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Abstract

Objective

Zhigancao Decoction (ZGCD), a classic traditional Chinese formula, is widely used for cardiovascular disorders, but its role in myocardial ischemia–reperfusion injury (MIRI) is unclear. This study evaluated the cardioprotective effects of ZGCD in rats and investigated the association with JAK2/STAT3/HIF-1α signaling.

Methods

A left anterior descending coronary artery ligation–reperfusion model was established in rats, which were randomized to sham, MIRI, low- and high-dose ZGCD, JAK2 inhibitor, or JAK2 inhibitor + ZGCD groups. After 4 weeks of gavage, cardiac function and infarct size were assessed, and myocardial histology, inflammatory and oxidative stress markers, and JAK2/STAT3/HIF-1α protein expression were measured.

Results

ZGCD improved left ventricular ejection fraction and reduced infarct size, attenuated histological damage, decreased serum TNF-α, IL-1β, malondialdehyde and myocardial reactive oxygen species, and increased IL-10 and superoxide dismutase activity compared with MIRI alone. ZGCD also enhanced myocardial p-JAK2/JAK2 and p-STAT3/STAT3 ratios and HIF-1α expression, whereas the JAK2 inhibitor largely counteracted these functional and molecular benefits.

Conclusion

ZGCD attenuates MIRI in rats, which is accompanied by coordinated anti-inflammatory and antioxidant effects and enhanced JAK2/STAT3/HIF-1α signaling, supporting its potential as an adjunctive cardioprotective strategy.

Keywords

Zhigancao decoction myocardial ischemia–reperfusion injury JAK2/STAT3/HIF-1α oxidative stress inflammation traditional Chinese medicine

Introduction

Cardiovascular disease remains the leading cause of death in China. Epidemiological data indicate that the disease burden and mortality risk associated with acute myocardial infarction (AMI) are still increasing.¹ For patients with ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI), early reperfusion strategies such as percutaneous coronary intervention (PCI) constitute the cornerstone of contemporary management, aiming to rapidly restore blood flow in the infarct-related artery and salvage jeopardized myocardium.² However, a substantial proportion of high-risk patients continue to develop heart failure, malignant arrhythmias, and other complications after PCI, indicating that post-reperfusion myocardial injury has not been adequately controlled and that myocardial ischemia–reperfusion injury (MIRI) remains a critical barrier to further improvement of prognosis.³

MIRI refers to secondary tissue damage that occurs in previously ischemic myocardium during the restoration of blood flow, which can aggravate myocardial necrosis and offset part of the benefit of reperfusion. Its core pathological substrates are an inflammatory cascade and dysregulated oxidative stress. In the early phase of reperfusion, inflammatory cell infiltration and cytokine release disrupt the microcirculation and promote excessive generation of reactive oxygen species (ROS), thereby triggering cardiomyocyte necrosis and apoptosis.^4,5 Clinical observations have shown that persistent elevation of inflammatory markers is closely associated with increased risks of death and cardiovascular adverse events in patients with acute coronary syndromes.⁶ Thus, effective modulation of inflammation and oxidative stress during the reperfusion phase is crucial for limiting MIRI and preserving cardiac function. However, the clinical benefits of currently available antioxidants, calcium channel blockers, and certain anti-inflammatory agents are limited, and their adverse effects are non-negligible.⁷ There is an urgent need to explore safer adjunctive strategies capable of providing integrated, multi-target intervention.

Within the framework of traditional Chinese medicine (TCM), acute myocardial infarction and reperfusion-related injury are classified under the syndromes of “chest impediment” and “heart pain”, which are typically characterized by deficiency of qi and yin with heart yang insufficiency, accompanied by phlegm-turbidity and blood stasis obstructing the collaterals. This pattern of “deficiency in origin and excess in superficiality” shows conceptual parallels to the inflammatory activation, oxidative stress and microcirculatory disturbances described in modern medicine. Multi-herb TCM formulas are thought to exert multi-component, multi-target and multi-level regulatory effects: they can tonify qi and nourish yin, warm yang and unblock the vessels, and resolve phlegm and blood stasis. These properties provide a theoretical basis for their use as adjunctive therapies in MIRI. Consistent with this view, recent experimental studies have reported that several compound formulas can concomitantly attenuate oxidative stress, suppress inflammatory responses, limit cardiomyocyte apoptosis and improve mitochondrial function, thereby ameliorating ventricular remodelling and clinical outcomes.^8,9

Zhigancao Decoction (ZGCD), first recorded in the Treatise on Cold Damage (Shang Han Lun), consists of honey-fried licorice (Glycyrrhizae Radix et Rhizoma Praeparata cum Melle), fresh ginger, ginseng, Rehmanniae Radix, cinnamon twig, donkey-hide gelatin, Ophiopogonis Radix, hemp seed, and jujube. It is traditionally prescribed to tonify qi and nourish yin, warm yang and restore the pulse, and relieve palpitations by alleviating acute spasmodic conditions, and is indicated for “intermittent and irregular pulse” and “palpitations” due to deficiency of heart yang, qi, and yin. In contemporary clinical practice, ZGCD and its modified formulas are widely used as adjunctive therapy for arrhythmias, chronic heart failure, and coronary heart disease, where they have been reported to improve symptoms, modulate electrocardiographic abnormalities, and reduce serum markers of myocardial injury when added to standard care.⁹ Pharmacological studies further suggest that ZGCD and several representative constituents can improve endothelial function, inhibit the release of pro-inflammatory cytokines, attenuate oxidative stress, and reduce apoptosis, highlighting a multi-target cardioprotective profile.^10–13 Nevertheless, the key molecular pathways through which ZGCD exerts cardioprotection in the specific context of MIRI remain to be clarified.

The Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) axis is a pivotal endogenous cardioprotective pathway. Experimental studies have demonstrated that activation of JAK2/STAT3 reduces infarct size, suppresses pro-inflammatory cytokine production, and enhances antioxidant defenses in MIRI models.¹⁴ STAT3 can also promote the expression and stabilization of hypoxia-inducible factor-1α (HIF-1α),¹⁵ and, by regulating glycolysis, angiogenesis, and cell-protective gene expression, participates in adaptive protective responses in reperfused myocardium.¹⁶ The JAK2/STAT3/HIF-1α signaling module may therefore represent one of the key axes with cardioprotective potential in the setting of myocardial reperfusion.

On this basis, the present study integrated TCM pathophysiological concepts with modern pharmacology by first using network pharmacology to predict the core targets and signaling pathways through which ZGCD may act in MIRI, and then performing in vivo validation in a rat MIRI model. We focused on the JAK2/STAT3/HIF-1α axis and on indices of inflammation and oxidative stress to delineate their changes in response to ZGCD treatment, with the aim of providing experimental evidence for the adjunctive use of ZGCD in MIRI and of exploring its potential mechanistic underpinnings. (Figure 1).

Figure 1.

Schematic illustration of the integrated research workflow. Network pharmacology and bioinformatics analyses were combined with in vivo validation to investigate the mechanism of ZGCD in alleviating MIRI.

Materials and Methods

Analysis Based on Network Pharmacology

Collection and Screening of Active Compounds in ZGCD

Putative active compounds of ZGCD were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) based on ADME parameters. In line with commonly used criteria,¹⁷ compounds were filtered using oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. The following herbs were queried: Glycyrrhizae Radix et Rhizoma Praeparata cum Melle, Zingiberis Rhizoma Recens, Panax Ginseng Radix et Rhizoma, Rehmanniae Radix, Cinnamomi Ramulus, Cannabis Semen, and Jujubae Fructus. For herbs not fully covered in TCMSP, candidate compounds were supplemented from the HERB database. Duplicate entries were removed, compound identifiers were harmonized, and canonical Simplified Molecular Input Line Entry System (SMILES) strings were obtained from PubChem for downstream target prediction.

Predicting Potential Drug Targets

Canonical SMILES strings retrieved from PubChem were submitted to SwissTargetPrediction, with the organism set to Homo sapiens. For each compound, we retained predicted targets with non-zero probability and discarded predictions with probability = 0.¹⁸ Predicted targets were then merged across compounds, deduplicated, and mapped to UniProt official gene symbols; only human protein-coding genes were kept for downstream analyses.

Acquisition of Targets Related to MIRI

To comprehensively characterize the molecular landscape of MIRI, we employed a multi-source data integration strategy. Transcriptomic profiling was performed using two mouse myocardial tissue microarray datasets (GSE61592 and GSE83472) retrieved from the GEO database.^19,20 Raw data processing involved log2-transformation and normalization (RMA or quantile) within the R 4.4.2 environment. Differentially expressed genes (DEGs) were identified using the limma package, with significance defined as an adjusted P < 0.05 and |log2FC| ≥ 1. In parallel, established MIRI-associated targets were curated from GeneCards, OMIM, and the Therapeutic Target Database (TTD). These transcriptomic DEGs were subsequently merged with the database-derived targets to construct a unified MIRI disease target library.

Candidate Therapeutic Targets

To ensure cross-species compatibility, mouse genes derived from the microarray analysis were mapped to their homologous human protein-coding genes prior to integration. Candidate therapeutic targets of ZGCD were then defined by intersecting the predicted drug targets with the unified MIRI disease target library established above. This approach effectively isolated genes that were both targeted by ZGCD components and implicated in MIRI pathology (Intersection = ZGCD Targets ∩ [GSE DEGs ∪ Database Targets]). The resulting overlapping targets were tabulated and visualized via a Venn diagram.

PPI Network and hub Genes

Protein–protein interactions (PPI) were retrieved from STRING for Homo sapiens at the highest confidence threshold (minimum required interaction score = 0.900). Disconnected nodes were removed. Hub genes were ranked with cytoHubba, and the top 30 nodes were visualized in Cytoscape 3.10.²¹

Functional Enrichment Analysis

Functional over-representation of candidate genes derived from the PPI network was assessed in DAVID with Homo sapiens as the background set. Gene Ontology (GO) enrichment covered biological process (BP), molecular function (MF), and cellular component (CC) categories, and KEGG pathway enrichment was performed in parallel.²² Enrichment significance was evaluated with DAVID's modified Fisher exact test (EASE), and terms with Benjamini–Hochberg–adjusted P < 0.05 (false discovery rate, FDR) were considered significant.

