Yixinshu attenuates myocardial infarction via SHP1/JAK2/STAT3-mediated regulation of mitochondrial function and apoptosis
Xueting Wang, Xinrui Wang, Yang Liu, Yang Cui, Keyi Chen, Hongkun Wu, Mingshan Zhang, Ming Liao, Linyun Fu, Xiangchun Shen
TL;DR
Yixinshu, a traditional Chinese medicine, protects the heart after a heart attack by improving mitochondrial function and reducing cell death through a specific molecular pathway.
Contribution
The study identifies the SHP1/JAK2/STAT3 pathway and dihydrotanshinone I as key mediators of Yixinshu's cardioprotective effects.
Findings
Yixinshu improved cardiac function and reduced infarct size in MI models.
Dihydrotanshinone I suppressed SHP1 and activated STAT3 to mitigate mitochondrial injury.
SHP1 overexpression reversed the protective effects of dihydrotanshinone I.
Abstract
Yixinshu (YXS), a traditional Chinese formula, is applied for coronary artery diseases in clinic, however, its cardioprotective mechanisms remain unclear. At present, the role and underlying mechanisms of YXS is to elucidate in enhancing post-myocardial infarction (MI) recovery. MI mouse models and hypoxia-injured cardiomyocytes were reproduced to evaluate YXS efficacy. The pharmacological targets and blood-absorbed compounds of YXS were determined by network pharmacology and LC–MS, respectively. Bioactive components were screened via molecular docking and surface plasmon resonance (SPR), and mechanisms were validated by molecular assays. YXS improved cardiac function and reduced infarct size. These effects were linked to preserved mitochondrial homeostasis and reduced apoptosis through the SHP1/JAK2/STAT3 pathway. Dihydrotanshinone Ⅰ (DHT), a key compound identified in plasma,…
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Figure 7- —Special subject of scientific and technological research on traditional Chinese medicine and ethnic medicine in Guizhou Province
- —High-level Talent Start-up Fund Project of Guizhou Medical University
- —Guizhou Provincial Health Commission Science and Technology foundation
- —Guizhou Provincial Science and Technology Projects
- —National Natural Science Foundation of China
- —High-level Innovation Talents
- —the Guizhou province health high-quality development medical research joint fund
- —Project with the Leader Appointed after the Results are Announced of Guizhou Educational Department
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Taxonomy
TopicsTraditional Chinese Medicine Analysis · Cytokine Signaling Pathways and Interactions · Cardiac Fibrosis and Remodeling
Introduction
Cardiovascular diseases (CVDs) remain the leading cause of global mortality, with ischemic heart disease (IHD) identified as the primary etiological contributor [1, 2]. Myocardial infarction (MI), the most severe clinical manifestation of IHD, is pathologically defined by abrupt myocardial hypoperfusion resulting from thrombotic occlusion of coronary arteries, which ultimately progresses to heart failure and cardiogenic death [3, 4]. As terminally differentiated cells, cardiomyocytes are highly susceptible to irreversible loss under MI-induced pathological conditions, including hypoxic stress, metabolic dysregulation, and oxidative damage. This cardiomyocyte loss is predominantly driven by the aberrant activation of programmed cell death (PCD), a process that accelerates ventricular remodeling and compromises cardiac function [5].
Mitochondria serve as central hubs for energy metabolism and intracellular signaling, and their dysfunction plays a pivotal role in the pathogenesis of age-related disorders, including cardiovascular and neurodegenerative diseases [6]. In the context of MI, mitochondria-dependent apoptosis represents the predominant form of PCD, morphologically characterized by cell shrinkage, nuclear condensation, and DNA fragmentation, culminating in the formation of apoptotic bodies. This apoptotic cascade is tightly regulated by the caspase family of cysteine proteases, which are activated via proteolytic cleavage to initiate downstream signaling events [7].
The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is an evolutionarily conserved transmembrane signaling cascade consisting of cell surface receptors, receptor-associated tyrosine kinases (JAKs), and downstream transcription factors (STATs). In the heart, cytokines such as interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and ciliary neurotrophic factor (CNTF) activate JAK2, which in turn phosphorylates STAT3 (e.g., at the Tyr705 residue). Phosphorylated STAT3 dimerizes, dissociates from the receptor complex, and translocates to the nucleus to upregulate the expression of anti-apoptotic genes, including BCL2 and Mcl-1 [8, 9]. Furthermore, activated STAT3 exerts a direct mitochondrial protective effect by inhibiting mitochondrial permeability transition pore (mPTP) opening, reducing reactive oxygen species (ROS) accumulation, and sustaining electron transport chain (ETC) activity [8–10]. Targeting key regulatory nodes within the JAK/STAT signaling cascade to preserve mitochondrial function and suppress apoptotic signaling thus holds considerable promise as a therapeutic strategy for MI [11].
