that oral administration of 12 mg·kg^-1·d^-1 STA for 4 weeks significantly improved ejection fraction and shortening fraction, enhanced the end-systolic pressure-volume relationship, and inhibited collagen deposition and fibroblast activation. Toxicological studies show that in acute toxicity tests on mice, the LD₅₀ of STA ranges from 1.8 to 2.0 g·kg^-1, about 150 to 200 times the effective dose, indicating a wide safety margin. ⁶² However, these findings have only been validated in preclinical animal models, and human dose-response data are lacking. Consequently, this combination therapy has not yet been widely adopted in clinical practice.

3.3. Natural Compounds Modulating the Wnt/β-Catenin Signaling Pathway

3.3.1. Triptolide (TP)

TP is derived from Tripterygium wilfordii Hook.f. (Celastraceae, Tripterygium Hook.f.) and exhibits anti-inflammatory, antiproliferative, immunomodulatory, and proapoptotic effects. In CVDs research, TP significantly alleviates stress-induced cardiac injury in rats by downregulating pro-inflammatory cytokines (IL-1β and IL-6) and profibrotic factors, while effectively suppressing the abnormal activation of CFs. ⁶³ The Wnt/β-catenin signaling pathway plays a pivotal role in cardiac development, particularly in cardiomyocyte proliferation and differentiation. Aberrant activation of this pathway is closely associated with the progression of MF.

Research shows that TP synergistically blocks Wnt/β-catenin signaling through dual binding sites. First, TP covalently binds to the PDZ domain of DVL2, preventing CK1δ phosphorylation at Ser143/Thr224 and inhibiting GSK-3β dissociation from the destruction complex, which maintains β-catenin in a phosphorylated, degradation-prone state. Simultaneously, TP occupies the Arg235-Glu290 site within the β-catenin Arm repeat 3-4 region, disrupting its high-affinity binding to TCF4/LEF1, thereby weakening transcriptional activation of downstream profibrotic genes. Second, TP inhibits c-Myc’s transcriptional repression of Smad7 by blocking c-Myc transcription, which is β-catenin/TCF4-dependent. This elevates Smad7 levels, promoting the ubiquitination and degradation of Smad3, thereby synergistically inhibiting the Wnt/β-catenin and TGF-β/Smad signaling pathways and attenuating their profibrotic effects. In vitro studies show that this mechanism effectively counteracts ISO-induced fibroblast proliferation and differentiation. In a rat model of cerebral ischemia/reperfusion injury, TP reduces the stability of intracellular free β-catenin, inhibits its nuclear translocation, and blocks its binding to TCF/LEF transcription factors, downregulating the transcriptional activation of c-Myc and Cyclin D1. Additionally, TP may promote β-catenin degradation by activating GSK-3β, reducing its abnormal intracellular accumulation. The Wnt/β-catenin agonist BML-284 partially reverses TP’s inhibitory effect on this pathway, upregulating the protein levels of β-catenin, c-Myc, and Cyclin D1⁴⁹.

In rodent models of MF, the minimum effective dose of TP was 50 μg·kg^-1 (administered via intraperitoneal injection every other day for 4 weeks), which significantly inhibited β-catenin nuclear translocation and reduced myocardial collagen deposition. However, safety studies show that the single-dose oral LD₅₀ in mice is only 0.83 mg·kg^-1, with a therapeutic index of approximately 16. When administered at doses ≥ 0.2 mg·kg^-1·d^-1 via oral gavage for 28 consecutive days, reversible liver and kidney damage occurs. ²⁷ These findings provide theoretical support for the potential use of Tripterygium glycosides in treating MF, though their poor water solubility and high toxicity limit clinical applications.

3.3.2. Salidroside (SAL)

SAL, derived from Rhodiola rosea L. (Crassulaceae, Rhodiola L.), is a plant that primarily grows in extreme environments, such as high altitudes and areas with low oxygen levels. As the main active compound of Rhodiola, SAL exhibits a broad range of pharmacological effects, including anti-fatigue, anti-tumor, anti-inflammatory, anti-aging, antioxidant, and immunomodulatory properties. Pharmacokinetic studies show that SAL is rapidly absorbed after oral administration, but it has low bioavailability. When administered intravenously, it primarily distributes to the liver, lungs, and heart. Approximately half of the compound is excreted unchanged in urine after undergoing deglycosylation and sulfation in the liver. Recent research has demonstrated that using whole cells of Aspergillus oryzae for region-selective acylation at the 6′-OH position of SAL, in a non-aqueous phase, significantly enhances its lipophilicity. The acylated product exhibits prodrug and sustained-release characteristics, which improve membrane permeability and oral absorption. ⁶⁴

Regarding the mechanism of action against MF, SAL regulates two key signaling pathways: TGF-β1/Smad3 and Wnt/β-catenin. Research has shown that SAL binds to the Armadillo repeat domain (repeats 5-6) of β-catenin, disrupting the β-catenin/Smad3 transcriptional complex. Additionally, SAL occupies the hydrophobic pocket of the Wnt1/3a ligand-FZD receptor, inhibiting downstream phosphorylation of DVL2. As a result, SAL suppresses Wnt/β-catenin signaling activation at multiple levels. Consequently, it not only downregulates the expression of TGF-β1 and interleukin-1 to reduce cardiomyocyte inflammatory responses but also alleviates oxidative stress and excessive ECM deposition, thereby reducing MF. In diabetic cardiomyopathy models, β-catenin acts as a co-activator for several transcription factors, showing synergistic expression with Smad3. This suggests significant cross-regulation between the TGF-β1/Smad3 and Wnt/β-catenin signaling pathways. During both embryonic development and pathological conditions, SAL reduces the release of inflammatory factors and collagen secretion by inhibiting the abnormal activation of the Wnt1/3a/β-catenin signaling pathway. It also blocks β-catenin-Smad3 synergy, stabilizes the GSK-3β complex, and accelerates β-catenin degradation, significantly alleviating diabetes-induced cardiac injury and MF. ²⁸

In vivo studies showed no apparent adverse effects following continuous oral administration of 40 mg·kg^-1·d^-1 of SAL for 7 weeks. Toxicology studies indicate that the single-dose LD₅₀ in rats is 2.65 g·kg^-1. Primary toxic effects were reversible, including serum transaminase elevation and renal tubular epithelial swelling, but no significant cardiac damage was observed at up to 30 times the therapeutic dose. This suggests a broad safety margin for Rhodiola rosea extract in cardiac disease applications. ⁶⁵ Notably, the Wnt/β-catenin signaling pathway has dual regulatory effects during fibrosis: it directly modulates the expression of profibrogenic genes via β-catenin nuclear translocation and indirectly amplifies or suppresses the fibrotic response through interactions with the TGF-β1 signaling pathway. This cross-talk plays a crucial role in maintaining tissue homeostasis and regulating fibrosis.

3.3.3. Saikosaponin a (SSA)

SSA, derived from Bupleurum chinense DC. (Apiaceae, Bupleurum L.), exhibits multiple pharmacological activities, including anti-inflammatory and antioxidant effects, and plays a crucial role in regulating cardiac remodeling. During hepatic stellate cell activation, SSA modulates the expression of bone morphogenetic protein 4. In chemically induced rat models of hepatic inflammation and fibrosis, SSA demonstrates significant protective effects. ⁶⁶

Mechanistic studies have shown that SSA prevents the phosphorylation of Ser1490 in the intracellular domain of low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) induced by Wnt3a. This inhibition disrupts the GSK-3β/CK1ε-mediated phosphorylation of the PDZ domain of downstream DVL2, enhancing the activity of the APC/Axin/GSK-3β complex. Consequently, sustained phosphorylation of β-catenin at Ser33/Ser37/Thr41 triggers beta-transducin repeat-containing protein and proteasomal degradation. The absence of ligand-bound β-catenin in the nucleus prevents TCF4/LEF1 from initiating the expression of epithelial-mesenchymal transition-related transcription factors such as Snail1, Slug, and Twist, thus blocking the transcriptional reprogramming required for EndoMT. Furthermore, SSA inhibits TGF-β type I receptor (ALK5)-mediated phosphorylation of Smad2 in the linker domain (Ser245/250/255), weakening Smad3-Smad4 complex formation and its synergistic transcriptional activity with β-catenin. This dual molecular disruption severs the positive feedback loop between TGF-β/Smad and Wnt/β-catenin pathways, ultimately reducing fibroblast activation and collagen deposition. ²⁹ Under conditions of classic Wnt signaling inactivation, cytoplasmic β-catenin associates with Adenomatous polyposis coli, Axis inhibition protein, and Glycogen synthase kinase-3β, leading to its phosphorylation, ubiquitination, and degradation. Binding of Wnt ligands to FZD and LRP receptors inhibits the destruction complex, stabilizing β-catenin and promoting its nuclear translocation. Nuclear β-catenin then interacts with TCF/LEF transcription factors to facilitate EndoMT. TGF-β further enhances canonical Wnt signaling by downregulating AXIN2 expression in fibroblasts. In vitro and in vivo experiments show that SSA significantly alleviates HF and MF induced by long-term pressure overload. High-dose SSA blocks the conversion of CFs to myofibroblasts, inhibiting TGF-β-induced fibroblast proliferation, activation, and collagen secretion. In contrast, low-dose SSA suppresses TGF-β-induced EndoMT and pressure overload-induced cardiac remodeling by inhibiting the activation of the Wnt/β-catenin signaling pathway in mouse myocardial endothelial cells. ²⁹

Animal studies show that oral administration of SSA (300 mg·kg^-1·d^-1) for one week induces liver damage and hepatocyte apoptosis in mice. ⁶⁷ Overall, SSA exerts a protective effect against MF and remodeling by modulating the Wnt/β-catenin signaling pathway and synergistically inhibiting TGF-β-related fibrotic signaling, highlighting its potential as a novel therapeutic agent for MF.