Enrichment outputs were exported and plotted in R 4.4.2 using ggplot2 and circlize. GO terms were visualized as a multi-track circular chart: the outer track listed GO IDs, BP MF and CC categories were color coded, a radial bar track showed gene counts, an inner track showed the rich factor from 0 to 1, and color intensity represented minus log10 of the FD.KEGG pathways were displayed as a bubble plot with the gene ratio on the x-axis, bubble size representing gene count, and color indicating minus log10 of the FD.Animals

All animal procedures were approved by the Institutional Animal Welfare and Ethics Committee of Guangxi University of Chinese Medicine, China (approval No. DW20240507-080). Experiments complied with the Regulations for the Administration of Affairs Concerning Experimental Animals (China) and the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).

The ethical protocol approved the use of up to 72 rats to account for potential attrition; however, 60 rats were sufficient for the study design and were ultimately used. Sixty male Sprague–Dawley rats (200 ± 20 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd and maintained under specific-pathogen-free (SPF) conditions. Animals were housed in a temperature-controlled facility on a 12-h light/12-h dark cycle with ad libitum access to standard chow and water. All rats were acclimatized for 7 days before experiments. All in vivo experiments were designed, conducted and reported in accordance with the ARRIVE 2.0 guidelines.²³

Rat MIRI Model

Rats were randomized into six groups (n = 10 per group): Sham (sham surgery group), MIRI (model group), ZLG (low-dose ZGCD group), ZHG (high-dose ZGCD group), IG (JAK2 inhibitor group), and IZG (inhibitor + ZGCD group); randomization used an online generator (randomizer.org). Among them, three rats per group were randomly selected for echocardiographic assessment of LVEF followed by TTC staining to determine infarct size, while the remaining animals were used for biochemical, histological and Western blot analyses.

Anesthesia was induced with pentobarbital sodium (50 mg/kg, intraperitoneal). After tracheal intubation, animals were mechanically ventilated with a rodent ventilator and maintained normothermic on a heating pad. Through a left thoracotomy, the heart was exteriorized. In the Sham group, a 6–0 silk suture was passed under the left anterior descending (LAD) coronary artery but left untied, and no ischemia was induced. In the remaining groups, the LAD was occluded with a 6–0 silk suture to induce ischemia; successful ischemia was confirmed by blanching of the anterior left ventricular wall and ST-segment elevation on ECG. After 30 min, the ligature was released to allow reperfusion.²⁴

Drug administration by oral gavage was initiated 24 h after reperfusion and continued once daily for 4 weeks. ZGCD pills were purchased from Beijing Tong Ren Tang (Beijing, China), pulverized, and dissolved in distilled water to prepare a stock solution at 80 mg/mL, which was stored at 4 °C until use. The ZGCD pills were purchased from Beijing Tong Ren Tang (Beijing, China; Batch No. 231105) and produced in accordance with the Chinese Pharmacopoeia (2020 Edition), ensuring standardized quality control of the herbal constituents. The equivalent clinical dose of ZGCD for rats was calculated from the human dose using the standard body surface area conversion formula, and this value was defined as the clinically equivalent dose. Rats in the ZLG received 0.8 g/kg/day ZGCD (1× clinical equivalent dose), whereas those in the ZHG received 1.6 g/kg/day (2× clinical equivalent dose).^25,26 These dose levels fall within the range commonly used for TCM formulas in rat cardiovascular models.

The IG group was treated with the JAK2 inhibitor AZD1480 at 10 mg/kg/day (Selleck, China), and the IZG group received AZD1480 10 mg/kg/day combined with ZGCD 1.6 g/kg/day. Sham and MIRI model groups were gavaged with an equal volume of normal saline. After 4 weeks of treatment, rats were sacrificed; blood was collected from the abdominal aorta, serum was separated by centrifugation and stored at −80 °C for subsequent assays. Hearts were rapidly excised; part of the tissue was snap-frozen in liquid nitrogen and stored at −80 °C for molecular analyses, and the remaining portion was fixed in 4% paraformaldehyde for histological evaluation.

Echocardiography

At 4 weeks after I/R and before sacrifice, rats were lightly anesthetized with isoflurane (1-2% in oxygen) to maintain stable respiration. The left ventricular ejection fraction (LVEF) was assessed using an Esaote MyLab Six ultrasound system (Esaote, Genoa, Italy). All measurements were performed by an operator blinded to the experimental grouping.

Pathology

Rat hearts were fixed in 4% paraformaldehyde for 24 h, processed, and embedded in paraffin. Transverse sections (4-5 µm) were cut at the mid-ventricular level. After deparaffinization and rehydration, sections were stained with hematoxylin for 4 min, differentiated, blued, and counterstained with eosin for 2 min. Slides were dehydrated, cleared, and mounted with neutral resin. Myocardial histopathological changes— including myofiber disarray, inflammatory cell infiltration, and interstitial edema—were examined under a light microscope (×200 and ×400 magnification), and representative fields were photographed.

