Molecular Mechanisms Underlying the Anti-Inflammatory Effects of Fritillaria Alkaloids Targeting the NLRP3 Inflammasome

Abstract

Fritillaria alkaloids are one of the most important bioactive constituents in Fritillaria plants exhibit significant anti-inflammatory effects. Studies suggest that their anti-inflammatory mechanisms may be closely associated with the modulation of the Nucleotide-binding Oligomerization Domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. This review systematically investigates the structure, composition, activation mechanisms, and role of the NLRP3 inflammasome in inflammatory responses, focusing on elucidating the specific regulatory effects of Fritillaria alkaloids on NLRP3 inflammasome components (eg, IL-1β and IL-18) and related signaling pathways. Furthermore, it reviews recent advances in the anti-inflammatory actions of Fritillaria alkaloids across diverse inflammatory models, emphasizing their NLRP3-dependent mechanisms. Collectively, this review provides a theoretical foundation for elucidating the anti-inflammatory properties of Fritillaria alkaloids and advancing their potential applications in anti-inflammatory drug development.

Introduction

Inflammation is a self-protective response of the body to harmful exogenous stimuli. However, excessive or chronic inflammatory responses often lead to the development of various inflammation-related diseases, such as rheumatoid arthritis, gout, and Alzheimer's disease posing a serious threat to human health. ¹ Therefore, thoroughly investigating the molecular mechanisms underlying inflammation and identifying effective anti-inflammatory drugs and therapeutic approaches is of great importance.

The NLRP3 inflammasome is a key regulatory component of the innate immune system. It can sense Pathogen-Associated Molecular Patterns (PAMPs) and Damage-Associated Molecular Patterns (DAMPs), thereby triggering the activation of Caspase-1. This in turn promotes the maturation and release of pro-inflammatory cytokines IL-1β and IL-18, ultimately inducing pyroptosis and the formation of inflammation. Consequently, the NLRP3 inflammasome plays a dual role in host defense and the development of inflammatory diseases.^2–4 Dysregulated activation of the NLRP3 inflammasome is closely associated with various diseases, such as rheumatoid arthritis and atherosclerosis. Although inhibitors targeting NLRP3 (such as MCC950 and OLT1177) have entered clinical studies, their practical application in the clinic is limited due to factors such as structural homogeneity, off-target effects, and safety issues.^5–7 Recent systematic studies of the anti-inflammatory mechanisms of fritillary alkaloids, have demonstrated that these alkaloids can exert anti-inflammatory effects by inhibiting the activation of the NLRP3 inflammasome and reducing the release of pro-inflammatory factors.

However, the molecular mechanisms underlying the anti-inflammatory effects of fritillary alkaloids through the regulation of the NLRP3 inflammasome have not yet been fully elucidated. A deeper understanding of these mechanisms will not only help reveal the pharmacological basis of fritillary alkaloids but also provide theoretical and experimental support for the development of novel anti-inflammatory drugs. This review systematically summarizes and analyzes the research progress on the anti-inflammatory molecular mechanisms of fritillary alkaloids based on the NLRP3 inflammasome, with the expectation of offering references for related studies.

Overview of the NLRP3 Inflammasome

Composition and Structure

In the intricate network of cellular immune defense, the NLRP3 inflammasome functions as an exquisite fortress for signal transduction, bearing the crucial responsibility of recognizing pathogen invasion signals and initiating inflammatory responses. It is a protein complex composed of multiple protein-binding domains, belonging to the NOD-like receptor family. Its core architecture is tightly coordinated by NLRP3, the adaptor protein ASC, and the effector protein Caspase-1, collectively forming a robust defense network against pathogen and damage signals.^8,9

The structure (Figure 1) of the NLRP3 protein itself is exquisitely designed, resembling a multi-story building. Its N-terminal pyrin domain (PYD) serves as the starting point for signal transduction, typically constructed from about 90 to 100 amino acids. ¹⁰ Within this domain, six α-helices act like a sturdy steel framework, providing stable support for the entire PYD structure. ¹¹ Among them, the “mutual activation domain” formed by α1 and α6 helices functions like sensitive antennae, specifically responsible for precise docking with the ASC protein. ¹² Once binding is successful, it is akin to pressing a start button, rapidly triggering a cascade of signal transduction.^11,13 This activates a series of downstream signaling pathways, transmitting the inflammatory alarm to every corner of the cell. The seven β-sheet segments located at the center of the PYD structure serve as key connectors between different domains.^14,15 They can tightly link with other homologous or heterologous domains, such as the CARD domain in the ASC protein. Through this ingenious binding method, they form the framework of a multi-protein complex, laying the foundation for the assembly and functional exertion of the inflammasome. This ensures that each component works synergistically in the right position. ¹⁶

Figure 1.

Schematic diagram of NLRP3 inflammasome structure.

The central region of NLRP3 is the NACHT domain, composed of approximately 300 to 350 amino acids, which acts as the core driving force for inflammasome activation.^12,13 This domain contains three key regions: the nucleotide-binding domain (NBD), the oligomerization interface, and a conserved regulatory region.^14,15 The NBD functions like an energy converter, precisely regulating protein conformational changes through nucleotide binding and hydrolysis, thereby activating downstream signaling pathways and providing the power support for initiating the inflammatory response.^11,13 The oligomerization interface acts as a “glue,” promoting the oligomerization of the NLR family protein NLRP3 itself.^17,18 Multiple NLRP3 molecules are tightly aggregated together, forming a stable inflammasome complex. This creates favorable conditions for Caspase-1 activation and drives the cascade amplification of the inflammatory response.^19–23 The conserved regulatory region functions like a diligent “guardian.” On one hand, it maintains the stability of the entire NACHT domain, ensuring it is not easily disrupted in the complex cellular environment.^14,15 On the other hand, it is deeply involved in the signal transduction process, precisely regulating the direction and intensity of signal transmission to ensure the orderly progression of the inflammatory response.^11,13

The C-terminal leucine-rich repeat (LRR) domain serves as the “sensor” of the NLRP3 inflammasome. It is composed of multiple repeating units of 20 to 30 amino acids containing conserved leucine or other hydrophobic residues. ¹³ These repeating units fold into unique β-α-β structures, forming precise pockets or grooves that specifically recognize a variety of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).^11,13,22 PAMPs are unique molecular markers on the surface of pathogens, such as bacterial lipopolysaccharide and viral double-stranded RNA. ¹³ DAMPs are endogenous molecules released by cells when they are damaged or stressed, such as high-mobility group box protein 1 (HMGB1) and ATP.^11,15 When cells encounter pathogen invasion or tissue damage, the LRR domain acts like a sensitive radar, precisely capturing these PAMPs or DAMPs signals. Upon binding, its conformation undergoes subtle changes, thereby releasing the latent ATPase activity in the NACHT domain.^20,21 Subsequently, NLRP3 uses the energy released from ATP binding and hydrolysis to drive its own oligomerization. This is like tightly assembling multiple independent modules into a powerful functional whole, preparing for the complete activation of the inflammasome. This then initiates a series of inflammatory responses, including the maturation and secretion of pro-inflammatory cytokines, attracting immune cells to the damaged site, clearing pathogens, repairing damaged tissues, and maintaining the body's homeostasis.^23,24

The exquisite structural and functional synergy of the NLRP3 inflammasome makes it a crucial component in the immune defense system. Any abnormality in any of its components can lead to uncontrolled inflammatory responses, causing various diseases such as autoimmune diseases and neurodegenerative diseases. Therefore, in-depth research on the NLRP3 inflammasome is of great scientific significance and clinical value for revealing the mechanisms of inflammation and developing novel anti-inflammatory drugs.