3.3.4. Resveratrol

Resveratrol is a polyphenolic compound derived from the grape species Vitis vinifera L. (Vitaceae, Vitis L.) and is also found in the skins of blueberries, raspberries, and mulberries, as well as in peanuts. It is naturally synthesized by plants in response to biological or environmental stressors, exhibiting significant biological properties, including anti-inflammatory and antioxidant effects. Despite its potential, resveratrol’s rapid metabolism and low bioavailability have historically limited its clinical application. However, recent advancements in nanotechnology have significantly improved its stability and tissue absorption, enhancing its distribution in organs such as the brain, liver, and kidneys.

Research indicates that resveratrol exerts cardioprotective effects by modulating the Wnt/β-catenin signaling pathway. Secreted frizzled-related proteins are key regulators in this pathway, influencing Wnt ligand-receptor interactions by binding to Frizzled (Fz) receptors. In studies on organ fibrosis, sFRP2 has been found to be overexpressed. Blocking sFRP2 with specific antibodies and downregulating myocardial Wnt signaling significantly reduced MF and improved cardiac function. Further studies show that resveratrol downregulates sFRP2 expression in a SIRT1-dependent manner. This reduces the competitive binding between sFRP2 and Wnt3a, decreasing the amount of Wnt3a available to bind FZD and thereby inhibiting the initiation of the canonical Wnt signaling pathway. Compared to untreated controls, resveratrol treatment significantly decreased immunofluorescence staining for sFRP2, Wnt3a, and β-catenin in diabetic rat myocardial tissue. Additionally, the expression of downstream target genes Cyclin D1 and c-Myc was reduced, further inhibiting the abnormal activation of the Wnt/β-catenin pathway. ⁶⁸ In the context of dilated cardiomyopathy, resveratrol preconditioning combined with mesenchymal stem cell administration has shown superior therapeutic efficacy, mainly by suppressing sFRP2 expression in fibroblasts and reducing MF. Elevated sFRP2 levels are closely associated with fibrosis and activation of the Wnt3a/β-catenin pathway. Resveratrol-preconditioned mesenchymal stem cells significantly downregulate sFRP2, Wnt3a, and β-catenin, alleviating MF. In an ex vivo mouse preantral follicle model, resveratrol demonstrated protective efficacy within the concentration range of 5-10 μM (approximately 1.1-2.3 μg·mL^-1), with no observed DNA fragmentation. However, when the resveratrol concentration was increased to 10 μM without co-administration of doxorubicin, follicle survival rates were lower than in the control group, suggesting a potential pro-oxidative effect. Therefore, a safety upper limit of 10 μM is recommended for ovarian protection, with concurrent monitoring of oxidative stress markers. ³⁰

The clinical efficacy of resveratrol in patients with hypertension complicated by MF has been validated in a prospective RCT. The study enrolled 80 patients with primary hypertension, who received standardized antihypertensive therapy in addition to either 400 mg of resveratrol daily (n = 43) or continued baseline therapy (n = 37) for 6 consecutive months. Results demonstrated that resveratrol significantly reduced the cardiac MRI delayed enhancement area, improved the E/e' ratio, and enhanced global longitudinal strain, with minimal adverse effects. A twelve-month follow-up revealed significant antifibrotic benefits, providing evidence-based support for the long-term clinical application of resveratrol. ⁶⁹ Additionally, resveratrol has shown significant cardiovascular protective effects, good oral tolerability, and high bioavailability, particularly when combined with other drugs. In rat models of MI, resveratrol, whether administered alone or in combination with valsartan, significantly reduced left ventricular dilatation, improved ejection fraction, and decreased levels of oxidative stress markers and inflammatory cytokines. Furthermore, resveratrol inhibits collagen deposition and reduces MF, improving cardiac structure and function. Despite its safety, co-administration with warfarin increases bleeding risk, contraindicating its use in patients on anticoagulants, antiplatelet agents, or non-steroidal anti-inflammatory drugs.

3.3.5. Berberine

Berberine, an isoquinoline alkaloid derived from Coptis chinensis Franch. (Ranunculaceae, Coptis Adans.), is a widely used natural compound with a broad range of therapeutic applications. It has been shown to reduce the incidence of CVDs and is associated with several beneficial effects, such as improving endothelial function, regulating dyslipidemia, inhibiting LDL oxidation, and lowering blood pressure. Despite its potent biological effects, berberine suffers from low oral bioavailability in vivo, primarily due to poor intestinal absorption and rapid metabolic degradation. To overcome these limitations, novel approaches, including nanodelivery systems and structural modifications, are being explored to enhance its bioavailability and therapeutic efficacy.

Research indicates that the Wnt 5a/β-catenin signaling pathway plays a crucial role in myocardial protection and cardiac remodeling. Abnormal activation of this pathway promotes MF and exacerbates remodeling. Thus, targeting myocardial Wnt signaling has emerged as a promising strategy for improving ischemic cardiac injury. After MI, significant fibrotic responses in cardiac tissue are linked to dysregulated activation of the Wnt pathway. Berberine has shown potent antifibrotic effects in MI mouse models, mainly by inhibiting the aberrant activation of the Wnt/β-catenin pathway. Berberine exerts its antifibrotic effects through two mechanisms: First, it binds directly to the lipid-modifying pocket (Cys90-Thr94 region) of the Wnt5a ligand, blocking its interaction with the Frizzled2 receptor and co-receptor LRP6. This inhibition prevents the phosphorylation of DVL2, maintaining the activation of GSK-3β. Consequently, β-catenin undergoes sustained phosphorylation, ubiquitination, and proteasomal degradation. Second, berberine binds to the Armadillo repeat domains (Arm repeats 5-6) of β-catenin, preventing its nuclear translocation and inhibiting its interaction with TCF/LEF transcription factors. This reduces the transcriptional activity of profibrotic genes such as c-Myc and Cyclin D1, further attenuating fibrosis. Experimental results show that berberine significantly reduces Wnt5a protein expression, blocks β-catenin nuclear translocation, and suppresses the secretion of proinflammatory factors, leading to reduced MF and ventricular remodeling, ultimately improving cardiac function. ³¹

In a clinical study involving 130 patients with acute coronary syndrome, berberine (300 mg, three times daily for 30 days) was added to standard therapy. Compared to baseline values, patients showed significantly reduced serum levels of intercellular adhesion molecule-1, MMP-9, and vascular cell adhesion molecule-1. These findings suggest that berberine inhibits the inflammation-fibrosis cascade and reduces myocardial interstitial collagen deposition, providing clinical evidence for its antifibrotic effects. ⁷⁰ Regarding safety, clinical studies primarily report mild gastrointestinal reactions, such as diarrhea and constipation. In a study involving 36 diabetic patients, treatment with berberine (500 mg·d^-1, divided into three doses) for 13 weeks resulted in adverse reactions in 34.5% of patients. These included abdominal distension (19.0%), diarrhea (10.3%), abdominal pain (3.4%), and constipation (6.9%). Most adverse reactions were transient and resolved within the first 4 weeks of treatment. ⁷¹ Concerning drug interactions, studies on the combination of berberine and irbesartan show that the two drugs enhance berberine’s bioavailability by inhibiting the glycoprotein P-gp in the intestine, prolonging its exposure to CYP3A4. Conversely, berberine increases plasma concentrations of irbesartan with minimal impact on its metabolism. Co-administration results in elevated plasma levels of both drugs compared to monotherapy, suggesting a pharmacokinetic interaction that may optimize therapeutic outcomes.

3.4. Natural Compounds Modulating the NLRP3 Signaling Pathway

3.4.1. Artemisinin

Artemisinin is a sesquiterpene lactone compound with an endoperoxide bridge, primarily derived from Artemisia annua L. (Asteraceae, Artemisia), a traditional Chinese medicinal plant. It is one of the most effective antimalarial drugs, following pyrimidine, chloroquine, and primaquine in efficacy. As the core active ingredient in artemisinin, it not only rapidly clears malaria parasites but also induces tumor cell apoptosis, inhibits the cell cycle, and suppresses tumor angiogenesis and metastasis. Despite its beneficial effects, artemisinin has poor water solubility, low oral absorption, and instability, with a short half-life and significant hepatic first-pass metabolism. These factors result in a rapid decline in blood concentration, limiting its sustained therapeutic effects. To enhance its bioavailability, forming inclusion complexes with β-cyclodextrin or γ-cyclodextrin at a 1:1 molar ratio improves its solubility and reduces the risk of relapse in malaria treatment.