Assessment of Myocardial Infarct Size

Myocardial infarct size was assessed by 23,5-triphenyltetrazolium chloride (TTC) staining (Servicebio, China). After euthanasia, hearts were excised, rinsed in ice-cold 0.9% saline, and briefly frozen at −20 °C for 10 min. The left ventricle was cut transversely into 2 mm slices from apex to base and incubated in prewarmed 2% TTC at 37 °C for 15 min in the dark. Viable myocardium stained red, whereas infarcted tissue remained pale. Slices were then fixed in 4% paraformaldehyde to stabilize staining. Digital images of both sides of each slice were acquired and analyzed using ImageJ. Infarct size was expressed as the infarct area fraction of the left ventricular myocardium.

Measurement of Biochemical Markers

Serum was isolated, and malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured by a Chemray 800 fully automated biochemical analyzer. The levels of serum tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-10 (IL-10) were quantified using ELISA kits (Servicebio). The reactive oxygen species (ROS) levels in myocardial tissue were measured using a myocardial tissue reactive oxygen species detection kit (BestBio, China).

Western Blotting

Myocardial tissue from the left ventricle was rinsed in ice-cold PBS and homogenized in RIPA lysis buffer containing 1% phenylmethylsulfonyl fluoride (PMSF). Lysates were centrifuged at 12,000 × g for 15 min at 4 °C, and supernatants were collected. Protein concentrations were determined using a BCA assay kit (Biosharp, BL52A). Equal amounts of protein (30-50 µg per lane) were separated by 10% SDS–PAGE and transferred onto PVDF membranes (0.45 µm, Servicebio, G6015-0.45). Membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against JAK2 (Bioswamp, PAB53356, 1:1000), phospho-JAK2 (Tyr221) (Bioswamp, PAB30711, 1:1000), STAT3 (Bioswamp, PAB30835, 1:1000), phospho-STAT3 (Tyr705) (Bioswamp, RMAB54407, 1:1000), HIF-1α (Bioswamp, PAB55591, 1:500), and GAPDH (Biosharp, BL006B, 1:5000). After washing, membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Servicebio, GB23303, 1:5000) for 30 min at room temperature. Signals were developed using an enhanced chemiluminescence substrate (Biosharp, BL520A) and captured with a chemiluminescence imaging system. Band intensities were quantified using ImageJ, normalized to GAPDH, and phospho-proteins were additionally expressed as p-/total protein ratios. For each group, samples from n = 3 independent biological replicates (tissue samples obtained from 3 distinct rats per group) were analyzed to ensure reproducibility.

Statistical Analysis

Data processing and figure generation were performed using SPSS 25.0, GraphPad Prism 9.0, and ImageJ 1.54. Continuous variables are expressed as mean ± standard deviation (SD), where n denotes the number of animals per group. Data distribution was assessed for normality using the Shapiro–Wilk test, and homogeneity of variance was examined with Levene's test. For normally distributed data with homogeneous variance, one-way analysis of variance (ANOVA) was used for intergroup comparisons, followed by the least significant difference (LSD) test for post hoc multiple comparisons. When comparing two independent groups, Student's t-test was applied. All tests were two-sided, and a P < 0.05 was considered statistically significant.

Results

Identification of Active Compounds and Candidate Targets

We mined TCMSP and HERB for ZGCD constituents and identified 205 nonredundant putative active compounds. Using PubChem-derived SMILES and SwissTargetPrediction, 1074 predicted targets corresponding to these compounds were obtained.

Based on the GSE61592 and GSE83472 datasets and thresholds of |log2FC| ≥ 1 and adjusted P < 0.05, differential expression analysis yielded 1785 and 92 DEGs, respectively (totaling 1877 DEGs), which were visualized as heatmaps and volcano plots (Figure 2). To establish a comprehensive MIRI-related target pool, we merged these 1877 DEGs with 1715 MIRI-related targets retrieved from GeneCards, TTD, and OMIM. After removing redundant entries and mapping to human protein-coding genes, a total of 3243 unique MIRI-related genes were obtained. This gene set was then intersected with the 1074 ZGCD-predicted targets, identifying 467 overlapping genes as potential candidate targets for ZGCD in the treatment of MIRI (Figure 3A).

Figure 2.

DEGs identified from GEO datasets (total DEGs = 1877). (A, B) Volcano plot and heatmap of DEGs in GSE61592. (C, D) Volcano plot and heatmap of DEGs in GSE83472.

Figure 3.

Network pharmacology–derived candidate targets of ZGCD for MIRI. (A) Overlap between ZGCD-predicted targets (n = 1074) and the MIRI-related gene set (n = 3243); intersection (n = 467) defined as candidate targets. (B) Protein–protein interaction network of the top 30 candidate targets ranked by cytoHubba in Cytoscape.