Traditional Chinese medicine (TCM) emphasizes the free flow of Qi and blood through meridians as essential to life. Myocardial infarction involves key pathological changes of "blood stasis" and "qi stagnation," which obstruct heart meridians and cause chest pain. The efficacy of TCM in managing ischemic diseases is deeply rooted in millennia of practice and extensive empirical evidence [12]. Yixinshu (YXS), a traditional Chinese medicinal formula deriving from the classical formula as named ShengMaiSan, is including seven Chinese medicines: Salvia miltiorrhiza, Ophiopogon japonicus, Astragalus membranaceus, Crataegus pinnatifida, Ligusticum chuanxiong, Schisandra chinensis, and Panax ginseng. It has been widely applied for angina pectoris in clinic [13]. According to TCM theory, YXS achieves its therapeutic aim of symptom relief and cardiac protection through integrated effects including replenishing qi, nourishing yin, activating blood circulation, resolving stasis, dispelling phlegm, and unblocking collaterals. Recently accumulating evidence indicated that YXS could alleviate chronic heart failure and mitigate myocardial ischemia–reperfusion injury by enhancing energy metabolism, attenuating oxidative stress, and suppressing inflammatory responses [14–17]. A promising modern strategy is to identify key genes targeted by TCM components and clarify their synergistic mechanisms for developing new ischemic therapies [18] However, the key bioactive ingredients and pharmacological targets of YXS are unclear to date.
In the present study, we integrated serum pharmacochemistry with network pharmacology to identify the key blood-absorbed bioactive compounds of YXS. Our primary objective was to elucidate the cardioprotective effects of YXS and its underlying mechanisms, with a specific focus on the regulation of apoptotic signaling and preservation of mitochondrial functions to facilitate cardiac functional recovery following MI.
Material and methods
Chemicals and reagents
Yixinshu Capsule (Approval No. Z52020038) was provided by Guizhou Xinbang Pharmaceutical Co., Ltd. (Guiyang, China). Compound Danshen Dripping Pill (Approval No. Z10950111) was provided by Tasly Pharmaceutical Group Co., Ltd. 5-Brdu-Dihydrotanshinone I (≥ 98% purity) was purchased from MedChemExpress (Princeton, NJ, USA), and salvianolic acid B from Shanghai Yuanye Bio-Technology Co., Ltd. DMEM and 0.25% trypsin were obtained from Gibco (Grand Island, NY, USA), and fetal bovine serum from Every Green (Hangzhou, China). Primary antibodies against JAK2 (AF1489), STAT3 (AF1492), phospho-STAT3 (Tyr705, AF1276), and phospho-JAK2 (AF1486) were purchased from Beyotime (Shanghai, China); anti-SHP1 from Abcam (Cambridge, UK); and anti-BAX, anti-BCL2, anti-cleaved caspase-3, and anti-β-actin from Proteintech (Wuhan, China). Protein phosphatase inhibitor (Cat. P1260), MTT, and DMSO were supplied by Solarbio (Beijing, China). The LDH assay kit was from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and the mouse CK-MB ELISA kit from Shanghai Fanyin Biotechnology Co., Ltd. (Shanghai, China).
Drug preparation and administration
For animal experiments, YXSC was freshly dissolved in double-distilled water (ddH₂O) and administered via gavage 48 h after ischemia. For cell-based assays, dihydrotanshinone I (DHT) was dissolved in DMSO to prepare a 5 mM stock solution, which was stored at − 80 °C. DHT was diluted to working concentrations (0.01 and 0.02 μM) in culture medium and applied to cardiomyocytes for 18 h prior to oxygen–glucose deprivation (OGD) treatment.