Research has shown that artemisinin’s protective effect against MF is largely due to its dual inhibition of the inflammasome and NF-κB signaling pathways. The inflammasome is a critical intracellular multiprotein complex, composed of ASC, nucleotide-binding oligomerization domain, and caspase-1. Among these components, the NLR family—including NLRP1, NLRP3, NLRC4, and NLRC5—plays a vital role in inflammasome activation, with NLRP3 being involved in various inflammation-related diseases such as Alzheimer’s disease, atherosclerosis, and ischemia/reperfusion injury. In rat cardiomyocytes after MI, artemisinin inhibits the NF-κB pathway through two molecular mechanisms. First, the peroxide bridge of artemisinin, activated by heme-Fe²⁺, covalently binds to Cys279 in the NLRP3-NACHT domain, obstructing ATP hydrolysis and NLRP3 oligomerization, which prevents inflammasome scaffold formation. Second, the radical oxidizes Cys99 in the activation loop of IKKβ, an upstream kinase in the NF-κB pathway, blocking IKKβ-mediated phosphorylation of IκBα. This prevents the degradation of IκBα via the ubiquitin-proteasome pathway, trapping the NF-κB p65/p50 heterodimer in the cytoplasm. Consequently, the heterodimer cannot translocate into the nucleus to bind NLRP3 and the κB site on the pro-IL-1β promoter. As a result, artemisinin reduces both NLRP3 protein expression and its functional activity, inhibiting caspase-1 cleavage of pro-IL-1β and the release of mature IL-1β. This weakens the autocrine loop of IL-1β signaling to TGF-β1, reducing Smad2/3 phosphorylation and collagen I/III deposition, thereby mitigating MF. Further studies show that following ischemia-reperfusion injury, levels of NLRP3, ASC, IL-1β, TNF-α, IL-6, and other inflammatory mediators significantly increase in cardiac tissue. Artemisinin treatment markedly reduces the expression of these mediators, including IL-1β and TNF-α. Additionally, artemisinin also activates the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling axis, suppressing NLRP3 inflammasome activation and inflammation, thereby preventing fibroblast transdifferentiation into myofibroblasts. ³² Concurrently, artemisinin significantly improves MF by inhibiting the TGF-β1/Smad3 pathway and reducing collagen I/III deposition.

Toxicological studies have shown that artemisinin analogs (e.g., artemether) induce embryonic lethality at doses ≥12 mg·kg^-1·d^-1 with continuous administration for ≥12 days. This toxicity is linked to depletion of embryonic erythrocyte precursors, impaired myocardial wall development, and delayed ossification of long bones. Its toxic concentration is only 1.2-2.4 times that of the effective antifibrotic dose (10 mg·kg^-1·d^-1 in mice), indicating a low therapeutic index. In contrast, adult animals administered 40 mg·kg^-1·d^-1 showed only a reduction in reticulocytes and mild anemia, with no structural damage to major organs (liver, kidney, heart). ⁷² Currently, research on drug interactions involving artemisinin in the treatment of MF is limited. Its clinical application requires careful administration, guided by a thorough assessment of the therapeutic window and potential toxicity risks.

3.4.2. Celastrol

Celastrol is a bioactive compound extracted from the root bark of Tripterygium wilfordii Hook.f. (Celastraceae) and other plants in the same family, such as southern snake vine. It demonstrates significant anti-inflammatory activity. Celastrol, the primary active component, exhibits broad pharmacological effects across multiple inflammation-related disease models. However, its clinical application is limited by pharmacokinetic challenges, including low bioavailability, poor water solubility, and substantial toxicity toward non-target cells. To address these issues, strategies like direct covalent modification have been employed to enhance Celastrol’s solubility and stability, significantly improving its bioavailability.

In inflammation and MF, NLRP3 plays a key role as a molecular mediator. Celastrol suppresses MF by downregulating NLRP3, ASC, IL-1β, and IL-18. Research shows that Celastrol homofuranone covalently modifies Cys279 in the NLRP3-NACHT domain, inhibiting ATP hydrolysis and oligomerization, while also preventing ASC recruitment. This, in turn, suppresses IKKβ Ser177/181 phosphorylation, reduces NF-κB p65 nuclear translocation, and lowers transcription levels of NLRP3 and pro-IL-1β. Additionally, inhibiting ASC spot formation decreases caspase-1 self-cleavage activity and reduces the release of mature IL-1β/IL-18. These effects disrupt downstream signaling pathways, including the TAK1-JNK/ERK-c-Jun and JAK-STAT pathways, inhibit TGF-β1 transcription, and suppress fibroblast proliferation, ultimately blocking the inflammation-fibrosis cascade. ³³

Toxicological and clinical studies have shown that Celastrol inhibits the human ether-à-go-go-related gene potassium channel (IC₅₀ = 0.83 μM), leading to QT interval prolongation and an increased risk of torsades de pointes. This concentration overlaps with its effective anti-inflammatory range (0.5-1 μM), indicating a narrow cardiac safety margin. In animal models, doses of ≥1-3 mg·kg^-1·d^-1 induced multiorgan toxicity, including decreased heart rate, tubular vacuolar degeneration, and reduced testicular weight. Additionally, zebrafish embryo models showed pericardial edema and slowed heart rate at Tripterygium glycoside concentrations ≥0.5 μM⁶⁵. Despite its broad pharmacological activity, Celastrol’s poor solubility, low bioavailability, and significant multi-organ toxicity necessitate structural modifications or advanced delivery systems to mitigate toxicity and facilitate clinical translation.

3.4.3. Oridonin

Oridonin, a natural diterpenoid compound derived from Isodon rubescens (Hemsl.) H.Hara (Lamiaceae, Isodon (Schrad. ex Benth.) Kudo), exhibits diverse pharmacological activities, including anticancer, anti-inflammatory, hepatoprotective, and cardioprotective effects. As its primary active component, oridonin exerts significant biological effects in various inflammation-related diseases. Following oral administration, it is rapidly absorbed, though its bioavailability remains low due to significant first-pass metabolism, primarily mediated by hepatic CYP3A4 and CYP2C9 enzymes, along with a high plasma protein binding. To enhance its aqueous solubility and oral bioavailability, various structural modifications and delivery systems, such as cyclodextrin inclusion complexes, nanocrystals, and solid dispersions, have been explored.

Mechanistically, oridonin directly interacts covalently with NLRP3 inflammasome components, inhibiting its activation without affecting ATPase activity. Both in vitro and in vivo studies have demonstrated its potent anti-inflammatory properties. Proper modulation of inflammation can facilitate myocardial healing post-infarction, while excessive inflammation can lead to MF, impairing cardiac function and potentially resulting in HF. After MI, elevated IL-1β levels within the infarct zone stimulate pro-inflammatory responses in monocytes, which primarily originate from the bone marrow and spleen—both of which rapidly respond to inflammation. Studies have shown that oridonin exhibits significant anti-inflammatory activity in both in vitro and in vivo models. In animal models, it reduces MF induced by myocardial inflammation, improving cardiac remodeling and protecting cardiac function. In comparison to untreated animals, oridonin significantly suppresses NLRP3 inflammasome activation in mice following MI, inhibiting the expression of caspase-1, IL-1β, and IL-18. This anti-inflammatory and antifibrotic effect occurs through the following molecular mechanism: oridonin covalently binds to NLRP3, occupying the Gly227-Phe279 segment of its NACHT domain, blocking the hydrophobic-electrostatically complementary interface, and disrupting nucleotide-dependent oligomerization of the NLRP3-ASC complex. This inhibits the recruitment of the caspase-1 precursor and its autocatalytic cleavage at Loop 3 (Tyr360-Phe381), reducing the maturation and release of IL-1β and IL-18, and attenuating the “inflammation-fibrosis” cascade. ³⁴

In terms of toxicology and clinical safety, studies using zebrafish models show that low concentrations (115-190 mg/L) of rabdosia rubescens promote spontaneous movement and hatching, while higher concentrations exert inhibitory effects. The half maximal effective concentration (EC₅₀) is 412 μg·kg^-1, and the toxic concentration causing malformations is only 2-3 times higher than the effective concentration, indicating a narrow therapeutic index. In rodent models, a 28-day repeated-dose toxicity study revealed reversible weight loss and elevated serum ALT levels at doses exceeding 30 mg·kg^-1·d^-1. The liver and kidneys were identified as the primary target organs for toxicity, with recovery observed within 4-6 weeks after discontinuation. ⁷³ When combined with cardiovascular drugs, liposomal oridonin co-delivered with nicorandil prolongs its half-life fourfold in rat ischemia-reperfusion models. This combination synergistically suppresses NLRP3 inflammasome activation, reduces oxidative stress, and mitigates reperfusion-induced MF, providing anti-inflammatory, antioxidant, and antifibrotic protection.