Construction of a PPI network followed by cytoHubba-based topological ranking yielded 30 hub genes. Among these, STAT3, HIF1A (HIF-1α), and JAK2 were identified as top-ranking hub nodes with high degree centrality (Figure 3B). Considering their high topological importance and their well-documented roles as core components of the “Survival Activating Factor Enhancement” pathway—a key endogenous cardioprotective mechanism—these targets were prioritized for subsequent in vivo validation.

Enrichment Analysis

GO enrichment of the candidate targets (Figure 4A) prioritized cytokine-mediated signaling, leukocyte migration and chemotaxis, regulation of the inflammatory response, processes related to oxidative stress such as response to reactive oxygen species and regulation of lipid peroxidation, and apoptotic signaling pathways (FDR < 0.05). KEGG pathway analysis (Figure 4B) identified significant enrichment of HIF-1 signaling, PI3 K/Akt signaling, TNF signaling, apoptosis, AGE–RAGE signaling in diabetic complications, fluid shear stress and atherosclerosis, “lipid and atherosclerosis”, efferocytosis, and proteoglycans in cancer (FDR < 0.05). Although some pathways are annotated in the context of cancer, many of the underlying components participate in inflammatory, vascular, and hypoxia-responsive networks that are pertinent to MIRI pathophysiology.

Figure 4.

Enrichment analysis of intersecting genes. (A) GO enrichment analysis showing BP, CC, and MF. (B) KEGG pathway enrichment analysis.

These findings suggest that the putative actions of ZGCD against MIRI involve inflammatory, oxidative, and hypoxia-related mechanisms. On the basis of these enrichment results and the identification of STAT3 as a high-ranking hub, the subsequent in vivo experiments were directed towards assessment of the inflammatory mediators IL-1β, TNF-α, and IL-10, as well as examination of the JAK2/STAT3/HIF-1α signaling axis in the rat MIRI model.

ZGCD Improves Cardiac Function and Limits Infarct Size in MIRI Rats

As shown in Table 1, LVEF in the MIRI group was significantly reduced compared with the Sham group (45.17 ± 6.35% vs 79.30 ± 5.00%, P < 0.05). Administration of ZGCD increased LVEF to 66.56 ± 1.79% in the ZLG group and 70.00 ± 2.09% in the ZHG group, both significantly higher than in the MIRI group (P < 0.05). Notably, no statistically significant difference was observed between the low- and high-dose groups, indicating that both doses produced comparable cardioprotective effects. LVEF values in the IG (51.68 ± 6.57%) and IZG (50.02 ± 6.44%) groups did not differ significantly from that in the MIRI group; however, LVEF in the IZG group was significantly lower than in the ZHG group (P < 0.05) (Table 1; Figure 5A,B).

Figure 5.

Evaluation of cardiac function and infarct size. (A) LVEF assessed by echocardiography. (B) Myocardial infarct area measured by TTC staining.

Table 1.

The Effect of ZGCD on Cardiac Function (LVEF) and Myocardial Infarction Area (TTC) in MIRI Rats.

Group	LVEF(%)	Infarct Area Fraction (TTC)
Sham	79.30 ± 5.00	0
MIRI	45.17 ± 6.35*	0.78 ± 0.06*
ZLG	66.56 ± 1.79^#	0.62 ± 0.06^#
ZHG	70.00 ± 2.09^#	0.58 ± 0.06^#
IG	51.68 ± 6.57	0.71 ± 0.13
IZG	50.02 ± 6.44^△	0.75 ± 0.03^△

Note: *P < 0.05 versus Sham; #P < 0.05 versus MIRI; △P < 0.05 versus ZHG; n = 3.

TTC staining was used to evaluate myocardial infarct size, which was expressed as the infarct area fraction of the left ventricular myocardium (unitless, range 0-1). No infarct area was detected in the Sham group, whereas the infarct area fraction in the MIRI group reached 0.78 ± 0.06 (P < 0.05 vs Sham). ZGCD treatment reduced the infarct area fraction to 0.62 ± 0.06 in ZLG and 0.58 ± 0.06 in ZHG, both significantly lower than in the MIRI group (P < 0.05), with no significant difference between the two ZGCD doses. In contrast, infarct area fractions in the IG (0.71 ± 0.13) and IZG (0.75 ± 0.03) groups were not significantly different from that in the MIRI group; the IZG group, however, exhibited a significantly larger infarct area fraction than the ZHG group (P < 0.05) (Table 1; Figure 5C,D).

H&E staining of myocardial tissue was consistent with these quantitative findings (Figure 6). Myocardium from the Sham group showed preserved architecture with regularly arranged myofibers, clear cross striations and narrow interstitial spaces. In the MIRI, IG and IZG groups, myocardial fibers were markedly disorganized with widened interstitium, prominent inflammatory cell infiltration, interstitial edema, and myocyte swelling, degeneration and necrosis; lesions appeared particularly extensive in the IG group. In the ZLG and ZHG groups, these histopathological alterations were attenuated, with relatively preserved fiber alignment and striation, reduced inflammatory infiltration and edema, and fewer necrotic myocytes compared with the MIRI group.

Figure 6.

Histological assessment of myocardial tissue using H&E staining. ZGCD treatment reduced myocardial damage and inflammatory infiltration compared with the MIRI group.