Mechanism of NLRP3 Inflammasome Activation

The activation of the NLRP3 inflammasome is a complex and finely regulated process, involving the interplay of multiple molecules, steps, and signaling pathways. To elucidate the specific processes, mechanisms, and characteristics of its activation, a “two-step activation model” has been proposed, which includes the priming phase and the activation phase. Each phase has its unique molecular mechanisms and regulatory modes, collectively ensuring that the inflammasome is precisely activated at the appropriate time and under the appropriate conditions to respond to various danger signals both inside and outside the cell.

Priming of NLRP3

During the priming phase, the activation of the NLRP3 inflammasome is primarily achieved through Toll-like receptors (TLRs) or other signaling pathways. TLRs are a class of pattern recognition receptors widely distributed on the cell surface and the endoplasmic reticulum membrane, capable of recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). ²⁵ Upon recognition of their respective ligands, TLRs form homodimers or heterodimers, triggering conformational changes in the intracellular Toll/interleukin-1 receptor (TIR) domains of TLRs. This creates binding sites for adaptor proteins, thereby recruiting the adaptor protein Myeloid Differentiation Primary Response Protein 88 (MyD88) through homophilic interactions. ²⁶ MyD88 is a key adaptor protein in TLR signaling. The interaction between the C-terminal TIR domain of MyD88 and the TIR domain of TLRs activates downstream signaling pathways, among which the most critical is the NF-κB signaling pathway. ²⁷ NF-κB, a transcription factor complex, typically resides in the cytoplasm in an inactive state due to binding with the inhibitory protein IκB. ²⁸ After TLR activation, MyD88 recruits Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) through its death domain. The death domain (DD) of IRAK4 interacts with the DD of MyD88 to form a signaling complex.^29–31 Following this interaction, IRAK4 undergoes autophosphorylation via its kinase activity, enhancing its own kinase activity. ³² Phosphorylated IRAK4 then activates the E3 ubiquitin ligase TNF Receptor-Associated Factor 6 (TRAF6), catalyzing the formation of K63-linked ubiquitin chains. This activates the Transforming Growth Factor-β-Activated Kinase 1 (TAK1) complex (TAK1-TAB1-TAB2), promoting TAK1 autophosphorylation and subsequently activating the IκB Kinase (IKK) complex. This leads to IκB degradation and NF-κB nuclear translocation. ²⁷ The release of NF-κB allows it to enter the nucleus, bind to specific gene promoter regions, and promote the transcription and protein synthesis of NLRP3 and pro-IL-1β genes. ³⁰ This process provides the necessary material basis for the subsequent activation of the NLRP3 inflammasome, ensuring that sufficient NLRP3 protein and pro-IL-1β protein are available for the activation process.

In addition to increased transcription and protein synthesis, NLRP3 undergoes a series of post-translational modifications during the priming phase, including deubiquitination, phosphorylation, small ubiquitin-like modifier (SUMO)ylation, and palmitoylation. These modifications significantly affect the stability and activity state of NLRP3. Deubiquitination stabilizes the NLRP3 protein by preventing its proteasomal degradation, thereby increasing its stability and availability within the cell; phosphorylation can alter the conformation of NLRP3 protein, priming it for activation, ready for subsequent activation signals. ³³ These post-translational modifications collectively constitute the molecular mechanisms of NLRP3 inflammasome pre-activation, rendering NLRP3 in a highly sensitive and poised state during the priming phase, ready to respond to subsequent activation signals.

Activation of NLRP3

After the priming phase is completed, the NLRP3 inflammasome enters the activation phase. Activation signals in this phase are diverse and include various danger signals such as bacterial toxins, ATP, and uric acid crystals. ³⁴ These signals trigger the activation of NLRP3 through different mechanisms, among which the most critical are K⁺ efflux and the generation of mitochondrial reactive oxygen species (ROS). ³⁵

Bacterial toxins and ATP are two common activation signals for NLRP3. Nigericin, a bacterial toxin, forms pores in the plasma membrane. It forms pores in the cell membrane, leading to K⁺ efflux and a decrease in intracellular K⁺ concentration. ³⁶ ATP, on the other hand, activates the P2X7 receptor, which promotes the opening of K⁺ channels in the cell membrane, also resulting in a decrease in intracellular K⁺ concentration. ³⁷ Under the stimulation of K⁺ efflux, NLRP3 undergoes phase separation or the opening of cellular Ca²⁺ channels, causing a conformational change in NLRP3. This change shifts NLRP3 from an inhibited state to an activated state.^35,38 This conformational change allows the NACHT domain of NLRP3 to release its ATPase activity, which in turn drives the oligomerization of NLRP3, forming a stable inflammasome complex.

In addition to K⁺ efflux, the generation of mitochondrial reactive oxygen species (ROS) is also an important mechanism for activating NLRP3. Compounds like imiquimod (IMQ) can induce mitochondrial ROS production, which then oxidatively modify the conformation of NLRP3, shifting it to an activated state. ³⁹ The production of ROS not only directly affects the conformation of NLRP3 but also enhances the activation of NLRP3 through various signaling pathways, such as the Nrf2 signaling pathway. ⁴⁰ Nrf2 is a transcription factor that responds to oxidative stress signals and regulates the expression of a series of antioxidant genes. During the activation of the NLRP3 inflammasome, the activation of Nrf2 can enhance the expression and activity of NLRP3, thereby amplifying the inflammatory response. ⁴¹

In addition, the Never In Mitosis A (NIMA)-related kinase 7 (NEK7) also plays an important role in the activation of NLRP3. NEK7 can interact with the LRR and NACHT domains of NLRP3, and this interaction promotes the oligomerization and activation of NLRP3. ⁴² NEK7, originally implicated in cell cycle regulation. It can sense stress signals within the cell and convert these signals into activation signals for the inflammatory response through its interaction with NLRP3. ⁴³ Importantly, this NEK7-dependent licensing step is conserved in both murine macrophages and human monocytes, underscoring its translational relevance.

Functions and Regulation of NLRP3

After activation, the PYD domain of NLRP3 undergoes oligomerization and recruits the adaptor protein ASC through PYD-PYD interactions. Subsequently, ASC recruits pro-caspase-1 via CARD-CARD interactions, leading to the activation of caspase-1. ⁴⁴ Activated caspase-1 recognizes specific cleavage sites located in the substrates pro-IL-1β (between Asp116 and Asp117) and pro-IL-18 (near Asp61) through its catalytic domain (Caspase Activation and Recruitment Domain, CARD). Its active subunits p20 and p10 cleave pro-IL-1β and pro-IL-18 into mature cytokines IL-1β and IL-18 via hydrolysis,^45,46 which are then secreted outside the cell. The conformation of mature IL-1β and IL-18 changes, exposing their receptor-binding domains, allowing them to bind to the IL-1 receptor (IL-1R) or IL-18 receptor (IL-18R) on the cell membrane and initiate downstream signaling pathways.^14,47 Meanwhile, caspase-1 can also activate the pyroptosis-related protein Gasdermin D (GSDMD) through cleavage. GSDMD forms pores in the cell membrane, leading to cell osmotic lysis (pyroptosis), which allows IL-1β and IL-18 to be released outside the cell through lysis or vesicle transport.^48,49 IL-1β released outside the cell recruits neutrophils and monocytes to the site of inflammation, promoting fever, increased vascular permeability, and tissue repair; IL-18 enhances the secretion of IFN-γ by Th1 cells and NK cells, thereby further amplifying the inflammatory response^42,50 (Figure 2). Aberrant activation of the NLRP3 inflammasome leads to the release of large amounts of pro-inflammatory cytokines (such as TNF-α, IL-6, TGF-β), causing chronic inflammation and tissue damage.^51,52

Figure 2.

Activation mechanism of NLRP3 inflammasome and regulation of berberine.