3.5. Natural Compounds Modulating the PPAR-γ Signaling Pathway

3.5.1. Curcumin

Curcumin, a lipophilic polyphenolic compound derived from the rhizomes of Curcuma longa L. (Zingiberaceae, Curcuma L.), is the primary active component responsible for the wide range of pharmacological effects attributed to this plant. These include antiproliferative, antioxidant, anti-inflammatory, anticancer, and anti-infective activities, making curcumin a promising candidate for therapeutic applications, especially in cardiovascular protection. Pharmacokinetically, curcumin undergoes extensive metabolism in the small intestine, liver, and kidneys, where it is converted into curcumin glucuronide, curcumin sulfate, and methylated curcumin. These metabolites are rapidly excreted in urine and feces. However, its clinical use is limited by poor solubility, low intestinal absorption, and rapid systemic metabolism. Recent research has shown that piperine, the primary alkaloid in black pepper, can increase curcumin’s bioavailability by approximately 20-fold. Additionally, nanoencapsulation technology has significantly improved curcumin’s therapeutic efficacy by increasing its solubility and prolonging its half-life, addressing key challenges in its clinical application. ⁷⁴

At the mechanistic level, PPAR-γ regulates multiple physiological processes. Its ligands can inhibit excessive ECM production and cell proliferation in Ang II-induced CFs. Studies on curcumin’s effects on MF in spontaneously hypertensive rats and in rat CFs in vitro have demonstrated that curcumin dose-dependently inhibits Ang II-induced expression of type III collagen and FN1. Notably, the PPAR-γ antagonist GW9662 partially reverses this inhibitory effect. Curcumin binds to the hydrophobic pocket of the PPAR-γ ligand-binding domain (LBD), inducing a conformational change in helix-12 and recruiting coactivator SRC-1 to form a transcriptionally active PPAR-γ/SRC-1 complex. This complex inhibits the binding of NF-κB (p65) and Smad3 to the TGF-β1 promoter region, blocking Smad3 nuclear translocation. It also upregulates IκBα expression and inhibits IKKβ-mediated phosphorylation of Ser32/Ser36, attenuating the NF-κB downstream kinase cascade. As a result, the expression of connective tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1) is downregulated, significantly alleviating MF. CTGF and PAI-1, both profibrotic factors, are strongly induced by TGF-β, whose expression is partially regulated by PPAR-γ. In a spontaneously hypertensive rat model, both mRNA and protein levels of CTGF and PAI-1 were significantly elevated, suggesting their involvement in hypertension-associated MF. Curcumin’s upregulation of PPAR-γ activity led to a marked reduction in CTGF and PAI-1 expression, both in vivo and in vitro, exerting a protective effect against hypertension-induced MF. ³⁵

In a double-blind, randomized clinical trial involving 33 patients diagnosed with coronary heart disease, participants received either curcumin or a placebo. The curcumin group was given 500 mg curcumin capsules four times daily for 8 weeks. Only two patients experienced mild gastrointestinal symptoms, such as diarrhea. No significant changes in urea, creatinine, or high-density lipoprotein cholesterol levels were observed when compared to the placebo group. These results suggest that 4 g/day of curcumin is well-tolerated in humans. However, potential cumulative toxicity with longer durations, doses ≥8 g/day, or intravenous formulations warrants further investigation. ⁷⁵ Regarding drug interactions, the combined use of curcumin and metformin activates Nrf2, inducing HO-1 expression and enhancing cellular antioxidant defenses. This combination also inhibits the phosphorylation of JAK2 and signal transducer and activator of STAT3, reducing the release of pro-inflammatory cytokines such as TGF-β1 and IL-6, thereby attenuating inflammatory responses and providing cardioprotective effects.

3.5.2. Propolis Extract (PE)

PE, derived from the Western honeybee (Apis mellifera), contains a complex array of active compounds with diverse pharmacological effects, including antioxidant, antibacterial, antiviral, antiparasitic, anti-inflammatory, and wound-healing properties. However, its clinical application is hindered by pharmacokinetic challenges, such as low bioavailability, poor solubility, insufficient effective concentrations, and rapid metabolism. Nanocarriers can improve the pharmacokinetic profile of PE by enhancing its bioavailability, solubility, absorption, and half-life, and by enabling targeted release. The utilization of nanocarriers can prolong its half-life in therapeutic contexts, improve pharmacokinetic profiles, and enhance bioavailability, solubility, absorption, and targeted release properties.

PE upregulates PPAR-γ, a transcription factor in the nuclear hormone receptor superfamily, providing cardioprotection after MI. This protection occurs through mechanisms such as reducing inflammation, decreasing sympathetic nervous system activity, and alleviating ER stress. The PPAR-γ antagonist GW9662 reverses these effects. The main active component of PE, caffeic acid phenethyl ester (CAPE), binds to the ligand-binding domain of PPAR-γ, stabilizing its activated form and promoting coactivator SRC-1 binding, which enhances PPAR-γ transcriptional activity. Additionally, CAPE inhibits phosphorylation of the NF-κB p65 subunit at Ser536 by binding to PPREs, preventing NF-κB from binding to κB sites on the promoters of inflammatory cytokines such as IL-1β and TNF-α. CAPE also inhibits IKKβ kinase activity, preventing IκBα degradation and further blocking NF-κB nuclear translocation. This multi-tiered inhibition of NF-κB-mediated inflammation is completely reversed by GW9662 in LPS-induced macrophage models, confirming the crucial role of PPAR-γ in PE’s anti-inflammatory effects. In MI rat models, PE significantly reduced IL-1β and TNF-α mRNA expression levels and suppressed NF-κB-mediated inflammation. Subsequent studies showed that the reduction of these inflammatory markers delayed ventricular remodeling after MI and improved cardiac function. Moreover, PE directly binds to the Rel homology domain of the NF-κB p65 subunit, inhibiting its DNA-binding activity and reducing Ang II-mediated Ang II type 1 receptor activation. This mechanism decreases IL-1β, TNF-α, plasma C-reactive protein (CRP), and Ang II levels, alleviating inflammation and cardiac injury post-MI. ³⁶

In vitro safety studies demonstrated that concentrations of 0.25-0.5 mg·mL^-1 effectively killed 90% of staphylococci and inhibited biofilm formation. At 1 mg·mL^-1, no significant toxicity was observed in human dermal fibroblasts (cell survival >90%), with mild inhibition of keratinocytes (survival rate ∼60%). Significant cytotoxicity (survival rate ∼20%) occurred only at 2.5 mg·mL^-1, yielding a therapeutic index ≥4. No damage to the liver, kidneys, or hematopoietic system was observed, suggesting the safety of PE for short-term topical use. ⁷⁶ Clinically, nano-formulated PE significantly reduces serum levels of CK-MB, cardiac troponin I, and lactate dehydrogenase, reversing doxorubicin-induced myocardial damage and exhibiting potent cardioprotective effects.

3.5.3. Procyanidin B2

Procyanidin B2 is a natural food pigment derived from the Vitis vinifera L. (Vitaceae) seeds. It is widely distributed in the seeds, fruits, and leaves of various plants, such as grapes, hawthorn, black beans, and blueberries. Procyanidin B2 exhibits several biological activities, including anti-inflammatory, antioxidant, and anti-apoptotic effects, and shows promise in preventing and treating diseases like diabetes, its complications, atherosclerosis, and non-alcoholic fatty liver disease. Additionally, Procyanidin B2 modulates multiple signaling pathways.

Research indicates that Procyanidin B2 binds to the hydrophobic pocket of the PPAR-γ (LBD, aa 206-477) in a concentration-dependent manner. It forms hydrogen bonds and π-π stacking interactions with key residues (His323, Tyr327, Cys285, and Phe363), stabilizing the activation function-2 helix within the PPAR-γ activation domain in its activated conformation. This interaction blocks the Ang II-induced PKC-ERK1/2 kinase cascade, inhibits the nuclear translocation of the transcription factors Elk1 and c-Jun, and reduces their transcriptional activation at the activator protein-1 site of the TGF-β1 promoter. Simultaneously, Procyanidin B2 upregulates the recruitment of the PPAR-γ coactivator SRC-1, enhancing the binding activity of PPREs in the Smad7 promoter region. This, in turn, reverses the inhibition of Smad3 phosphorylation and the expression of profibrotic genes like COL1A1 and FN1, ultimately suppressing Ang II-induced cell proliferation, differentiation, and collagen accumulation. These effects can be blocked by the PPAR-γ antagonist GW9662 or RNA interference. ³⁷ Moreover, Procyanidin B2 alleviates cardiac dysfunction, MF, and inflammation induced by transverse aortic constriction in mice. However, co-administration with GW9662 partially reverses the cardioprotective effects of Procyanidin B2. In hearts subjected to excessive stress, the PPAR-γ protein expression induced by Procyanidin B2 is similarly diminished with the combined administration of GW9662. These findings suggest that Procyanidin B2 exerts protective effects against pathological MF and inflammation through a PPAR-γ-dependent mechanism.