ZGCD Attenuates Inflammation and Oxidative Stress in MIRI Rats

As shown in Table 2, compared with the Sham group, rats in the MIRI group exhibited significantly increased serum MDA, TNF-α and IL-1β levels and myocardial ROS, while SOD activity and IL-10 levels were significantly decreased (P < 0.05), a pattern consistent with enhanced inflammatory activation and oxidative stress after ischemia–reperfusion. Compared with the MIRI group, both the ZLG and ZHG groups showed significantly lower TNF-α and IL-1β levels and significantly higher IL-10 levels and SOD activity, accompanied by significant reductions in MDA and ROS levels (P < 0.05), suggesting that both low and high doses of ZGCD were associated with significant improvements in inflammatory and oxidative stress indices. No statistically significant difference was observed between the low- and high-dose groups for any of these parameters, further confirming comparable efficacy within the tested dose range.

Table 2.

Effects of ZGCD on Inflammatory Factors and Oxidative Stress Indicators in MIRI Rats.

Group	SOD (U/ml)	MDA (nmol/ml)	TNF-α (pg/ml)	IL-1β (pg/ml)	IL-10 (pg/ml)	ROS (*10⁶ RFU/mg Protein)
Sham	145.39 ± 9.10	12.34 ± 3.97	161.13 ± 13.29	110.55 ± 7.09	297.84 ± 9.46	8.75 ± 0.83
MIRI	41.98 ± 9.39*	89.32 ± 7.35*	327.94 ± 24.94*	542.95 ± 27.47*	208.64 ± 13.77*	20.59 ± 1.31*
ZLG	65.70 ± 4.28^#	66.35 ± 5.61^#	234.65 ± 10.78^#	488.30 ± 27.59^#	259.89 ± 12.74^#	14.01 ± 1.46^#
ZHG	72.26 ± 4.59^#	62.63 ± 4.59^#	227.31 ± 20.26^#	368.70 ± 30.00^#	265.80 ± 9.68^#	13.98 ± 1.22^#
IG	36.41 ± 9.19	91.69 ± 9.19	311.17 ± 18.74	541.03 ± 22.57	208.36 ± 14.48	20.70 ± 0.87
IZG	38.17 ± 9.73^△	94.38 ± 6.22^△	329.43 ± 24.51^△	528.56 ± 26.78^△	205.29 ± 10.95^△	20.42 ± 0.77^△

Note: *P < 0.05 versus Sham; #P < 0.05 versus MIRI; △P < 0.05 versus ZHG; n = 10.

In the IG group, all parameters were comparable to those in the MIRI group, indicating that JAK2 inhibition alone did not modify the baseline extent of MIRI-induced injury. Indices in the IZG group also did not differ significantly from those in the MIRI group. However, compared with the ZHG group, the IZG group showed significantly lower SOD activity and IL-10 levels and significantly higher MDA, TNF-α, IL-1β and ROS levels (P < 0.05), suggesting that JAK2 inhibition blunted the anti-inflammatory and antioxidant changes observed with ZGCD.

ZGCD Modulates JAK2/STAT3/HIF-1α Signaling in MIRI Myocardium

Western blot results are shown in Figure 7. Compared with the Sham group, the MIRI group exhibited significantly lower myocardial p-JAK2/JAK2 and p-STAT3/STAT3 ratios and markedly reduced HIF-1α protein expression (P < 0.05), indicating suppression of JAK2/STAT3/HIF-1α signaling after ischemia–reperfusion. In both the ZLG and ZHG groups, the p-JAK2/JAK2 and p-STAT3/STAT3 ratios were significantly increased and HIF-1α expression was significantly upregulated relative to the MIRI group (P < 0.05). Consistent with the functional and biochemical results, no statistically significant difference was observed between the ZLG and ZHG groups regarding the phosphorylation levels of JAK2/STAT3 or the expression of HIF-1α.

Figure 7.

Effect of ZGCD on the JAK2/STAT3/HIF-1α signaling pathway in MIRI rats. (A) Representative Western blot bands showing the protein expression of p-JAK2, JAK2, p-STAT3, STAT3, and HIF-1α in myocardial tissue. GAPDH was used as the loading control. (B) Quantitative analysis of the ratio of phosphorylated JAK2 to total JAK2 (p-JAK2/JAK2). (C) Quantitative analysis of the ratio of phosphorylated STAT3 to total STAT3 (p-STAT3/STAT3). (D) Quantitative analysis of HIF-1α protein levels normalized to GAPDH. Data are presented as mean ± SD. n = 3 biological replicates (samples from 3 distinct rats per group). * P < 0.05, ** P < 0.01 vs. Sham group; # P < 0.05, ## P < 0.01 vs. MIRI group.