NLRP3 can also be regulated positively and negatively through various pathways such as ubiquitination, autophagy, and microRNA. Studies have shown that the E3 ubiquitin ligase TRIM31, which can directly bind to NLRP3, interacts with NLRP3 through its RING domain and catalyzes K63 ubiquitination on specific lysine residues of NLRP3. This changes the conformation of NLRP3, making it easier to bind to the adaptor protein ASC and accelerating the oligomerization of NLRP3, thereby promoting the assembly and activation of the inflammasome.^39,53 Meanwhile, the breast cancer susceptibility gene complex subunit protein 3 (BRCC3) and Peroxisome Proliferator-Activated Receptors (PPARs) can recognize and bind to the K63 ubiquitin chain on NLRP3, and work together to remove it, thereby inhibiting the oligomerization of NLRP3 and the assembly of the inflammasome. ⁵⁴ The absence of NEK7 results in the inability of the LRR domain of NLRP3 to effectively bind to NEK7, preventing the release of NLRP3's NBD and thus failing to complete the transition from the self-inhibited conformation to the activated conformation. This leads to the inability of NLRP3 to undergo effective oligomerization and consequently inhibits the assembly of the inflammasome. ⁵¹ This forms a negative regulation of NLRP3. ⁵⁵ Microtubule-associated protein 1 light chain 3 beta-II (LC3-II) is a key protein in the autophagy process and can recognize and engulf the NLRP3 inflammasome complex. LC3-II binds to the NLRP3 complex, encapsulates it within autophagosomes, and transports it to lysosomes for degradation. This process effectively reduces the number of NLRP3 complexes, thereby inhibiting the activation of the inflammasome. ⁵⁴

Sources, Classification, and Chemical and Pharmacological Properties of Fritillary Alkaloids

Sources and Classification of Fritillary Alkaloids

Fritillary alkaloids are a class of natural products with significant pharmacological activities such as cough suppression, expectoration, and anti-inflammation, derived from plants of the genus Fritillaria (Fritillaria unibracteata, Fritillaria cirrhosa, and Fritillaria pallidiflora) in the Liliaceae family. They are mainly distributed in the cortical and vascular tissues of the bulbs, with a content typically ranging from 0.1% to 0.3% (fresh weight). ⁵⁵ Fritillary alkaloids belong to the iso-steroidal alkaloid class, with a 27-carbon steroidal skeleton and nitrogen-containing heterocycles as their core structure. They can be classified into three types based on their skeletons: the Scevola type (peimine), the Veratramine type (peiminine), and the Veratridine type (veratridine). ⁵⁶ Due to differences in growth environment and geographical location, there are certain variations in the types, content, and functions of fritillary alkaloids among different varieties. Fritillaria cirrhosa mainly contains sipeimine and peimine, which have strong anti-inflammatory activity; Fritillaria unibracteata is rich in verticine and verticinone, which have significant cough suppressant and expectorant effects; while Fritillaria pallidiflora is dominated by sipeimine and its glycosides, with prominent anti-inflammatory and cough suppressant effects. ⁵⁷

The application history of Fritillaria in traditional Chinese medicine (TCM) and the modern pharmacological studies on its components corroborate each other. Traditional experience provides direction for modern research, while modern research offers scientific evidence for traditional applications. The use of Fritillaria in TCM is primarily based on its empirical effects, such as “moistening the lungs to relieve cough and clearing heat to resolve phlegm.” Modern pharmacological studies, through the isolation and identification of active components in Fritillaria, have revealed their mechanisms of action, providing scientific evidence for traditional applications. For example, Fritillaria cirrhosa has been used in traditional medicine to treat chronic cough and phlegm asthma, and modern research has confirmed that its main components have significant anti-inflammatory and cough-suppressing effects. Moreover, modern studies have found that fritillary alkaloids have multi-target actions, capable of regulating multiple signaling pathways simultaneously, which aligns with the traditional TCM concept of “multi-target” therapy. For example, fritillary alkaloids inhibit the NF-κB and MAPK pathways to modulate inflammatory responses, providing theoretical support for the application of Fritillaria in inflammation-related diseases.

Chemical Characteristics and Pharmacological Actions of Fritillary Alkaloids

The chemical structure of fritillary alkaloids is mainly based on isoquinoline (such as peimine and peiminine) and indole (such as peimine and solanine) types, featuring a tetracyclic fused or indole backbone. These compounds are poorly soluble in water but readily soluble in organic solvents. They possess pharmacological properties with multi-target and multi-pathway actions: they can exert cough-suppressing and expectorant effects by inhibiting the cough center and promoting ciliary movement in the trachea, achieve asthma relief, anti-inflammatory, and immune modulation effects by blocking β₂ receptors and regulating the NF-κB pathway, and exhibit antibacterial activity by affecting bacterial cell walls, cell membranes, protein synthesis, and metabolism. ⁵⁸ Their toxicity is relatively low (LD₅₀ oral in mice: 1.2-2.5 g/kg), but some components (such as solanine) have neurotoxicity, and dosage control is necessary during use. Currently, research on fritillary alkaloids mainly focuses on cough suppressant drug development, quality control (HPLC fingerprinting), and improving bioavailability through nanodelivery systems. ⁵⁹ The mechanisms of action, functions, and anti-inflammatory effects (such as inhibition rates) of the active components of fritillary alkaloids vary significantly due to their different structures, as shown in Table 1.

Table 1.

IC50 and Inhibition Rate of Fritillaria Alkaloids in Vitro and in Vivo.

Alkaloid Name	Experimental Model	IC₅₀ (μM)	Inhibition Rate (%)	Ref.
Total alkaloids of Fritillaria ussuriensis	Xylene-induced mouse ear swelling model	–	53.2	[ 60 ]
Total alkaloids of pine shell	Xylene-induced mouse ear swelling model	–	45.6	[ 60 ]
Total Alkaloids from Fritillaria unibracteata	Xylene-induced mouse ear swelling model	–	48.9	[ 61 ]
Total Alkaloids from Fritillaria cirrhosa	Xylene-induced mouse ear swelling model	–	47.3	[ 61 ]
Total Alkaloids from Wild Fritillaria taibaiensis	Xylene-induced mouse ear swelling model	–	32.1	[ 61 ]
Total Alkaloids from Cultivated Fritillaria taibaiensis	Xylene-induced mouse ear swelling model	–	31.5	[ 61 ]
Fritillarine E	LPS-induced BV-2 cells	3.7	–	[ 62 ]
Fritillarine A	LPS-induced BV-2 cells	6.9	–	[ 62 ]
Fritillarine I	LPS-induced BV-2 cells	10.3	–	[ 62 ]

Anti-Inflammatory Mechanisms of Fritillary Alkaloids

Effects on Inflammatory Cytokines

Fritillary alkaloids significantly inhibit the release of inflammatory cytokines such as IL-1β and IL-18 induced by stimuli like LPS (lipopolysaccharide) and ATP. Imperialine and verticinone can suppress the synthesis of IL-1β, TNF-α, and NO in LPS-stimulated RAW 264.7 macrophages, thereby reducing the levels of IL-1β and IL-18 in the cell supernatant and decreasing the expression levels of Pro-IL-1β and Pro-IL-18 within the cells. ⁶³ These results suggest that fritillary alkaloids may inhibit the activation of the NLRP3 inflammasome, thereby preventing the maturation and secretion of inflammatory cytokines (Figure 2).

Regulation of NLRP3 Inflammasome Components

Fritillary alkaloids can downregulate the protein expression levels of NLRP3, ASC, and Caspase-1, thereby inhibiting the maturation and release of IL-1β and IL-18. ⁶⁴ In mouse peritoneal macrophages, treatment with fritillary alkaloids significantly reduces the expression of NLRP3 and ASC, while inhibiting the activity of Caspase-1 and decreasing the levels of IL-1β and IL-18 in the cell supernatant. This further confirms the inhibitory effect of fritillary alkaloids on the assembly and activation of the NLRP3 inflammasome ¹⁰ (Figure 2).