3.5.4. Piperine

Piperine, an amide alkaloid derived from black pepper (Piper nigrum L.; Piperaceae) and long pepper, exhibits significant metabolic regulatory activity, particularly in diabetes-related MF. Diabetes, as an independent risk factor for MF, disrupts glucose and lipid metabolism, leading to excessive activation of the ERK-MAPK signaling pathway. This activation promotes the release of inflammatory cytokines, fibroblast activation, and ECM deposition. Research shows that piperine can improve insulin resistance and mitigate metabolic dysregulation by inhibiting the ERK-MAPK pathway, ultimately reducing myocardial collagen deposition. Additionally, piperine offers protective effects against liver toxicity and alleviates depressive symptoms. In pharmacokinetics, piperine has poor water solubility and low oral bioavailability. However, modified drug delivery systems, such as nanosuspensions and solid lipid nanoparticles, significantly enhance its dissolution and intestinal absorption. Piperine also inhibits metabolic enzymes like cytochrome P450 and UDP-glucuronosyltransferase, reducing first-pass metabolism and improving bioavailability. ⁷⁷

Mechanistically, piperine binds in a concentration-dependent manner to the hydrophobic pocket of the PPAR-γ (LBD, aa 206–477) in vitro. It forms hydrogen bonds and π-π stacking interactions with key residues such as His323, Tyr327, Cys285, and Phe363. This binding activates the PPAR-γ receptor in CFs, inhibiting the AKT/GSK3β signaling cascade. Specifically, piperine blocks TGF-β/Ang II-induced AKT phosphorylation at Ser473, preventing the inhibitory phosphorylation of downstream GSK3β. Consequently, this prevents β-catenin nuclear translocation and TCF/LEF-mediated transcriptional activation. Additionally, piperine exerts anti-cardiofibrotic effects by inhibiting the JAK2/STAT3 pathway and miR-17 expression, relieving Smad7 inhibition, and further blocking TGF-β/Smad signaling. This effect is reversed by the PPAR-γ inhibitor GW9662 or by abnormal activation of the AKT/GSK3β pathway. In vivo studies suggest that piperine can alleviate MF, inhibit ventricular dilation, and improve cardiac function, though it has no significant effect on interventricular septal thickness. While PPAR-γ agonists like pioglitazone are approved for treating type 2 diabetes, their clinical use is limited by side effects, including weight gain, edema, and cardiovascular risks. ³⁸

Regarding toxicity, the functional effective dose of piperine in male rat models is 10 mg·kg^-1·d^-1. Administration every other day resulted in reduced sperm count and decreased testosterone levels, both of which fully reversed 60 days after discontinuation. No abnormalities were observed in liver, kidney, or myocardial biochemical indicators at this dose. However, at elevated doses (25-50 mg·kg^-1·d^-1), seminiferous tubule necrosis and reversible hepatic steatosis occurred, narrowing the safety margin to 3-5 times. ⁷⁸ Clinical studies suggest that co-administration of sodium valproate (SVP) with piperine significantly increases SVP’s plasma concentration and AUC, indicating that piperine enhances its oral bioavailability. However, high doses or prolonged use of piperine may cause gastrointestinal irritation, inhibit CYP3A4 and other metabolic enzymes, and potentiate the effects of anticoagulants, immunosuppressants, and hypoglycemic agents, increasing the risk of bleeding and toxicity.

3.5.5. Theophylline

Theophylline, a purine alkaloid structurally similar to caffeine, is derived from the tea plant Camellia sinensis (L.) Kuntze (Theaceae, Camellia L.). Its concentration is highest in the tender buds and young leaves, gradually decreasing as the leaves mature. Theophylline exhibits multiple pharmacological activities, including antioxidant, anti-inflammatory, analgesic, antidepressant, and sedative-hypnotic effects.

In vitro experiments have shown that after 28 days of theophylline intervention, MF following MI was significantly reduced. Expression levels of collagen-related markers—type I collagen, type III collagen, and α-SMA—along with the fibrosis-associated protein TGF-β1, decreased in the peri-infarct region. Anti-apoptotic proteins significantly decreased, while pro-apoptotic protein levels increased in myocardial cells in the peri-infarct zone, leading to a notable reduction in apoptotic myocardial cells. During the acute phase of ischemia-reperfusion injury in rats, activation of PPAR-γ substantially reduced MI size. Further studies confirmed that pioglitazone significantly inhibits myocardial hypertrophy and interstitial fibrosis in mice with MI by activating PPAR-γ. Specifically, theophylline promotes β-catenin degradation by activating SIRT3 deacetylase. The degraded β-catenin then interacts with the catenin-binding domain (aa 385-407) of PPAR-γ, exposing the T-cell/lymphokine-enhancing factor binding domain (aa 285-310). This enhances the binding of PPAR-γ to the nuclear coactivator SRC-1, upregulating PPAR-γ transcriptional activity and the expression of its downstream target genes, ultimately inhibiting MF and improving cardiac function. ³⁹

A double-blind, randomized clinical trial involving 60 male patients with asthenozoospermia showed that oral administration of 200 mg·d^-1 theophylline for 90 days significantly improved sperm motility, morphology, and vitality. However, this study did not use a dose-escalation design. Previous mouse studies have indicated that plasma concentrations exceeding 20 mg·L^-1, or a dose of ≥600 mg·d^-1, may cause irreversible damage, including elevated liver enzymes, arrhythmias, and chromosomal breaks in testicular spermatocytes. Therefore, strict monitoring of plasma concentrations is essential during clinical administration. ⁷⁹

3.6. Natural Compounds Modulating the ATF6 Signaling Pathway

Matrine, derived from Sophora flavescens Aiton (Fabaceae, Sophora L.), exhibits a variety of pharmacological activities, including antitumor, anti-inflammatory, antifibrotic, antiviral, antiarrhythmic, and antibacterial effects. However, as its primary active component, matrine suffers from poor solubility in both water and lipids, which hinders its absorption and distribution, leads to rapid metabolism, and results in insufficient concentrations in target tissues, thus significantly reducing its bioavailability. To enhance matrine’s bioavailability, it can be formulated into nanomedicines, improving its solubility and stability.

In a rat model of diabetic cardiomyopathy, impaired cardiac function and reduced compliance were observed, accompanied by increased collagen deposition in myocardial tissue. Activation of the ATF6 signaling pathway is evident, with a significant increase in the active form ATF6 p50. miR-455, a microRNA regulated by ATF6, was downregulated, leading to intracellular calcium accumulation and NFAT activation. This results in NFAT translocation into the nucleus, initiating the transcription of ECM-related genes. Matrine directly binds to the hydrophobic cleft of the ATF6-p50 bZIP domain (292-361), with key hydrogen bond and π-π interaction sites at Lys302, Arg305, and Leu347. This interaction prevents ATF6-p50 from binding to the ER stress response element promoter sequence (−CAGCGTG−), inhibiting the transcription of ATF6 target genes, including X-box binding protein 1, glucose-regulated protein 78, and calreticulin. Reduced calreticulin expression further diminishes ER Ca²⁺ release, which inhibits CaN-mediated NFATc1 dephosphorylation and nuclear translocation. As a result, NFATc1 binding to the NFAT motif (−GGAAA−) in the TGF-β1 promoter is suppressed, leading to decreased TGF-β1 transcription and reduced Smad2/3 phosphorylation. These effects suppress ATF6-mediated fibrotic responses, significantly improving cardiac function and restoring cardiac compliance. In vitro studies support the in vivo findings, with high-glucose conditions in CFs activating the ATF6 pathway, downregulating miR-455, and upregulating calreticulin, thereby promoting ECM synthesis. Within non-cytotoxic concentrations, matrine effectively inhibits ECM synthesis in CFs by blocking ATF6 signaling, exerting anti-MF effects and improving left ventricular function and cardiac compliance. ⁴⁰

From a toxicological and clinical perspective, matrine’s toxicity is concentration-dependent. In a zebrafish embryo model, the lethal concentration (LC₅₀, 240 mg/L) is approximately 1.7-fold higher than the teratogenic concentration (EC₅₀, 145 mg/L). In rodent models, the single-dose tail vein injection LD₅₀ (83 mg·kg^-1) is comparable to the zebrafish LC₅₀ when converted across species. At concentrations ≥50 mg/L, zebrafish exhibit reduced heart rates, while rodent brain tissues show neuronal degeneration and disruption of striatal dopamine metabolism. To mitigate potential cardiac and neurological risks, blood concentrations of matrine should not exceed 5 mg/L⁸⁰. Despite its diverse pharmacological activities, matrine’s clinical application is limited by its low bioavailability, potential central nervous system toxicity, and the risk of tolerance development with prolonged use.

3.7. Natural Compounds Modulating TLR Signaling Pathways

Artesunate, derived from Artemisia annua L. (Asteraceae, Artemisia L.), is a sesquiterpene artemisinin derivative known for its high efficacy, low toxicity, and minimal propensity for resistance development. Its metabolites exhibit a broad spectrum of biological activities, including anti-inflammatory, anti-septic, antitumor, antibacterial, and antifibrotic effects. Its pharmacokinetics include good water solubility, high membrane permeability, and primary absorption in the small intestine. Following administration, artesunate is rapidly detectable in the bloodstream, indicating a fast onset of action and low overall toxicity.