Animal experiments
Male C57BL/6 J mice (6–8 weeks old) and Sprague–Dawley rats (8 weeks old, 180–220 g) were purchased from from Sibeifu Biotechnology Co., Ltd. (Beijing, China). All animals were housed under standard laboratory conditions (12/12-h light/dark cycle, temperature 21 ± 1 °C, relative humidity 60%) with free access to food and water. All experimental procedures were approved by the Animal Care and Use Committee of Guizhou Medical University (Approval No. 2303412). Healthy male C57BL/6 J mice (18–22 g, specific pathogen-free, SPF) were randomly divided into the following groups (n = 10 per group): Sham, acute myocardial infarction (AMI), positive control (Danshen Dripping Pill, DDP, 105 mg·kg-1·day-1), low-dose YXSC (350 mg·kg-1·day-1), and high-dose YXSC (700 mg·kg-1·day-1). The drug dose was converted to the mouse equivalent dose based on the recommended dose in the drug package insert using the body surface area conversion method. The administration duration was 28 consecutive days.
Mouse model of MI
Male mice were anesthetized with 1.25% tribromoethanol. After intubation and thoracotomy, the left anterior descending (LAD) coronary artery was permanently ligated using 8–0 silk sutures to induce myocardial ischemia. Successful modeling was confirmed by ECG monitoring for 2 min post-ligation, evidenced by regional myocardial blanching and characteristic ECG changes. Sham-operated mice underwent the same procedure without LAD ligation.
Echocardiography
Echocardiography was performed at 24 h, 14 days, and 28 days after MI using a VINNO6LAB small animal ultrasound system (VINNO, Suzhou, China). After depilation of the left chest area, cardiac geometry was assessed from the parasternal long-axis view. Left ventricular function was evaluated using M-mode echocardiography, and ejection fraction (EF) and fractional shortening (FS) were calculated as the average of three consecutive cardiac cycles.
Histological staining
Mouse heart samples were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 µm slices. Paraffin sections were stained with hematoxylin and eosin (H&E) for cardiac tissue morphology. Masson’s trichrome staining was performed to assess collagen content and infarct size, while Wheat Germ Agglutinin (WGA) staining was used to evaluate cardiac remodeling. Images were captured and analyzed with ImageJ software.
Cardiac mass index
Following anesthesia, blood samples were collected. The heart was excised, weighed, and used to calculate the cardiac mass index (heart mass/body weight, mg/g) and cardiac hypertrophy index (heart mass/tibial length, mg/cm). Serum LDH and CK-MB levels were measured using commercial assay kits.
UPLC-MS-based serum metabolomics
Extraction of drug samples
The drug was ground into powder and transferred to a tube. One milliliter of methanol–water solution was added, followed by vortex mixing and sonication in a water bath for 30 min. The mixture was then centrifuged at 16,000 × g for 10 min at 4 °C. The supernatant was collected and vacuum freeze-dried. The dried residue was reconstituted in 40% methanol–water, vortexed, and centrifuged again at 16,000 × g for 15 min at 4 °C. The resulting supernatant was collected as the final extract.
Collection and preparation of serum samples
After echocardiography, mice were sacrificed under anesthesia and blood was collected via the abdominal aorta. Serum was isolated by centrifugation at 3500 × g for 10 min at 4 °C and stored at − 80 °C until analysis. An appropriate volume of serum was mixed with methanol, vortexed for 60 s, and incubated at − 20 °C for 30 min. After centrifugation at 16,000 × g for 20 min at 4 °C, the supernatant was collected and vacuum-dried. The residue was reconstituted in 100 μL of 40% methanol–water, vortexed, and centrifuged at 16,000 × g for 15 min at 4 °C. The final supernatant was used for analysis.
Extraction of blank serum and drug samples
An appropriate amount of blank serum was mixed with the drug extract, followed by the addition of methanol. The mixture was vortexed for 60 s and incubated at − 20 °C for 30 min. After centrifugation at 16,000 × g for 20 min at 4 °C, the supernatant was collected and vacuum dried. The residue was reconstituted in 100 μL of 40% methanol–water, vortexed, and centrifuged at 16,000 × g for 15 min at 4 °C. The final supernatant was used for further analysis.