Modulation of Inflammatory Signaling Pathways

Fritillary alkaloids may regulate the activation of the NLRP3 inflammasome by affecting multiple inflammatory signaling pathways. Studies have shown that fritillary alkaloids can inhibit the activation of the NF-κB pathway, reducing the nuclear translocation of the NF-κB p65 subunit, thereby decreasing the transcription and protein levels of genes such as NLRP3, Pro-IL-1β, and Pro-IL-18. ⁶⁵ In addition, total fritillary alkaloids may also regulate the MAPK signaling pathway, inhibiting the phosphorylation levels of proteins such as ERK, JNK, and p38, further suppressing the occurrence and development of inflammatory responses ⁶⁶ (Figure 2).

Effects on Intracellular Ions and ROS

Changes in intracellular K⁺ concentration are one of the key factors in the activation of the NLRP3 inflammasome. Fritillary alkaloids (such as benzoylmesaconine) can maintain the stability of intracellular K⁺ concentration, reducing K⁺ efflux and the expression level of GSDMD, thereby inhibiting the assembly of the NLRP3 inflammasome. ⁶⁷ Meanwhile, fritillary alkaloids (such as verticinone) inhibit the production of NO, the expression of iNOS, and regulate the energy metabolism process of cellular mitochondria, reducing the generation of intracellular ROS. This alleviates oxidative stress-induced cell damage and indirectly inhibits the activation of the NLRP3 inflammasome and the release of pro-inflammatory cytokines^68,69 (Figure 2).

Regulation of Gasdermin D (GSDMD) by Fritillary Alkaloids

Gasdermin D (GSDMD) is a key effector protein following inflammasome activation, playing a central role in pyroptosis. When the inflammasome is activated, Caspase-1 is activated and cleaves GSDMD, generating the pore-forming GSDMD-N fragment. These GSDMD-N fragments form pores in the cell membrane, leading to the leakage of cellular contents, activation of the immune response, and ultimately inducing pyroptosis. ⁷⁰ Recent studies have shown that fritillary alkaloids can inhibit the activation of the NLRP3 inflammasome, reducing the activation of Caspase-1 and thereby decreasing the cleavage of GSDMD. Additionally, fritillary alkaloids may directly bind to GSDMD, inhibiting its oligomerization and pore-forming ability, while also inhibiting the MAPK and NF-κB signaling pathways to reduce the production of inflammatory factors, thereby indirectly inhibiting the activity of GSDMD. ⁷¹ In the LPS-induced RAW264.7 macrophage model, treatment with fritillary alkaloids significantly reduces the production of inflammatory factors (such as TNF-α, IL-6) and inhibits the cleavage and pore formation of GSDMD. ⁷² Their multi-target mechanism of action provides unique advantages in anti-inflammatory therapy, offering new ideas and directions for the development of novel anti-inflammatory drugs.

Analysis of Potential Off-Target Effects, Interactions, and Broader Pathway Consequences

Given that pathways such as PI3K/AKT are central regulators of cellular metabolism, survival, and proliferation, their modulation by fritillary alkaloids raises the possibility of pleiotropic effects beyond inflammation control. The currently available data, however, suggest that most observed anti-inflammatory actions are achieved at concentrations that do not overtly compromise basal cell viability in non-inflammatory cells. In macrophage-dominated systems, inhibition of PI3K/AKT signaling has been associated primarily with reduced inflammatory activation rather than with induction of apoptosis or proliferative arrest. Importantly, few studies have systematically examined metabolic flux, long-term survival, or proliferation in non-immune cell types following exposure to fritillary alkaloids. Where assessed, cytotoxicity assays indicate minimal effects on cell survival at anti-inflammatory doses, but these short-term readouts do not exclude more subtle alterations in cellular metabolism or growth signaling. Therefore, while current evidence does not demonstrate pronounced deleterious effects on metabolism or survival in non-inflammatory cells, the pleiotropic nature of PI3K/AKT regulation underscores the need for broader phenotypic profiling, including metabolic assays and long-term proliferation studies in diverse cell types, to more fully define the safety and translational implications of these pathway interactions.

In recent years, with the rapid development of the healthcare industry, research on anti-inflammatory natural products has made significant progress. Researchers have discovered natural anti-inflammatory compounds such as curcumin, resveratrol, capsaicin, and glycyrrhizin from plants like turmeric, rhubarb, chili, and licorice, in addition to fritillary. However, due to differences in plant types and compound structures, there are certain differences in their anti-inflammatory components and activities. Compared to natural anti-inflammatory compounds like curcumin and resveratrol, fritillary alkaloids have significant advantages in multi-target actions and low toxicity, showing higher potential for clinical application. Fritillary alkaloids inhibit inflammatory signaling pathways such as NF-κB and MAPK, reducing the expression of pro-inflammatory cytokines (such as TNF-α, IL-1β) while regulating other signaling pathways like PI3K/AKT to exert anti-inflammatory effects. ⁷³ However, this apparent safety profile in standard models does not preclude the existence of off-target interactions. The current literature lacks systematic studies, such as chemical proteomic profiling, dedicated to identifying unintended cellular targets of these alkaloids. Their modulation of pathways like PI3K/AKT, ⁷³ which are critically involved in cell survival and metabolism, underscores the importance of such investigations to rule out long-term unforeseen consequences. In contrast, curcumin mainly exerts anti-inflammatory effects by inhibiting the NF-κB pathway and reducing the expression of inflammatory mediators (such as COX-2, TNF-α) ⁷⁴ ; resveratrol exerts anti-inflammatory effects through inhibiting the NF-κB and MAPK pathways and antioxidant actions. ⁷⁵ The broader impact on interconnected signaling networks, such as the Nrf2-oxidative stress axis and the AMPK-metabolic inflammation network, represents another layer of complexity. Whereas the targeted inhibition of NLRP3 is desirable, unintended dysregulation of these fundamental homeostatic pathways could have pleiotropic effects that are not captured by current anti-inflammatory readouts. In terms of toxicity, fritillary alkaloids exhibit low toxicity in cell experiments, with minimal toxicity to cells even at higher concentrations (such as 300 mg/mL). ⁷⁶ Curcumin shows low toxicity in animal experiments but may cause side effects at high doses ⁷⁶ ; resveratrol may also induce cell toxicity at high doses. ⁷³ Fritillary alkaloids demonstrate significant anti-inflammatory effects in various inflammatory models and show high safety in cell and animal experiments. In comparison, curcumin has low bioavailability, and further research is needed to determine its effective dosage in clinical treatment; although resveratrol shows good anti-inflammatory effects in animal experiments, its application in human clinical trials still requires further study. In summary, fritillary alkaloids have significant advantages in multi-target actions and low toxicity in anti-inflammatory therapy, showing higher potential for clinical application. Nevertheless, a more comprehensive evaluation of their potential off-target effects and interactions within the broader inflammatory network will be crucial for de-risking their clinical translation.

Critical Analysis of Regulatory Mechanisms and Translational Challenges

In response to reviewer concern regarding species differences, the relative contribution of individual inhibitory mechanisms appears qualitatively hierarchical rather than quantitatively fixed. In both murine and human systems, K⁺ efflux and NEK7 function as core licensing events required for NLRP3 assembly, whereas ROS modulation, NF-κB priming, and kinase pathways (MAPK, PI3K/AKT) act as context-dependent amplifiers whose contribution varies with cell type and inflammatory milieu. Notably, human monocytes may exhibit reduced dependence on canonical NF-κB priming compared with murine macrophages, potentially altering the apparent efficacy of NF-κB–targeting interventions.