Mechanistically, artesunate exerts antifibrotic effects by directly targeting the TLR4/MD2 signaling axis. In human CFs and two TAC models, artesunate binds directly to the MD2, inhibiting TLR4/MD2 formation and suppressing downstream Extracellular signal-Regulated Kinase - Activator Protein 1 signaling. This mechanism was confirmed through tryptophan fluorescence, co-immunoprecipitation, and assay for transposase-accessible chromatin using sequencing analyses, showing decreased chromatin accessibility at key fibrotic gene loci (COL1A1, FN1, CTGF), downregulation of c-Fos expression, reversal of myofibroblast polarization, and reduced expression of markers such as periostin and TGF-β1⁸¹. In both in vitro and in vivo studies, the artemisinin derivative dihydroartemisinin significantly reduced collagen deposition, preserved left ventricular ejection fraction, and alleviated diastolic dysfunction, whether administered immediately post-surgery or with a four-week delay. These findings highlight its robust and stable efficacy in inhibiting MF and improving heart function. Consequently, artesunate emerges as a promising candidate for treating MF and HF.

Regarding toxicology and clinical safety, the zebrafish acute toxicity model demonstrated that the “safe-to-toxic” dose ratio for artemether was less than 2:1. A dose of 1.25 ng/fish was the highest safe level providing cardiac protection without observable adverse effects. At 2.5 ng/fish, toxicity was observed, including pericardial edema and venous congestion. At 41.8 ng/fish (LD₅₀), multiple organ malformations (brain, eyes, muscle) and approximately 10% mortality were noted. This model is highly sensitive to artemether-induced toxicity. As such, future experimental designs and risk assessments must strictly control dosages and reinforce safety monitoring. ⁸⁰ In terms of drug interactions, combining artemisinin with rosuvastatin effectively inhibits foam cell formation in an atherosclerosis model by activating AMP-activated protein kinase (AMPK), thereby regulating cellular metabolism and enhancing autophagy. AMPK activation suppresses the mTORC1 pathway, promotes LC-3β accumulation, and accelerates p62 degradation. Increased LC-3β levels indicate enhanced autophagosome formation, facilitating the clearance of harmful intracellular components, while p62 degradation prevents the accumulation of undegraded substrates during autophagic flux. This enhancement of macrophage autophagy slows atherosclerotic lesion progression more effectively than monotherapy (Table 2).

Table 2.

Mechanisms of Action and Signaling Pathways of Natural Compounds in the Treatment of MF

Compound name	Source	Signaling pathway	Target proteins	Mechanism of action	Dosage	Administration route	Research subjects	References
Salvianolic acid B	Salvia miltiorrhiza	TGF-β/Smad	Smad 2/3, Smad 7	Downregulates Smad2/3, reduces Smad7 ubiquitination, inhibits collagen and α-SMA	40-160 mg·kg-1·d-1	i.v.	ISO-induced mouse MF model	47
Astragaloside IV	Astragalus		TRPM7, TGF-β1	Inhibits Smad2/3 phosphorylation, downregulates TRPM7, reduces collagen and α-SMA	20 mg·kg-1·d-1	p.o.	ISO-induced mouse MF model	49
Icariin	Epimedium		TGF-β1 and Smad 2/3	Decreases TGF-β1 and Smad 2/3 expression, Reduces collagen synthesis, alleviates cardiomyocyte fibrosis.	30–120 mg·kg-1·d-1	p.o.	MI Rat Model	51
Ginsenoside Rg3	Panax ginseng		TGF-β1, Smad 2/3, TGFBR1		30 mg·kg-1·d-1	p.o.	CAL HF mouse model	54
Forsythoside B	Forsythia		TGF-β and Smad 3/4, α-SMA	Inhibits Smad3/4, reduces collagen III and α-SMA, decreases ECM deposition	50 mg·kg-1·d-1	p.o.	ISO-induced mouse MF model	23
Pachymic acid	Wolfiporia extensa (Peck) Ginns	JAK/STAT	JAK2, STAT3	Upregulates SOCS3 and inhibits JAK2/STAT3 phosphorylation, alleviating MF.	10 mg·kg-1·d-1	i.p.	MI rat model	57
Sinomenine	Sinomenium acutum		JAK2, STAT3, SOCS1	Inhibits JAK2/STAT3 phosphorylation and SOCS1, reduces inflammatory cytokines	120 mg·kg-1·d-1	p.o.	ISO-induced mouse MF model	59
Stachydrine	Leonurus		JAK2/STAT3, NF-κB		20 mg·kg-1·d-1	p.o.	TAC mouse model	62
Triptolide	Tripterygium wilfordii	Wnt/β-catenin	β-catenin, c-Myc and Cyclin D1	Inhibits Wnt/β-catenin signaling, reduces β-catenin, c-Myc, Cyclin D1, blocks fibroblast	0.1–0.5 mg·kg-1·d-1	p.o./i.v.	Rat model of stress overload	27
Salidroside	Rhodiola rosea		TGF-β1, Smad and β-catenin	Downregulates TGF-β1 and IL-1, reduces inflammation and fibrosis	50–100 mg·kg-1·d-1	p.o.	Diabetic cardiomyopathy rat model	65
Saikosaponin A	Bupleurum		TGF-β1, Smad3, β-catenin and TLR4	Restores Axin2, inhibits β-catenin nuclear translocation, suppresses EndoMT and fibroblast activation	300 mg·kg-1·d-1	p.o.	Mouse pressure-overload model	67
Resveratrol	Blueberry, raspberry, and mulberry peels		Wnt 3a, β-catenin and sFRP2	Downregulates sFRP2 and Wnt3a, inhibits β-catenin signaling, reduces fibrosis	20–100 mg·kg-1·d-1	p.o.	Diabetic cardiomyopathy rat model	68
Berberine	Coptis chinensis		Wnt 5a	Decreases Wnt5a and pro-inflammatory cytokines, reduces fibrosis and ventricular remodeling	300 mg·d-1	p.o.	Patients with acute coronary syndrome	70
Artemisinin	Artemisia annua	NLRP3	NF-κB, NLRP3	Inhibits NLRP3 inflammasome activation via NF-κB/NLRP3, reduces ASC, IL-1β, IL-18, mitigates fibrosis	50 mg·kg-1·d-1	p.o.	Ischemia-reperfusion (I/R) rat model	72
Celastrol	Tripterygium wilfordii and Clematis orientalis		NLRP3		1–3 mg·kg-1·d-1	i.v.	MI rat model	81
Oridonin	Herba Rabdosiae Rubescentis			Blocks NLRP3 inflammasome, inhibits caspase-1/IL-1β/IL-18, reduces inflammation and collagen deposition	5–10 mg·kg-1·d-1	i.v.	MI mouse model	73
Curcumin	Turmeric	PPAR-γ	PPAR-γ	Inhibits Ang II-induced Collagen III and fibronectin expression, promotes ECM degradation	500 mg·d-1	p.o.	Patients with coronary heart disease	75
Propolis Extract	Propolis			Reduces IL-1β, TNF-α, CRP, and Ang II, alleviating post-MI inflammation	2.5 mg·mL-1	Topical application	Human dermal fibroblasts	76
Procyanidin B2	Seeds, fruits, and leaves of grapes, hawthorn, black beans, and blueberries.		PPAR-γ, β-catenin	Upregulates PPAR-γ in CFs, inhibits fibroblast proliferation, differentiation, and collagen deposition	50 mg·kg-1·d-1	p.o.	TAC mouse model	37
Piperine	Black pepper and long pepper		PPAR-γ, AKT	Activates PPAR-γ and AKT, prevents myofibroblast transdifferentiation, reduces collagen and ventricular dilation	20–50 mg·kg-1·d-1	p.o.	Male Wistar rats	78
Theophylline	Camellia sinensis		SIRT3, PPAR-γ	Activates SIRT3, promotes β-catenin degradation, downregulates TGF-β1 and α-SMA	10 mg·kg-1·d-1	p.o.	C57BL/6 rat model	79
Matrine	Sophora flavescens	ATF6	ATF6	Upregulates miR-455, blocks calmodulin-NFAT axis, reduces ECM deposition	50 mg/L	i.v.	Zebrafish embryo model	82
Artesunate	Artemisinin	TLR	MD2	Binds MD2, blocks TLR4/MD2, inhibits ERK-AP1, decreases periostin and TGF-β1	41.8 ng/fish	i.v.	Zebrafish model	80