Acquisition of UPLC-MS data
Samples were analyzed using a Vanquish UHPLC system (Thermo Scientific, Waltham, MA, USA) equipped with an ACQUITY UPLC HSS-T3 column (100 × 2.1 mm, 1.8 μm; Waters) and coupled to a TripleTOF 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA). The mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B; LC–MS grade, Fisher Chemical). Chromatographic separation was performed at a flow rate of 0.3 mL/min with the following gradient: 5% B (0–1 min), linear increase to 98% B (1–17 min), return to 5% B in 0.5 min, and held at 5% B for 2.5 min. A QC sample was run after every three analytical samples to assess system stability and data reliability.
Data processing
Raw LC–MS data were acquired in both positive and negative ion modes. Data were converted to mzXML format and processed using Progenesis QI software (Waters) for baseline correction, peak detection, integration, retention time alignment, and normalization. Compound identification was based on accurate mass, isotopic distribution, and MS/MS fragmentation patterns, using reference data from an in-house TCM database (Shanghai Applied Protein Technology Co., Ltd.) and public databases including GNPS, ReSpect, and MassBank.
Prediction and screening of targets
Potential targets of YXSC were predicted using the TCMSP, SymMap, GeneCards, and OMIM databases. Active components were screened from TCMSP and SymMap based on oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. Coronary heart disease-related targets were obtained by integrating data from GeneCards and the OMIM database (https://www.omim.org/). YXSC-related targets were then intersected with disease-related targets, and the overlapping genes were considered potential therapeutic targets of YXSC for coronary heart disease. A component–target network was constructed using Cytoscape software. The shared targets were further imported into the STRING database (version 11.0, http://string-db.org/) to build a protein–protein interaction (PPI) network. PPI data were subsequently visualized in Cytoscape, and core genes were identified based on Degree centrality.
Gene ontology and KEGG pathway enrichment analysis
Potential targets were uploaded to the Database for Annotation, Visualization, and Integrated Discovery (DAVID, v6.8; https://david.ncifcrf.gov/) for GO and KEGG pathway enrichment analyses. GO terms were categorized into Molecular Function (MF), Biological Process (BP), and Cellular Component (CC). A p-value < 0.05 was considered statistically significant.
Transmission electron microscopy (TEM)
Fresh left ventricular tissues were cut into 1–2 mm strips and fixed in 4% glutaraldehyde and 1% osmium tetroxide. After dehydration and embedding, 70 nm ultrathin sections were stained with uranyl acetate and lead citrate. Sections were imaged using a JEM-1230 transmission electron microscope at 10,000 × magnification. Three random fields per section were analyzed using ImageJ to quantify mitochondrial volume, cristae density, and mitochondrial count.
Western blot
Cardiac tissues or cardiomyocytes were lysed in RIPA buffer (Solarbio) supplemented with phosphatase inhibitor (1:1000) and PMSF. After 30 min incubation on ice, lysates were centrifuged at 12,000 × g for 20 min at 4 °C. Protein concentration was determined using a BCA kit (Solarbio). Equal amounts of protein were denatured, separated via SDS-PAGE, and transferred to 0.45 µm PVDF membranes (Millipore). Membranes were blocked with 5% skim milk for 2 h, incubated overnight at 4 °C with primary antibodies, and then with secondary antibodies (1:2000) for 2 h at room temperature. Bands were visualized using ECL (New Cell Life Science, China) and quantified using Image Lab software (v5.2, Bio-Rad).
Quantitative real-time polymerase chain reaction(qRT-PCR)
Total RNA was extracted using RNA-easy isolation reagent (Vazyme), and reverse transcription was performed with EasyScript^®^ One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech). qRT-PCR was carried out using PerfectStart^®^ Green qPCR SuperMix (TransGen Biotech) on a Bio-Rad CFX Manager 3.1 system. Relative mRNA expression was calculated using the 2^−ΔΔCt^ method. Primer sequences are listed in Table 1. Table 1. Primer sequences used for qRT-PCR analysisTarget geneSequenceSHP1Forward primerRat5′-ATCGCCCAGTTCATCGAAAC-3′Reverse primerRat5′-CTTCCTTCTTGTTCTTGCTATGC-3′STAT3Forward primerMice5′-CAATACCATTGACCTGCCGAT-3′Reverse primerMice5′-GAGCGACTCAAACTGCCCT-3′Forward primerRat5′-AGTGCTGCCCCTTACCTGAAG-3′Reverse primerRat5′-CCATGTCAAACGTGAGCGAC-3′BCL2Forward primerMice5′-ATGCCTTTGTGGAACTATATGGC-3′Reverse primerMice5′-GGTATGCACCCAGAGTGATGC-3′Forward primerRat5′-TGGTGGACAACATCGCTCT-3′Reverse primerRat5′-AGGCTGAGCAGCGTCTTCA-3′β-actinForward primerMice5′-GTGCTATGTTGCTCTAGACTTCG-3′Reverse primerMice5′-ATGCCACAGGATTCCATACC-3′Forward primerRat5′-TGTCACCAACTGGGACGATA-3′Reverse primerRat5′-GGGGTGTTGAAGGTCTCAAA-3′
Molecular docking
Protein structures were obtained from the UniProt database, and compound structures were downloaded from PubChem. All structures were processed using AutoDockTools (ADT). The docking grid box parameters were set using PyMOL 2.2.0, and binding energies were predicted with AutoDock 4.2.