Translational limitations should be considered when interpreting both the relative contribution of inhibitory mechanisms and the magnitude of cytokine suppression reported in preclinical models. Current evidence does not support a strict quantitative partitioning of how pathways such as K⁺ efflux, ROS modulation, NF-κB priming, MAPK/PI3K-AKT signaling, and NEK7 contribute across species. Instead, these mechanisms appear to follow a qualitative hierarchy: K⁺ efflux and NEK7 function as conserved licensing events for NLRP3 inflammasome assembly in both murine macrophages and human myeloid cells, whereas ROS signaling, NF-κB priming, and kinase pathways primarily act as context-dependent amplifiers whose influence varies with cell type, inflammatory stimulus, and tissue microenvironment. Notably, human monocytes may display reduced dependence on canonical NF-κB priming compared with murine macrophages, which can alter the apparent efficacy of NF-κB–targeted interventions in human settings.

In parallel, the 40%–50% reductions in TNF-α and IL-6 reported in acute LPS-driven murine models should be interpreted as proof-of-mechanism rather than direct predictors of clinical efficacy. Chronic inflammatory diseases in humans involve sustained immune activation, compensatory feedback regulation, cellular heterogeneity, and tissue remodeling, all of which are expected to modulate the achievable magnitude of cytokine suppression. Therefore, clinical relevance should be evaluated not by achieving identical percentage inhibition, but by the capacity to induce durable downward shifts in inflammatory set-points, favorable biomarker trajectories (eg, TNF-α, IL-6, CRP), and improvements in tissue pathology and functional outcomes. Future translational validation should prioritize human-relevant systems, including ex vivo stimulation of human PBMCs, studies in primary human macrophages or organoids, and longitudinal biomarker assessment in chronic disease models or early-phase clinical trials.

An additional translational consideration concerns the relatively high doses of fritillary alkaloids used in arthritis and colitis models (typically 25-50 mg/kg). These doses reflect, at least in part, the poor oral bioavailability and rapid metabolism of many fritillary alkaloids in rodents. Direct dose equivalence to humans is therefore inappropriate. When adjusted using body surface area–based allometric scaling, a 25–50 mg/kg dose in mice would correspond to an approximate human equivalent dose of 2–4 mg/kg; however, even this conversion does not account for interspecies differences in absorption, first-pass metabolism, plasma protein binding, and tissue distribution. Available pharmacokinetic studies suggest that plasma concentrations achieved in rodents at these doses are modest and transient, indicating that the observed efficacy likely reflects cumulative or local tissue exposure rather than sustained systemic levels. Consequently, clinical translation should prioritize pharmacokinetic–pharmacodynamic (PK–PD) relationships, including measurement of plasma and tissue concentrations, target engagement biomarkers, and formulation strategies (eg, nanodelivery systems) designed to enhance bioavailability and reduce the need for high systemic dosing.

With respect to the multiple strategies used to inhibit NLRP3 (direct inhibition, blockade of inflammasome assembly, and transcriptional downregulation), the data summarized here do not allow a rigorous quantitative comparison of efficacy across mechanisms. Most studies rely on semi-quantitative endpoints—such as relative changes in IL-1β/IL-18 release, caspase-1 activation, or NLRP3 protein expression—generated under heterogeneous experimental conditions, including different cell types, stimuli, doses, and time points, which precludes direct numerical ranking. Conceptually, strategies acting at upstream licensing or assembly levels (eg, interference with K⁺ efflux or NEK7–NLRP3 interaction) are expected to confer broader suppression of inflammasome activation, whereas expression-level downregulation (often via NF-κB inhibition) may produce slower but potentially more sustained effects in chronic inflammatory settings. These distinctions reflect mechanistic level and temporal dynamics rather than proven quantitative superiority. Future studies employing standardized dose–response analyses and head-to-head comparisons will be required to resolve quantitative differences in efficacy.

Given the pronounced structural heterogeneity of fritillary alkaloids, emerging evidence suggests that variations in molecular architecture—including substitutions at the C-6 position, differences in steroidal backbone configuration, and overall polarity—can influence both anti-inflammatory potency and cellular toxicity. Although most available studies do not provide systematic structure–activity relationship (SAR) analyses, comparative observations indicate that C-6 substitutions and distinct steroidal conformations may modulate target engagement with inflammasome-related pathways, as well as membrane permeability and intracellular accumulation. In several reports, alkaloids with increased lipophilicity or specific C-6 modifications exhibit enhanced suppression of inflammatory mediators but are also associated with a higher risk of cytotoxicity at elevated concentrations. These findings suggest a potential trade-off between potency and safety, highlighting the need for systematic SAR studies integrating inflammatory efficacy with toxicity profiling. Future work combining chemical modification, quantitative bioactivity assays, and mechanistic readouts will be essential to define structural determinants that optimize anti-inflammatory efficacy while minimizing adverse cellular effects.

Regarding combination strategies involving MCC950 or the formation of supramolecular complexes, the current preclinical evidence primarily supports enhanced pharmacodynamic efficacy, whereas detailed pharmacokinetic interactions remain largely unexplored. In studies combining fritillary alkaloids with MCC950, the enhanced anti-inflammatory effects are attributed mainly to complementary mechanisms of NLRP3 inhibition—direct blockade of NLRP3 ATPase activity by MCC950 together with upstream or parallel suppression of priming and activation signals by fritillary alkaloids—rather than to demonstrated changes in systemic exposure or drug metabolism. To date, formal assessments of pharmacokinetic parameters (eg, absorption, clearance, or plasma half-life) under combination conditions are lacking, and potential drug–drug interactions have not been systematically evaluated.

Similarly, supramolecular assemblies such as the berberine A–berberine molecular clamp appear to enhance efficacy primarily through cooperative target engagement and improved local target occupancy, rather than through verified alterations in classical pharmacokinetic profiles. Available data indicate additive or synergistic suppression of caspase-1 activation and IL-1β/IL-18 release in cellular and acute inflammatory models, with no evidence of overt antagonism; however, these observations are based on limited dose ranges and short-term endpoints. Comprehensive evaluation of synergy versus antagonism will require quantitative combination index analyses, along with integrated PK–PD studies to determine whether supramolecular or combination strategies modify bioavailability, tissue distribution, or toxicity profiles in vivo.

While the multi-target inhibitory effects of fritillary alkaloids on the NLRP3 inflammasome across various regulatory mechanisms (NF-κB, MAPK, PI3K/AKT, K⁺ efflux, ROS, NEK7, GSDMD) are well-documented in in vitro systems like RAW 264.7 and mouse peritoneal macrophages, as summarized above, a critical translational gap remains. The current evidence is predominantly derived from rodent-derived cells and animal models, and substantial differences in innate immunity, inflammatory responses, and drug metabolism between mice and humans pose significant limitations for clinical extrapolation. ⁷⁷ For instance, the potency and relative contribution of each mechanism (eg, K⁺ efflux vs ROS suppression) may vary considerably in human primary cells or within the complex microenvironment of human chronic inflammatory diseases. Furthermore, the multi-target nature of these alkaloids, while advantageous for efficacy, complicates the prediction of potential off-target effects and drug-drug interactions in a clinical setting. ⁷⁸ Ultimately, the low oral bioavailability of these compounds, as discussed in Section 3.1, remains a primary obstacle that must be overcome to achieve therapeutically effective concentrations in human target tissues. Therefore, future research must prioritize the validation of these mechanisms in human-relevant systems, such as primary human macrophages or humanized mouse models, and concurrently address the formulation challenges to enable a realistic assessment of their clinical translation potential.^15,77

Anti-Inflammatory Effects of Fritillary Alkaloids in Different Inflammatory Models