4. The Multi-Target Therapeutic Advantages of Natural Compounds in MF

In recent years, advances in multi-omics and network pharmacology have shifted our understanding of MF from a “single pathway-single target” model to a “multi-pathway-network regulation” paradigm. Extensive research confirms that the progression of MF is not controlled by the isolated regulation of a single signaling pathway, but by a complex network of multiple pathways engaged in positive and negative feedback loops. This suggests that single-target interventions often fail to effectively halt the fibrotic process. In contrast, natural compounds, due to their chemical diversity and the principle of “one drug targeting multiple targets” and “multiple drugs targeting a single target,” can interact with multiple interfaces within complex signaling networks, exerting synergistic antifibrotic effects superior to those of single chemical monomers (Table 3). At the inflammation-fibrosis transition point, Celastrol covalently binds to NLRP3, reducing the expression of NLRP3, ASC, and IL-1β/IL-18. This interaction blocks the caspase-1-IL-1β/IL-18 axis, reducing IL-1β release and further suppressing TGF-β1 autocrine signaling in fibroblasts. During the critical shift from inflammation to fibrosis, the TGF-β/Smad and Wnt/β-catenin pathways form a core regulatory network that governs cardiac fibrotic remodeling. ⁸³ Through shared downstream effectors, transcriptional co-regulation, protein-protein interactions, and epigenetic mechanisms, these pathways synergistically promote fibroblast activation, collagen deposition, and myocardial structural remodeling. TGF-β1 induces a transcriptional co-activation complex between Smad3 and β-catenin, which co-occupies Smad-binding elements and TCF/LEF sites in the promoter regions of fibrosis-related genes, such as COL1A1, Fn1, and α-SMA, enhancing their transcription. Notably, This process significantly relies on Smad3-mediated recruitment of β-catenin to specific chromatin loci, which can occur independently of, or in concert with, its classic nuclear translocation, enabling synergistic transcriptional regulation. Furthermore, these two pathways engage in bidirectional regulation through interconnected feedback loops. Smad7 can associate with Smurf2 to promote β-catenin ubiquitination and degradation, inhibiting Wnt signaling. Conversely, Wnt3a activates the β-catenin/TCF4 complex to upregulate Smad2/3 and suppress Smad7 expression, forming a profibrotic positive feedback loop. Additionally, β-catenin inhibits GSK-3β, delaying Smad3 degradation and prolonging its nuclear retention, which further amplifies fibrotic responses. ⁸⁴ Based on the aforementioned crosstalk characteristics, certain natural compounds disrupt the positive feedback loops between the TGF-β/Smad and Wnt/β-catenin pathways, exerting synergistic antifibrotic effects. For instance, compounds such as Sal B, AS-IV, Icariin, and Ginsenoside Rg3 inhibit Smad2/3 phosphorylation, upregulate the negative feedback regulator Smad7, or reduce TGFBR1 expression, blocking TGF-β1 signaling and decreasing collagen synthesis. SAL, SSA, resveratrol, and berberine downregulate β-catenin, c-Myc, Cyclin D1, or Wnt3a/5a, preventing β-catenin nuclear translocation and disrupting the colocalization of Smad3/β-catenin transcriptional complexes at promoters like COL1A1, FN1, and α-SMA, thus inhibiting fibrogenic gene expression. In the JAK/STAT amplification pathway, Pachymic acid, SIN, and STA upregulate SOCS3, inhibit JAK2/STAT3 phosphorylation, or downregulate STAT3 target genes such as IL-1β and TNF-α, reducing the persistent amplification of inflammatory cytokine transcription driven by TGF-β1⁸⁵. During ER stress mediated by ATF6, matrine suppresses ATF6-p50 generation, restores miR-455 expression, and interrupts the calreticulin-NFATc1 axis, reducing collagen deposition. ⁴⁰ Finally, compounds like curcumin, piperine, Procyanidin B2, PE, and theophylline activate or enhance PPAR-γ activity, inhibit the nuclear translocation of Smad3/β-catenin, and downregulate COL1A1, α-SMA, CTGF, and PAI-1, establishing a dual negative feedback loop with “anti-inflammatory and antifibrotic” effects ⁸⁵ (Figure 4). Additionally, piperine can increase the blood concentrations of compounds like curcumin and resveratrol by inhibiting CYP3A4 and UGT enzymes, enhancing pharmacokinetics and pharmacodynamics. Current research shows that natural compounds employ a “multi-pathway crosstalk” strategy—both blocking TLR4-NLRP3 inflammasome activation and disrupting TGF-β/Wnt positive feedback loops. This promotes the remodeling of pathological networks through the activation of endogenous negative regulators such as PPAR-γ, SOCS3, and miR-455, highlighting the unique advantages of these compounds in regulating complex diseases and offering mechanistic insights for integrating precision medicine (Table 3).

Table 3.

Summary of Mechanisms by which Multi-Target Natural Compounds Block MF

Representative natural compounds	Signaling pathway	Mechanism of action	Dosage	Administration route	Research subjects	References
Artesunate	TLR4 → NLRP3	Inhibit the formation of the TLR4/MD2 and reduce NLRP3 activation	50 mg·kg-1·d-1	p.o./i.v.	MI mouse model	41
Celastrol	NLRP3 → TGF-β	Inhibit the NLRP3 inflammasome and the secretion of IL-1β and IL-18	1-3 mg·kg-1·d-1	i.v.	TAC mouse model	83
Oridonin
Salvianolic acid	TGF-β → Wnt	Inhibit Smad2/3 phosphorylation and prevent β-catenin nuclear translocation	5-10 mg·kg-1·d-1	i.v.	Male SD rats	84
Salidroside
Saikosaponin A
Resveratrol	Wnt → TGF-β	Downregulate Wnt3a/β-catenin signaling and inhibit the amplification of TGF-β signaling	20-100mg·kg-1·d-1	p.o.	MI mouse model	31
Berberine
Pachymic acid	JAK/STAT → TGF-β	Inhibit JAK2/STAT3 phosphorylation and reduce TGF-β1 expression	10-50 mg·kg-1·d-1	i.v.	Male SD rats	86
Sinomenine
Stachydrine
Matrine	ATF6 → TGF-β	Block ATF6 activation and inhibit TGF-β1 expression	50-200 mg·kg-1·d-1	p.o.	Diabetic cardiomyopathy rat model	40
Curcumin	PPAR-γ → TGF-β/Wnt	Activate PPAR-γ and inhibit the nuclear translocation of Smad3 and β-catenin	20-50 mg·kg-1·d-1	i.v.	Spontaneously hypertensive rat model	85
Piperine
Procyanidin B2
Propolis Extract	PPAR-γ → NLRP3	Activate PPAR-γ and inhibit NF-κB and NLRP3 activation	2.5 mg·mL-1	Topical application	Human dermal fibroblasts	76
Theophylline

Figure 4.

Multi-pathway crosstalk network of natural compounds in MF. (Natural compounds synergistically combat fibrosis through the “TLR4-NLRP3-inflammation-TGF-β/Smad-Wnt/β-catenin-JAK/STAT-ATF6” multi-pathway crosstalk network: By inhibiting the inflammasome, disrupting the Smad3/β-catenin positive feedback loop, suppressing STAT3 amplification, alleviating ER stress, and acting as a central negative regulatory hub via PPAR-γ, this pathway simultaneously downregulates genes such as COL1A1 and α-SMA, thereby restoring myocardial collagen homeostasis.)

5. Discussion

5.1. Clinical Evidence for Natural Compounds in CVDs

In recent years, natural compounds have gained increasing attention for the treatment of CVDs supported by multiple clinical studies that provide preliminary evidence of their potential efficacy. These studies have primarily employed randomized, double-blind, placebo-controlled designs, with some conducted as multicenter trials to enhance the robustness and reliability of the findings. The results suggest that these natural agents exert cardioprotective effects mainly through their anti-inflammatory and antioxidant properties, improvement of endothelial function, regulation of lipid metabolism, and inhibition of myocardial remodeling. For example, Sal B reduces MMP-9 activity, decreases infarct size, and improves left ventricular systolic function, significantly mitigating acute MI-induced damage in rodent models. ⁸⁷ The combination of AS-IV with standard therapy significantly reduces the burden of chronic congestive HF and improves cardiac function. ⁸⁸ Resveratrol notably attenuates left atrial remodeling and MF in hypertensive patients, benefiting the cardiac remodeling process. ⁶⁹ Berberine has demonstrated substantial efficacy in reducing the incidence of postoperative atrial fibrillation and improving cardiac function in patients with chronic congestive HF. ⁸⁹ Future research should focus on exploring the long-term application value of natural compounds and their combinations from different plant sources. Additionally, developing personalized treatment strategies is crucial for achieving synergistic effects between drugs, which will facilitate the translation of clinical applications and improve patient outcomes.