Isolation of neonatal rat cardiomyocytes (NRCMs) and H9C2 cell culture
The H9C2 cell line was obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in a humidified incubator at 37 °C with 5% CO₂.
Neonatal rat cardiomyocytes were isolated from 1–2-day-old specific-pathogen-free (SPF). Under sterile conditions, the hearts were exercised, rinsed in PBS, minced, and digested with 0.125% trypsin at 4 °C for 12 h. Following this, tissue was further digested with 0.8% trypsin at 37 °C for 5 min across 4–6 cycles. The collected supernatants were pooled and neutralized with DMEM containing 10% FBS. To eliminate fibroblasts, differential attachment was applied, and cardiomyocytes (10^5^ cells/mL) were cultured with 5-bromo-2′-deoxyuridine (5-BrdU) for 48 h.
To establish an oxygen–glucose deprivation (OGD) model, cells were incubated for 12 h in glucose-free DMEM in a hypoxia incubator (94% N₂, 5% CO₂, 1% O₂) at 37 °C. For hypoxia pre-experiments, NRCMs were subjected to 18 h of hypoxia, and H9C2 cells to 12 h.
Cell viability assay
The viability of NRCMs was assessed using the MTT assay. After treatment with various concentrations of Sal B, DHT, YXSC, or CDDP, cells were incubated with 0.1 mg/mL MTT solution for 4 h. The resulting formazan crystals were dissolved in DMSO, and absorbance was measured spectrophotometrically. Relative cell viability was calculated and expressed as a percentage of control values.
Immunofluorescence staining
Myocardial tissue was fixed in 4% paraformaldehyde containing 0.2% Triton X-100 at 4 °C for 30 min, followed by blocking with 5% bovine serum albumin (BSA) for 1 h. Tissues were then incubated overnight at 4 °C with primary antibodies against SHP1, phosphorylated JAK2 (p-JAK2), and phosphorylated STAT3 (p-STAT3). After washing, samples were incubated with fluorescent-conjugated secondary antibodies at 4 °C in the dark for 1.5 h. Nuclei were counterstained with DAPI. Images were captured using an inverted fluorescence microscope (Leica, Wetzlar, Germany) and analyzed using ImageJ (NIH, Bethesda, MD, USA).
Measurement of mitochondrial respiratory function
NRCMs were cultured for 48 h and then treated with DHT and Sal B under hypoxic conditions for 18 h. Mitochondrial respiratory function was assessed by measuring the oxygen consumption rate (OCR) using the XF24 Extracellular Flux Analyzer (Agilent Seahorse Bioscience). The assay involved sequential addition of the following compounds at their respective working concentrations: oligomycin (40 µM), FCCP (4 µM), rotenone (5 µM), and antimycin A (5 µM). Data was analyzed using XF Cell Mito Stress Test Generator software (Agilent Seahorse Bioscience).
TUNEL assay
An in vitro Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) assay was performed using a commercial kit (Yeasen Biotechnology Co., Ltd., Shanghai, China), following the manufacturer’s protocol. Images were captured using a fluorescent microscope. The apoptotic index was calculated as the ratio of TUNEL-positive nuclei to total DAPI-stained nuclei.
Metabolite and enzyme activities assays
Cardiomyocytes were incubated with the test compounds for 18 h and then rinsed with PBS. Mitochondrial enzymatic activities and intracellular reactive oxygen species (ROS) levels were evaluated using commercial assay kits according to the manufacturer’s instructions. Specifically, Mito-Tracker Red CMXRos and Annexin V-FITC (Beyotime) were used for staining, JC-10 dye was used for mitochondrial membrane potential assessment, and a Reactive Oxygen Species Assay Kit (Beyotime) was employed for ROS quantification.