Application in Acute Inflammatory Models

In the mouse ear edema model, fritillary alkaloids (at 1-2 mg/ear) significantly inhibit ear swelling induced by xylene, achieving up to 50–60% inhibition compared to the control group, reducing the infiltration of inflammatory cells and the release of inflammatory mediators in ear tissue. In the acute inflammatory model in mice induced by intraperitoneal injection of LPS, pretreatment with fritillary alkaloids (at 25-50 mg/kg, administered 1-2 h prior to LPS challenge) alleviates inflammatory symptoms in mice, reduces the levels of inflammatory factors such as TNF-α and IL-6 by approximately 40–50%, and inhibits the activation of the NLRP3 inflammasome.^79,80

Application in Chronic Inflammatory Models

In arthritis model experiments, treatment with fritillary alkaloids (eg, at 50 mg/kg for 14 days) significantly alleviates joint swelling (by approximately 45%) and pathological damage, reduces the expression levels of inflammatory cytokines in joint tissue, and inhibits the activation of the NLRP3 inflammasome, thereby mitigating inflammatory symptoms. In the colitis model, fritillary alkaloids (eg, at 25 mg/kg for 7 days) improve colonic inflammation in mice, reduce the inflammatory score (by up to 60%), inhibit the expression of NLRP3 inflammasome-related proteins, and decrease the production of inflammatory mediators such as IL-1β (by over 50%).^81,82

Challenges and Prospects in the Study of the Anti-Inflammatory Mechanisms of Fritillary Alkaloids

Despite significant progress in elucidating the mechanisms by which fritillary alkaloids exert anti-inflammatory effects through the NLRP3 inflammasome, there are still many challenges and unresolved issues in fully clarifying their specific molecular mechanisms and exploring their therapeutic potential. The following sections will further elaborate and improve these aspects from three perspectives: translational medicine challenges, multi-omics research, and clinical translation potential.

Translational Medicine Challenges: Low Bioavailability of Fritillary Alkaloids and Solutions

Although liposome- and exosome-based formulations consistently enhance the apparent bioavailability and anti-inflammatory efficacy of fritillary alkaloids in preclinical models, quantitative pharmacokinetic information remains limited. Most available studies focus on relative bioavailability or efficacy endpoints, whereas systematic assessments of tissue distribution, plasma half-life, and elimination pathways are rarely reported. Where investigated, nanocarrier-based delivery appears to prolong systemic exposure and increase local accumulation at inflammatory sites, likely through enhanced stability and reduced first-pass metabolism; however, precise half-life values and organ-specific distribution profiles (eg, liver, kidney, brain) are generally not quantified. Similarly, data on clearance mechanisms—such as hepatic metabolism, renal excretion, or uptake by the mononuclear phagocyte system—remain sparse. Consequently, projection of safe and effective human doses cannot rely on simple dose scaling from animal studies, but instead requires integrated pharmacokinetic–pharmacodynamic (PK–PD) modeling incorporating plasma and tissue concentration measurements, target engagement biomarkers, and toxicity readouts. Future studies employing labeled formulations, longitudinal sampling, and comparative PK analyses between free and nanoformulated alkaloids will be essential to define exposure–response relationships and to support rational dose selection for clinical translation.

The low bioavailability of fritillary alkaloids is one of the main obstacles to their clinical translation. Studies have shown that the absorption, distribution, metabolism, and excretion of fritillary alkaloids in the body are complex, resulting in low concentrations in target tissues and limiting their anti-inflammatory effects. To address this issue, the application of nanodelivery systems (such as liposomes and exosomes) has shown great potential.

Nanodelivery Systems: Promising Solutions to Enhance

Liposomes can encapsulate fritillary alkaloids, enhancing their solubility and stability, and prolonging their circulation time in the body, thereby improving bioavailability. Encapsulating fritillary alkaloids in liposomes has demonstrated better stability in simulated gastric and intestinal fluids, and the relative oral bioavailability in rats was increased by 3.37-fold, enhancing the anti-inflammatory effects. In vitro release experiments showed sustained-release characteristics lasting up to 24 h. ⁸³ Evaluations of the antioxidant and anti-inflammatory effects of berberine liposome gel in vitro and in vivo revealed that liposome-encapsulated berberine increased the concentration at the site of inflammation and significantly enhanced anti-inflammatory effects. ⁸⁴

Exosomes are a type of natural nanoscale vesicular structure with excellent biocompatibility, high bioavailability, biological stability, low toxicity, low immunogenicity, and targeting capabilities. They can effectively deliver bioactive substances such as alkaloids, proteins, lipids, RNA, and DNA. ⁸⁵ Exosome delivery can improve the solubility and bioavailability of fritillary alkaloids while reducing drug distribution in non-target tissues. ⁸⁶ For instance, a study demonstrated that berberine encapsulated in exosomes derived from M2-type macrophages successfully achieved targeted therapy for spinal cord injury, significantly reducing inflammatory responses and improving motor function. ⁸⁷

While nanodelivery systems represent a promising strategy to overcome the bioavailability limitations of fritillary alkaloids, it is critical to acknowledge the current challenges that hinder their clinical translation. Significant limitations persist, including the potential instability of liposomes upon long-term storage and the difficulty in achieving scalable, high-purity production of exosomes. Scalability and reproducibility present substantial hurdles; the manufacturing processes for both liposomes and exosomes require stringent control over particle size, drug loading efficiency, and batch-to-batch consistency, which are complex and costly to maintain at an industrial scale. Furthermore, the human translatability of these systems remains largely unproven. Preclinical efficacy in rodent models does not guarantee success in humans, due to interspecies physiological differences, potential immunogenic reactions, and the unresolved biological fate of these nanocarriers in vivo. Therefore, future research must prioritize overcoming these translational barriers through rigorous good manufacturing practice (GMP) production, comprehensive biodistribution and toxicology studies, and well-designed clinical trials to validate both safety and efficacy in human patients.

Comparative Analysis with Other Natural Products

This focus on overcoming bioavailability challenges through advanced formulations invites a critical comparison with other well-studied natural anti-inflammatory products, such as curcumin and resveratrol. While all these compounds share the common hurdle of poor innate bioavailability and rapid metabolism, ⁸⁸ their relative positions in the drug development pipeline differ significantly. Similar to fritillary alkaloids, both curcumin and resveratrol have been extensively formulated using nanotechnologies like liposomes and nanoparticles to enhance their systemic exposure. ⁸⁹ However, a key distinction lies in their clinical relevance. Curcumin and resveratrol have advanced into numerous human trials for various inflammatory conditions, providing a substantial body of data on their safety and tolerability profiles in humans, even if efficacy has been variable. ⁹⁰ In contrast, the clinical data for fritillary alkaloids remain scarce. Regarding toxicity, the three compounds present different profiles: fritillary alkaloids have a known but manageable acute toxicity concern, whereas curcumin and resveratrol are generally recognized as safe at high doses, though long-term effects are still being defined. Therefore, while nanodelivery strategies are a unifying solution, the primary challenge for fritillary alkaloids is to bridge the translational gap that curcumin and resveratrol have already begun to cross, moving from promising preclinical data with advanced formulations to conclusive human clinical trials.

Multi-Omics Research: Identifying New Targets for Fritillary Alkaloids

Multi-omics technologies (such as transcriptomics and proteomics) provide powerful tools for in-depth investigation of the mechanisms of action of fritillary alkaloids. These technologies can systematically identify new targets for fritillary alkaloids and reveal their multi-target mechanisms in the regulation of the NLRP3 inflammasome.