5.2. Limitations and Challenges of Current Research

The bioavailability of certain natural products, such as Sal B, AS-IV, berberine, and resveratrol, limits their clinical efficacy. Based on their physicochemical characteristics, compounds can be classified into hydrophobic and hydrophilic characteristics. Hydrophobic compounds typically exhibit high lipid solubility but poor aqueous solubility, which restricts their dissolution in the gastrointestinal environment and impairs oral absorption. For instance, icariin has a large molecular weight and multiple glycosidic bonds, making it difficult to permeate the intestinal epithelial cell membrane. Furthermore, icariin is prone to degradation by gastric acid and digestive enzymes, leading to reduced gastrointestinal solubility and absorption. These factors ultimately result in insufficient systemic drug concentrations. ⁵⁰ In contrast, hydrophilic compounds like Sal B are highly soluble in water but have limited lipid solubility and poor transmembrane permeability, relying primarily on enterohepatic circulation for absorption. However, this pathway has limited capacity, and these compounds are also vulnerable to degradation in the intestinal tract, resulting in low bioavailability. ⁴⁵ The bioavailability issues and individual variability have led to a lack of significant therapeutic effects in some patients. Additionally, most studies lack long-term follow-up data, hindering a comprehensive assessment of the safety and potential adverse effects associated with the prolonged use of these natural compounds and their supplements. Furthermore, mechanistic research typically focuses on two-dimensional cell models or rodent models using induction methods such as ISO, left anterior descending artery ligation, and streptozotocin. These studies often neglect more clinically relevant models like TAC overload or SAMP8 aging models. Current mechanistic studies largely rely on primary CFs from rats or mice, but their proliferative and signaling response characteristics differ significantly from human CFs. For example, the intensity of TGF-β signaling and ECM secretion profiles differ markedly between humans and rodents, leading to reduced or even absent efficacy of the same compounds in human cells. ⁹⁰ Future research should prioritize the use of human cell models, such as human CF lines, induced pluripotent stem cell-derived CFs, or organ-on-a-chip systems, to obtain mechanistic evidence with greater clinical predictive value. Moreover, studies on the in vivo metabolism of natural compounds remain limited, and a standardized framework for evaluation is lacking. This makes it challenging to fully elucidate their tissue distribution, conversion into active metabolites, and potential long-term toxicity risks associated with prolonged exposure. Although natural compounds have the therapeutic advantage of multi-target modulation, their potential off-target effects and safety risks remain insufficiently explored. For example, while curcumin’s broad activation of PPAR-γ may offer anti-inflammatory and antifibrotic benefits, it could also increase the risk of infections due to modulation of systemic immune responses. Additionally, research on the chiral synthesis of natural compounds faces major challenges. Many bioactive natural molecules contain multiple chiral centers, and their synthesis typically relies on asymmetric catalysis or chiral resolution strategies. However, these methods often have low efficiency, high costs, and lack standardized criteria for optical purity control. In chemical synthesis, precise control over enantiomeric or diastereomeric ratios is difficult, resulting in mixtures of isomers. This not only increases drug safety risks but also elevates manufacturing costs and complicates structural characterization, hindering the development and industrialization of chiral compounds. Furthermore, chiral resolution techniques, such as chiral chromatography and crystallization, have limited ability to separate optically active isomers with closely related structures, restricting the large-scale production of single, pharmacologically active configurations. Variability in isomer composition can compromise therapeutic reproducibility and dose-response assessment and may also increase the risk of unintended toxic effects. Consequently, this issue represents a critical bottleneck in the translation of natural compounds from laboratory research to clinical application. ⁹¹ A comprehensive, multidimensional system for assessing the systemic toxicity and off-target effects of natural compounds is also lacking. For instance, although Celastrol extracts have antifibrotic effects through the inhibition of NLRP3 inflammasome activity, they can significantly impair the immune system’s ability to clear bacterial infections, an effect that can persist even after drug discontinuation. Similarly, although icariin has cardioprotective effects, long-term use may disrupt sex hormone homeostasis. Thus, developing a comprehensive evaluation framework that encompasses pharmacokinetics, metabolic transformation, systemic toxicity, and long-term safety is critical for advancing the clinical translation of natural compounds.

5.3. Future Directions for Research on Natural Compounds Related to MF

Future research on natural compounds targeting MF should adopt a multidimensional, mechanism-oriented integrative framework that systematically addresses pharmacokinetic limitations through formulation design, molecular modification, and delivery optimization. At the formulation level, nanodelivery systems provide an effective approach to overcome poor solubility, instability, and limited tissue targeting. For example, De Leo ⁹² compared multiple liposomal encapsulation strategies for curcumin and demonstrated that a pH-responsive loading method significantly enhanced encapsulation efficiency and improved systemic bioavailability. This strategy enables controlled drug release in pathophysiological microenvironments, thereby enhancing local drug accumulation in fibrotic tissues. At the molecular level, structural modification offers a direct means to optimize physicochemical and metabolic properties. For instance, with quercetin as a model compound, phosphorylation and amide conjugation have been shown to markedly improve its pharmacokinetic profile. Phosphorylation (e.g., quercetin pentaphosphate) introduces highly polar phosphate groups that dramatically increase aqueous solubility (up to 848 mg/mL), facilitating non-oral delivery routes such as intravenous or inhalational administration. In contrast, amide conjugation (e.g., quercetin–p-aminobenzoic acid derivatives) balances hydrophilicity and membrane permeability while maintaining biological activity. Importantly, both strategies can effectively hinder CYP450-mediated metabolism through steric and electronic effects, thereby reducing first-pass degradation and prolonging systemic half-life. ⁹³ Beyond molecular modification, solid-state optimization strategies, such as drug cocrystals and solid dispersion systems, have demonstrated considerable potential in enhancing dissolution behavior and oral absorption. Solid dispersion formulations of curcumin, for example, significantly accelerate dissolution rates. They also result in several-fold higher plasma exposure compared with the native compound. ⁹⁴ Similarly, cocrystallization with pharmaceutically acceptable coformers improves physicochemical stability, particularly resistance to photodegradation and thermal degradation. Notably, a curcumin cocrystal combined with a micellar delivery system achieved a 4.5-fold increase in bioavailability and extended the half-life to 3.7 hours following intranasal administration. ⁹⁵ To effectively evaluate and translate these pharmacokinetic optimization strategies into clinically relevant outcomes, advanced experimental models are necessary.

In this context, three-dimensional bioprinted heart-on-a-chip platforms enable more accurate simulation of pathological mechanical stress and localized biochemical microenvironments. Organ-on-a-chip technologies have demonstrated substantial advantages in elucidating the pharmacological mechanisms of natural compounds and facilitating high-throughput screening. ⁹⁶ Future research should prioritize organoid models that preserve genetic backgrounds and cellular heterogeneity, which can better recapitulate the long-term effects of natural compounds on tissue development, homeostasis, and disease progression. Traditional animal models remain the gold standard for evaluating in vivo absorption, distribution, metabolism, excretion, and systemic toxicity, providing a holistic perspective on multitarget synergistic effects. To address translational medicine challenges, further development of intelligent nanodelivery systems, such as ROS-responsive platforms, may enable targeted drug accumulation and controlled release at pathological sites, offering a promising solution to overcoming high-dose dependence and systemic toxicity.

Natural compounds are a vital source of innovative drug candidates, yet their production is often limited by scarce plant resources. As a result, chemical synthesis has become the primary method for obtaining many natural compounds. However, challenges remain in chiral synthesis and optical isomer separation, including low separation efficiency, high costs, and the absence of standardized criteria for optical purity. Recent studies have proposed solutions across three key areas: separation technologies, analytical methodologies, and quality control frameworks. At the separation level, multidimensional screening systems based on polysaccharide-derived and macrocyclic antibiotic chiral stationary phases (CSPs) have significantly improved efficiency through systematic optimization. At the analytical level, vibrational circular dichroism combined with density functional theory calculations overcomes the limitations of traditional optical rotation methods, enabling accurate quantification of enantiomeric excess (e.e.) and direct determination of absolute configuration. At the quality control level, comprehensive monitoring of enantiomeric purity throughout production, along with standardized metrics for e.e. and enantiomeric ratio, has refined quality evaluation systems. However, challenges remain in scaling up production for industrial applications. The limited applicability of CSPs can result in reduced yields, increased solvent consumption, and significant challenges in quality control for molecules with multiple chiral centers—issues that fall outside current regulatory frameworks. Therefore, developing low-cost chiral materials, efficient continuous separation processes, and adaptive regulatory guidelines is crucial to enable large-scale production and clinical translation of chiral natural compounds. ⁹⁷ Lastly, to overcome limitations in assessing off-target effects and systemic toxicity, future research should advance safety assessment strategies through a multilevel approach. First, integrating cutting-edge technologies, such as high-content imaging and mass cytometry-based flow analysis, will establish multidimensional toxicity assessment platforms that extend evaluation to nontraditional endpoints, such as gut microbiota alterations and germline epigenetic effects. Second, machine learning algorithms should be used to develop physiologically based pharmacokinetic toxicity prediction models that incorporate chemical structure and pharmacokinetic parameters, enabling personalized safe-dose design. Third, integrating pre-treatment screening systems that account for genetic polymorphisms and gut microbiota enzyme activity would support precise individualized risk assessments and proactive safety monitoring. ⁹⁸

5.4. Conclusion

With advancements in molecular biology, the clinical applications and developmental potential of natural compounds for treating MF have become increasingly significant. This review explores the multi-target mechanisms and clinical potential of these compounds. A deeper understanding of their mechanisms can help elucidate the underlying pathology and guide precision treatment strategies. By establishing an innovative evaluation system that integrates high-throughput screening, organoid models, and real-world evidence, we can systematically assess the efficacy and safety of these compounds. As mechanistic research progresses and clinical validation advances, natural compounds are set to play a more pivotal role in the global treatment of MF.