Flow cytometry
Flow cytometric analysis was conducted to assess apoptosis and mitochondrial membrane potential in H9C2 cells. Cells were harvested from 6-well plates via trypsinization and stained using the Annexin V-FITC/PI Apoptosis Detection Kit (Yeasen), Mito-Tracker Red CMXRos and Annexin V-FITC (Beyotime), and the JC-10 Mitochondrial Membrane Potential Assay Kit (Solarbio), in accordance with the manufacturers’ protocols. Fluorescence signals were measured using a FACStar Plus flow cytometer (Becton–Dickinson, NJ, USA).
Establishment of SHP1 over-expression cell line
Adenoviral vectors encoding SHP1 (Ad-Ndufs1) and an empty control vector (Ad-EV) were constructed and provided by GenePharma Co., Ltd. (Shanghai, China).
Surface plasmon resonance (SPR)
The activator solution was freshly prepared by mixing 400 mM 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 100 mM N-Hydroxysuccinimide (NHS) immediately before injection. The CM5 sensor chip was activated with this mixture for 420 s at a flow rate of 10 μL/min. SHP1 protein was diluted to 20 μg/mL in immobilization buffer and injected into the sample flow cell (Fc2) at a flow rate of 10 μL/min, typically achieving immobilization levels of approximately 12,600 response units (RU). The reference flow cell (Fc1) was left unmodified and did not undergo ligand immobilization. The sensor chip was subsequently deactivated using 1 M ethanolamine hydrochloride at 10 μL/min for 420 s. Dihydrotanshinone I (DHT) was diluted in analyte buffer to eight different concentrations (1–100 μM). Each concentration was sequentially injected into both Fc1 and Fc2 at a flow rate of 20 μL/min, with an association phase of 100 s and a dissociation phase of 180 s. All injections were conducted in the analyte buffer. The eight analyte concentrations were tested in ascending order. After each interaction cycle, the sensor chip was regenerated to restore baseline conditions.
Co-immunoprecipitation (Co-IP)
A total of 18 μL of Protein A-Agarose suspension was added to a spin column containing a filter membrane, followed by four washes with PBS buffer through centrifugation at low speed. Next, 0.7 mL of prepared cell lysate was transferred into a sterile 1.5 mL centrifuge tube. Then, 7 μL of 100 mM PMSF and 1 μg of a specific primary antibody were added, and the mixture was incubated at 4 °C for 12–16 h. The antibody–antigen complex was transferred to the pre-washed Protein A-Agarose column and incubated for an additional 2 h to overnight at 4 °C. After incubation, the column was placed into a new 2 mL collection tube and centrifuged to collect the precipitate. The agarose beads were then washed several times with cold IP buffer. Finally, 1 × SDS loading buffer was added, and the samples were heated at 95 °C for 5 min to elute the protein complexes. After centrifugation at 12,000 × g, the supernatant was collected for subsequent Western blot analysis.
Statistical analysis
All experiments were independently repeated a minimum of three times, with more repetitions for animal experiments. Data are expressed as mean ± SD. Statistical analyses were performed using one-way ANOVA, wherein post-hoc pairwise comparisons were conducted using Tukey’s test under homogeneity of variance or the Games-Howell test when this assumption was violated. A p-value < 0.05 was deemed to indicate statistical significance.
Results
YXS improved heart failure induced by myocardial infarction in mice
Following ligation of the left anterior descending (LAD) coronary artery, electrocardiography (ECG) exhibited prominent peaked T waves in the model mice (Fig. S1A–B). Echocardiographic assessment revealed significantly reduced ejection fraction (EF) and fractional shortening (FS) in all groups subjected to LAD ligation compared to the sham group, indicating compromised cardiac contractile function post-myocardial infarction (MI) (Fig. S1C–E). In parallel, serum levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase 1 (LDH1) were markedly elevated in the MI group (Fig. S1F–G), confirming successful model establishment.