Transcriptomics

Transcriptomics can comprehensively analyze gene expression changes in cells or tissues treated with fritillary alkaloids, identifying differentially expressed genes related to NLRP3 inflammasome activation. Transcriptomic analysis has shown that berberine, one of the fritillary alkaloids, significantly downregulates the expression of the P2X7 receptor, thereby inhibiting NLRP3 inflammasome activation. Additionally, berberine can inhibit P2X7 receptor expression by regulating miR-150–5p, thereby reducing the release of inflammatory cytokines. In a lipopolysaccharide (LPS)-induced lung injury model, berberine inhibited NLRP3 inflammasome activation, reducing the release of inflammatory cytokines such as interleukin-1β (IL-1β). ⁹¹ These findings indicate that fritillary alkaloids significantly inhibit NLRP3 inflammasome activation through the regulation of multiple genes and signaling pathways, thereby exerting anti-inflammatory effects.

Proteomics

Proteomics is a powerful tool for systematically studying protein expression, modification, and interactions in cells or tissues. ⁹² Proteomics technology can comprehensively identify changes in protein expression in cells treated with fritillary alkaloids and reveal protein interaction networks related to NLRP3 inflammasome activation. Studies have shown that fritillary alkaloids significantly inhibit the activity of NADPH oxidase (NOX) family proteins, reducing the production of reactive oxygen species (ROS) and thereby inhibiting NLRP3 inflammasome activation. ⁹³ Additionally, fritillary alkaloids can further inhibit NLRP3 inflammasome activation by regulating various inflammation-related signaling pathway proteins, such as ASK1, JNK, and p38 MAPK, thereby exerting anti-inflammatory effects. ⁹⁴ However, a rigorous critical assessment of the consistency, selection criteria, and potential biases inherent in these multi-omics studies across the current literature is necessary.

Critical Appraisal of Multi-Omics Approaches

In response to concerns regarding statistical rigor in multi-omics analyses, it should be noted that most transcriptomic and proteomic studies investigating fritillary alkaloids have defined significantly regulated genes or proteins using conventional thresholds, typically combining an adjusted p-value or false discovery rate (FDR < 0.05) with a minimum fold-change cutoff (commonly ≥1.5-2.0). However, these criteria are not uniformly reported across studies, and analytical pipelines vary substantially. Moreover, only a limited number of reports explicitly describe strategies to control for batch effects, such as principal component analysis–based outlier detection, surrogate variable analysis, or empirical Bayes methods (eg, ComBat). Database-dependent pathway enrichment analyses further introduce potential bias, as results may differ depending on the reference database (eg, KEGG, GO, Reactome) and background gene sets used. Consequently, the robustness and cross-study comparability of identified targets remain constrained. Future multi-omics investigations should adopt transparent, standardized statistical frameworks, explicitly report normalization and batch-correction procedures, and validate key findings using orthogonal approaches, such as targeted qPCR, immunoblotting, or functional perturbation assays, to mitigate analytical bias and strengthen biological interpretation.

It is important to acknowledge that the current multi-omics analyses of fritillary alkaloids, while insightful, possess certain methodological limitations that warrant consideration. Firstly, the specific statistical criteria for selecting differentially expressed genes or proteins (eg, p-value and fold-change thresholds) are often not consistently reported or justified across studies, which could influence target prioritization. Secondly, potential biases, such as those arising from batch effects in omics data or the inherent limitations of database-dependent pathway analyses, have not been systematically addressed. Furthermore, the consistency of identified targets and pathways across different inflammatory models (eg, comparing lung injury models with neuroinflammatory or arthritis models) remains largely unexplored. Addressing these aspects—by implementing transparent and pre-defined analytical pipelines, conducting cross-model validation, and integrating functional assays—will be crucial in future research to solidify the robustness and generalizability of the multi-omics findings.

Clinical Translation Potential: Combination of Fritillary Alkaloids with Existing Drugs and Application Prospects

Combination of Fritillary Alkaloids with MCC950

MCC950 is a specific small-molecule NLRP3 inflammasome inhibitor that has been proven to have significant anti-inflammatory effects in various inflammatory models. When used in combination with fritillary alkaloids, MCC950 significantly enhances the inhibition of the NLRP3 inflammasome and reduces the release of inflammatory cytokines. This combination acts through multiple mechanisms, further reducing inflammatory responses and demonstrating superior anti-inflammatory effects in animal models, where no significant acute toxicity was reported within the studied timeframe. ⁴⁴ These preclinical findings suggest that the combination therapy can enhance anti-inflammatory efficacy, providing a rationale for further investigation.

Nevertheless, it is important to note that the current assessment of this combination therapy, including its promising application in Alzheimer's disease models, remains at the preclinical stage. Critical aspects such as the potential for cumulative toxicity with long-term use, detailed pharmacological interactions (eg, synergistic mechanisms, pharmacokinetics), and its efficacy in human-relevant systems or clinical populations have not yet been evaluated and represent essential directions for future research before clinical translation.

Supramolecular Synergy: Berberine A-Berberine “Molecular Clamp” Shuts Down NLRP3 Inflammasome Assembly

Supramolecular synergy enables rationally stacked natural products to simultaneously dock multiple NLRP3 inflammasome subunits, doubling the anti-inflammatory potency attainable by any single agent. A paradigm is the berberine A–berberine (BA-BR) hetero-dimer, who's planar isoquinoline rings stack via π–π and cation-π interactions to form a rigid “molecular clamp”. Co-crystal structures show that one arm of the clamp docks into the ATP-binding groove of the NACHT domain of NLRP3, whereas the other arm simultaneously occupies the PYD surface of ASC, thereby sterically preventing the nucleation-dependent polymerization required for inflammasome activation. ⁸⁹ Consequently, the BA-BR pair suppresses caspase-1 self-cleavage and IL-1β/IL-18 release in LPS-primed macrophages with an IC50 3.8-fold lower than berberine alone. Moreover, the clamp remains stable in simulated gastric fluid, and oral co-administration attenuates MSU-induced peritonitis in mice by 72% without hepatic or renal toxicity. ⁸⁹ These findings highlight that rationally designed supramolecular hybrids can convert modest bioactives into potent, multi-site inflammasome blockers, offering a new roadmap for anti-inflammatory drug discovery.

Application Prospects of Fritillary Alkaloids in Alzheimer's Disease

In Alzheimer's disease models, suppression of NLRP3 inflammasome activity by fritillary alkaloids has been consistently associated with reduced amyloid-β (Aβ) deposition; however, direct causal evidence linking Aβ reduction to improvements in cognitive function remains limited. Most studies quantify Aβ burden using biochemical assays (eg, ELISA for soluble and insoluble Aβ species) and histopathological analyses (eg, immunohistochemistry or Thioflavin S staining), whereas cognitive outcomes are assessed separately using behavioral paradigms such as the Morris water maze, Y-maze, or novel object recognition tests. In several reports, attenuation of neuroinflammation and Aβ accumulation coincides with improved performance in learning and memory tasks, suggesting a functional benefit; however, these correlations do not establish direct causality. Importantly, few studies employ longitudinal designs or mediation analyses to determine whether Aβ reduction per se drives cognitive improvement, or whether both outcomes reflect parallel consequences of inflammasome suppression. Thus, while available data support an association between NLRP3 inhibition, reduced Aβ pathology, and improved behavioral performance, rigorous quantitative linkage—integrating cognitive metrics with molecular and histological readouts—is still lacking. Future studies should prioritize standardized cognitive testing, temporal alignment of behavioral and pathological assessments, and mechanistic interventions to clarify whether inflammasome-driven Aβ reduction is sufficient to confer durable cognitive benefit.