Compared with the MIRI group, the IG group showed further decreases in p-JAK2/JAK2, p-STAT3/STAT3, and HIF-1α protein levels (P < 0.05), consistent with additional downregulation of this signaling axis by the JAK2 inhibitor. In rats receiving high-dose ZGCD combined with AZD1480 (IZG), the p-JAK2/JAK2 and p-STAT3/STAT3 ratios were significantly lower than in both the MIRI and ZHG groups (P < 0.05), and HIF-1α expression was significantly lower than in ZHG (P < 0.05) but did not differ significantly from MIRI. Taken together, these findings are consistent with JAK2 inhibition markedly attenuating the ZGCD-associated increases in JAK2 and STAT3 phosphorylation and the accompanying changes in HIF-1α expression, with a particularly clear suppressive effect on upstream JAK2 and STAT3 phosphorylation.

Discussion

MIRI is tightly linked to an imbalance between inflammatory activation and oxidative stress. During early reperfusion, rapid accumulation of ROS can trigger inflammatory cascades and induce the release of multiple proinflammatory cytokines. In turn, infiltrating inflammatory cells and their cytokines further amplify ROS generation through mitochondrial dysfunction and activation of NADPH oxidases, forming a self-reinforcing positive feedback loop that perpetuates cardiomyocyte injury and worsens ventricular function.²⁷ Interrupting this vicious cycle and strengthening endogenous antioxidant defenses is therefore regarded as one of the key strategies to limit MIRI.²⁸

In the present study, both low and high doses of ZGCD significantly improved left ventricular systolic function and reduced infarct size in rats with MIRI, with no statistically significant differences between the two doses, suggesting a reproducible cardioprotective effect within the clinically equivalent dose range. ZGCD decreased serum MDA, TNF-α and IL-1β levels and myocardial ROS, while increasing SOD activity and IL-10, indicating a concomitant improvement in oxidative and inflammatory profiles. At the signaling level, MIRI was associated with reduced myocardial p-JAK2/JAK2 and p-STAT3/STAT3 ratios and downregulated HIF-1α expression, whereas ZGCD administration increased JAK2 and STAT3 phosphorylation and was accompanied by higher HIF-1α protein levels. Pharmacological blockade of JAK2 further depressed this signaling axis and, when combined with high-dose ZGCD, attenuated or abolished the functional and molecular benefits. Taken together, these in vivo findings are consistent with ZGCD-mediated cardioprotection being accompanied by modulation of the JAK2/STAT3/HIF-1α axis and by attenuation of the inflammation–oxidative stress feedback loop.

The network pharmacology and enrichment analyses are compatible with this interpretation. The intersection between predicted ZGCD targets and MIRI-related genes yielded a candidate target set in which JAK2, STAT3 and HIF-1α occupied central positions within the PPI network. Our rationale for prioritizing this specific axis for validation was twofold: statistically, they exhibited high degree centrality indicating their hub status; biologically, they constitute the classic SAFE signaling pathway, which is critical for limiting reperfusion injury and promoting cardiomyocyte survival.²⁹Gene Ontology enrichment primarily involved cytokine-mediated signaling, regulation of leukocyte migration and chemotaxis, oxidative stress responses, and apoptosis, while KEGG pathway analysis highlighted HIF-1 and PI3 K/Akt signaling together with TNF and other inflammation-related pathways. These bioinformatic findings point to a mechanistic landscape dominated by inflammatory and redox regulation, and the subsequent in vivo data showing modulation of JAK2/STAT3/HIF-1α signaling components are in line with these predictions.

ZGCD is a complex multi-component formula, and its cardioprotective effects are likely attributable to the synergistic action of its bioactive constituents. Based on our network pharmacology screening criteria (OB ≥ 30%, DL ≥ 0.18), several high-ranking compounds were identified as potential material bases. For instance, Kaempferol (OB = 41.88%, DL = 0.24) and Formononetin (OB = 69.67%, DL = 0.21) exhibit favorable pharmacokinetic profiles, ensuring their bioavailability to modulate myocardial targets. Previous studies have demonstrated that Kaempferol protects against myocardial ischemia-reperfusion injury by inhibiting oxidative stress and inflammation pathways,³⁰ while Formononetin has been shown to improve cardiac function and suppress apoptosis in ischemic hearts.³¹ These findings support the notion that the specific components of ZGCD with high bioavailability may act in concert to regulate the signaling modules observed in our study. Specifically, while Kaempferol may primarily target oxidative stress pathways, Formononetin could exert stronger effects on inflammatory modulation, thereby creating a synergistic protective shield against MIRI that surpasses the efficacy of single agents.

Previous studies have identified JAK2 as a non-receptor tyrosine kinase that transduces signals from multiple cytokine receptors and modulates inflammatory cell function.³² The JAK2/STAT3 axis forms part of the survivor activating factor enhancement pathway, an intrinsic cardioprotective mechanism. In reperfused myocardium, appropriate activation of JAK2/STAT3 signaling has been shown to limit proinflammatory cytokine production, augment antioxidant defenses, and support ventricular performance.^33,34 In the present model, ZGCD increased myocardial p-JAK2/JAK2 and p-STAT3/STAT3 together with reductions in TNF-α and IL-1β and an increase in IL-10, a pattern consistent with negative regulation of the inflammatory response potentially mediated by this axis.