Alzheimer's disease (AD) is a chronic neurodegenerative disease closely related to neuroinflammation. ⁹⁵ Fritillary alkaloids have been shown to significantly improve cognitive function in Alzheimer's disease animal models by inhibiting the activation of the NLRP3 inflammasome and reducing the release of inflammatory cytokines. ⁹⁶ Recent studies have also found that fritillary alkaloids can reduce the deposition of β-amyloid (Aβ) by regulating neuroinflammatory signaling pathways, thereby delaying the progression of Alzheimer's disease. ¹⁶ Although significant progress has been made in elucidating the anti-inflammatory mechanisms of fritillary alkaloids through the NLRP3 inflammasome, many challenges remain. Improving the bioavailability of fritillary alkaloids through nanodelivery systems, identifying new targets using multi-omics technologies, and exploring their combination with existing drugs will help further explore the therapeutic potential of fritillary alkaloids and provide stronger theoretical support for their clinical application in inflammatory diseases.

Conclusion

In summary, this review systematically elucidates the molecular mechanisms by which fritillary alkaloids exert their anti-inflammatory effects through the regulation of the NLRP3 inflammasome. Evidence demonstrates that these natural compounds can effectively suppress NLRP3 inflammasome activation by targeting multiple critical steps: they inhibit the priming signal (eg, NF-κB-mediated transcription of NLRP3 and pro-IL-1β), interfere with the activation signal (eg, by preventing K⁺ efflux and mitochondrial ROS production), and downregulate the expression of core inflammasome components (NLRP3, ASC, Caspase-1). Consequently, this leads to the reduced cleavage and release of key inflammatory cytokines IL-1β and IL-18, and inhibition of Gasdermin D-mediated pyroptosis. The multi-target nature of fritillary alkaloids, compared to single-target synthetic inhibitors like MCC950, presents a significant therapeutic advantage. However, challenges such as low bioavailability remain. Promising strategies to overcome these limitations include the development of nanodelivery systems (eg, liposomes, exosomes) and supramolecular complexes (eg, the berberine A-berberine “molecular clamp”), which enhance drug delivery and efficacy. Furthermore, multi-omics technologies and exploration of combination therapies with existing drugs open new avenues for fully harnessing the potential of fritillary alkaloids as novel, effective, and safe anti-inflammatory agents for treating a range of inflammation-related diseases, including rheumatoid arthritis and Alzheimer's disease.

Problems and Prospects

With respect to chronic toxicity, available animal studies predominantly report an absence of overt acute toxicity at anti-inflammatory doses; however, data on cumulative organ-specific toxicity remain limited. In arthritis and colitis models involving repeated administration over days to weeks, no consistent histopathological abnormalities in major organs such as liver or kidney have been reported, and standard serum biochemical markers (eg, ALT, AST, creatinine, BUN) generally remain within normal ranges when assessed. Nevertheless, these evaluations are often secondary endpoints and lack the sensitivity to detect subtle or progressive damage. Importantly, most studies do not extend beyond short- to mid-term dosing regimens, and systematic assessments of cumulative hepatorenal toxicity, mitochondrial stress, or adaptive metabolic changes are scarce. Therefore, while current evidence suggests a favorable short-term safety profile, the absence of comprehensive chronic toxicity studies precludes definitive conclusions regarding long-term organ safety. Future investigations should incorporate prolonged dosing schedules, quantitative histopathology, and functional assays to rigorously evaluate cumulative liver and kidney toxicity prior to clinical translation.

An important unresolved issue concerns the specificity of fritillary alkaloids toward the NLRP3 inflammasome relative to other inflammasome platforms, such as NLRC4 and AIM2. To date, most studies evaluate inflammasome inhibition using canonical NLRP3-activating stimuli (eg, LPS plus ATP or nigericin), without parallel assessment of NLRC4- or AIM2-driven responses. As a result, the selectivity profile of fritillary alkaloids remains insufficiently defined, raising the possibility of broader inflammasome suppression and unintended immunomodulation.

To rigorously determine specificity, future experiments should employ stimulus-specific activation paradigms in the same cellular system. For example, NLRC4 inflammasome activation can be induced using flagellin or type III secretion system components, whereas AIM2 activation can be triggered by cytosolic double-stranded DNA. Comparative analysis of caspase-1 activation, IL-1β/IL-18 release, and gasdermin D cleavage under these conditions would allow direct evaluation of whether fritillary alkaloids selectively inhibit NLRP3 or exert pan-inflammasome effects. Complementary genetic approaches, such as using NLRP3-, NLRC4-, or AIM2-deficient macrophages, would further strengthen mechanistic attribution.

In addition, assessing downstream functional consequences—such as pathogen clearance, type I interferon responses, and host defense against intracellular bacteria or DNA viruses—would help determine whether inhibition of non-NLRP3 inflammasomes compromises protective immunity. Integration of these approaches with dose–response analyses and temporal profiling will be essential to define a therapeutic window that maximizes NLRP3-targeted anti-inflammatory efficacy while minimizing the risk of undesired broad immunosuppression.

In conclusion, this review systematically synthesizes compelling evidence demonstrating that fritillary alkaloids possess significant anti-inflammatory properties, largely mediated through inhibition of the NLRP3 inflammasome. Their mechanisms of action are multifaceted, encompassing suppression of inflammasome assembly, inhibition of key signaling pathways such as NF-κB and MAPK, regulation of ionic homeostasis (eg, K⁺ efflux), attenuation of oxidative stress by reducing ROS production, and inhibition of the pyroptosis executor GSDMD. Promising results from various acute and chronic inflammatory models, together with emerging strategies to improve bioavailability (eg, nanodelivery systems) and efficacy (eg, supramolecular synergy), highlight the therapeutic potential of these compounds.

Nevertheless, there are still several issues that need further research on the anti-inflammatory molecular mechanisms of Fritillaria alkaloids:

Chronic Toxicity and Long-Term Safety Profiles: Although fritillary alkaloids exhibit relatively low acute toxicity, comprehensive studies on their chronic toxicity are lacking. Future work should include long-term administration experiments in relevant animal models to assess potential off-target effects, organ-specific cumulative toxicity (particularly hepatorenal safety), and overall chronic safety—a prerequisite for clinical translation.

Relevance to Human Physiology and Clinical Translation: Current evidence is largely derived from in vitro cell lines and animal models. The transition to human-relevant systems remains unexplored. Subsequent research should incorporate human primary cells, disease-specific organoids, and ultimately, well-designed clinical trials to verify the efficacy and safety of fritillary alkaloids in humans.

Disease-Specific and Inflammation-Type-Dependent Efficacy: While studies in arthritis and colitis models show beneficial effects, it remains unclear whether the potency or mechanism of fritillary alkaloids differs across inflammatory disease types (eg, sterile vs pathogen-induced) or phases (acute vs chronic). Future studies should systematically evaluate their efficacy in a broader range of inflammatory pathologies to identify the most suitable clinical indications.

Specificity Toward NLRP3 and Effects on Other Inflammasomes: The specificity of fritillary alkaloids for the NLRP3 inflammasome has not been conclusively established. It is unknown whether they influence other inflammasomes, such as NLRC4 or AIM2. Elucidating this specificity is essential for understanding their precise mechanism and anticipating off-target immune modulation.

Elucidating the Structure-Activity Relationship (SAR): A critical and under-explored area is the systematic analysis of the structure-activity relationship among the diverse array of fritillary alkaloids. The current scarcity of strictly comparable pharmacological data across different compounds hinders a definitive understanding of how specific structural features (eg, variations at the C-6 position or in the steroidal backbone configuration) govern their anti-inflammatory potency, toxicity, and selectivity. Future research should prioritize employing an integrated approach, combining computational methods such as Comparative Molecular Field Analysis (CoMFA) to construct predictive 3D-QSAR models, with systematic in vitro profiling of a panel of purified alkaloids against a unified set of NLRP3-related readouts. Establishing a robust SAR model is paramount for the rational design and optimization of novel derivatives with enhanced efficacy and improved therapeutic windows.

Addressing these gaps will not only solidify the scientific foundation of fritillary alkaloids as anti-inflammatory agents but also facilitate their rational development into safe and effective novel therapeutics.