STAT3, a key transcription factor downstream of JAK2, integrates inputs from IL-6, IL-10 and other cytokines and can exert context-dependent pro- or anti-inflammatory effects in the infarcted heart.^29,35 Activation of STAT3 has been reported to promote the expression of antioxidant enzymes such as SOD, reduce lipid peroxidation and ROS burden, and thereby attenuate reperfusion-related injury.^36,37 In agreement with these reports, ZGCD markedly increased SOD activity and reduced MDA and ROS levels in MIRI rats, supporting the possibility that a “JAK2/STAT3–anti-inflammatory/antioxidant” network contributes to its cardioprotective profile.

STAT3 also influences HIF-1α by enhancing its transcription and inhibiting its degradation, thus stabilizing HIF-1α protein.¹⁵ HIF-1α is a central transcription factor in the hypoxia response. While its role in MIRI can be complex and context-dependent, current evidence suggests that it functions primarily to promote glycolysis and angiogenesis, upregulate multiple protective genes, and participate in the suppression of excessive inflammation and regulation of cell survival.^38–40 In the present study, ZGCD increased myocardial HIF-1α expression, whereas this increase was substantially blunted when JAK2 was inhibited, in parallel with the loss of functional benefit. This pattern is consistent with HIF-1α acting as an important downstream effector node within the JAK2/STAT3 axis in the context of ZGCD treatment. However, given the context-dependent nature of HIF-1α signaling, the precise cell types and temporal dynamics involved in this protection warrant further clarification.

It is worth noting that the PI3 K/Akt signaling pathway was also significantly enriched in our KEGG analysis. The PI3 K/Akt axis is a well-established pro-survival pathway that plays a critical role in mitigating ischemia-reperfusion injury by inhibiting apoptosis and promoting cell survival.⁴¹ Although our current study focused primarily on the JAK2/STAT3 axis, it is plausible that ZGCD confers cardioprotection through a multi-target mechanism involving crosstalk between the JAK2/STAT3 and PI3 K/Akt pathways. This potential interaction warrants further investigation in future studies.

Our study has specific limitations. Mechanistically, we relied on pharmacological inhibition to implicate the JAK2/STAT3/HIF-1α axis. We did not employ genetic verification, such as cardiac-specific knockout or overexpression. As a result, the direct causality between ZGCD and this signaling pathway requires further confirmation. Another consideration is the treatment timing. We initiated dosing 24 h post-reperfusion and continued for 4 weeks. This protocol targets the subacute recovery phase rather than immediate acute protection. Additionally, while Western blot analyses were performed on independent biological replicates, the sample size (n = 3) is relatively small. Although statistical significance was achieved, this limited sample size may not fully capture the potential biological variability. Therefore, future studies with larger sample sizes are recommended to further validate the robustness of these molecular findings. Future work using transgenic mice will be necessary to strictly establish these causal mechanisms.

Conclusion

In a rat model of myocardial ischemia–reperfusion injury, ZGCD attenuated functional and structural myocardial damage and was associated with a more favorable inflammatory and oxidative stress profile, as well as modulation of JAK2/STAT3/HIF-1α signaling. Treatment reduced IL-1β and TNF-α, increased IL-10, lowered MDA and myocardial ROS, and restored SOD activity. The observation that these functional and molecular benefits were markedly blunted in the presence of a JAK2 inhibitor supports the involvement of the JAK2/STAT3/HIF-1α axis in the cardioprotective actions of ZGCD. Nevertheless, additional mechanistic studies—particularly targeted in vitro experiments and cell-specific analyses—are required to further delineate upstream targets, downstream effectors, and the precise contribution of this signaling axis to the overall protective effect.

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Footnotes

Acknowledgments

We are sincerely grateful to the Guangxi Key Laboratory of Molecular Biology of Preventive Medicine of Traditional Chinese Medicine for providing laboratory space and technical support for the experiments.

ORCID iDs

Weizhi Tang

Feifei Yang

Weibin Qin

Guixin He

Ethical Approval

All animal experimental procedures were reviewed and approved by the Guangxi University of Chinese Medicine Institutional Animal Welfare and Ethical Committee (Approval No. DW20240507-080).

Author Contributions

Weizhi Tang performed the network pharmacology and bioinformatic analyses, established the rat MIRI model, conducted echocardiography, TTC and histological assessments, carried out biochemical and molecular assays, analysed the data, and drafted the manuscript. Feifei Yang assisted with animal experiments, histological evaluation and Western blot analyses, contributed to data curation and statistical analysis, and helped revise the manuscript. Weibin Qin and Guixin He conceived and designed the study, provided methodological guidance and resources, supervised the research, critically revised the manuscript, and were responsible for project administration and funding acquisition. All authors read and approved the final manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Key Project of Guangxi Natural Science Foundation, Innovation Project of Guangxi Graduate Education, National Natural Science Foundation of China – Regional Project, (grant number 2020GXNSFDA297020, YCBXJ2025012, 81960861).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

All data are available from the relevant databases or from the corresponding author upon reasonable request.

Statement of Human and Animal Rights

All procedures involving animals were conducted in accordance with the approved protocols and in compliance with applicable institutional and national guidelines for the care and use of laboratory animals. This article does not contain any studies involving human participants, human data, or human tissue.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online.

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