At 24 h post-surgery, mice received daily oral gavage of either low-dose (350 mg·kg^−1^·day^−1^) or high-dose (700 mg·kg^−1^·day^−1^) Yixinshu Capsule (YXS), or the positive control drug, Compound Danshen Dripping Pills (CDDP) (Fig. 1A). Survival was monitored during the treatment period, and YXS significantly reduced MI-induced mortality (Fig. 1B). After 28 days of treatment, cardiac function and structure were re-evaluated. Both YXS and CDDP markedly improved EF and FS (Fig. 1C–E), reduced the heart weight-to-body weight ratio (heart index) (Fig. 1F–G), mitigated myocardial fibrosis (Fig. 1H), and alleviated pathological cardiac remodeling (Fig. 1I–J).Fig. 1YXS Improved Heart Failure Induced by Myocardial Infarction in Mice. A Schematic diagram of the experimental protocol and treatment schedule. B Kaplan–Meier curves. C Representative M-mode echocardiographic images of mice. Quantification of echocardiographic parameters, namely EF (D), and FS (E) (n = 10). F Representative macroscopic images of hearts. G The ratios of HW/TL (n = 7). H Representative Masson’s trichrome staining images of heart tissues (scale bar = 500 µm or 20 µm). Fibrotic area of mice hearts (n = 3). I WGA staining images of heart tissues (scale bar = 10 µm). J Quantification of the cross-sectional area of cardiomyocytes in mice. K Venn diagram of YXS and coronary heart disease. L GO functional analysis. M KEGG functional analysis. Data were shown as mean ± SD. *p < *0.05, *p < 0.01 vs. the sham group; ^#^p < 0.05, ^##^p < 0.01 vs. the MI group
To explore the underlying mechanism of YXS, 9,639 potential therapeutic targets related to coronary heart disease were retrieved from the OMIM and GeneCards databases. Additionally, 432 protein targets associated with the bioactive compounds of YXS were obtained from the TCMSP and SymMap databases. The intersection of these datasets identified 306 overlapping drug–disease targets (Fig. 1K). A comprehensive “Traditional Chinese Medicine–Compound–Target–Disease” interaction network was constructed using Cytoscape (Fig. S1H), where purple inverted triangles represent herbal medicines, orange triangles denote active ingredients, and squares represent common targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of these shared targets were conducted using the DAVID database (Fig. 1L–M). The results indicated that the cardioprotective effects of YXS may be mediated via regulation of mitochondrial metabolism and apoptosis-related pathways.
YXS promoted STAT3 phosphorylation to inhibit cardiomyocyte apoptosis
Transmission electron microscopy was employed to assess mitochondrial integrity in myocardial tissues post-MI (Fig. 2A). Compared with the sham group, the MI group showed pronounced mitochondrial cristae disruption and a significantly increased proportion of mitochondria damage. Treatment with both low and high doses of YXS effectively reduced the proportion of damaged mitochondria (Fig. 2B) and preserved the structural integrity of mitochondrial cristae (Fig. S2A). Given the central role of mitochondrial damage in triggering apoptosis, we further evaluated myocardial apoptosis following MI. Compared to the sham group, MI significantly increased the expression of pro-apoptotic proteins cleaved-caspase 3 and BAX, while decreasing the expression of the anti-apoptotic protein BCL2. YXS treatment reversed these changes, indicating its anti-apoptotic effects in infarcted myocardial tissue (Fig. 2C–D). To identify key regulatory targets underlying the cardioprotective effects of YXS, a protein–protein interaction (PPI) network was constructed from the overlapping drug–disease targets using Cytoscape software. The resulting network comprised 203 nodes and 615 edges, with central nodes including STAT3, AKT1, TP53, and HSP90AA1. Among them, STAT3 exhibited the strongest association with apoptosis regulation (Fig. 2E). We next assessed STAT3 expression in cardiomyocytes. YXS treatment did not significantly alter MI-induced STAT3 mRNA or total protein levels but significantly enhanced STAT3 phosphorylation (p-STAT3), thereby activating downstream signaling and upregulating the anti-apoptotic protein BCL2 (Fig. 2F–H). PPI network analysis further identified JAK2 as an upstream kinase responsible for STAT3 phosphorylation (Fig. 2I). The phosphatase SHP1 binds to phosphorylated JAK2 through its SH2 domain, promoting JAK2 dephosphorylation and thereby inhibiting continuous STAT3 activation (Fig. 2J). Co-immunoprecipitation (Co-IP) assays confirmed the interactions between STAT3 and JAK2, as well as between JAK2 and SHP1, in myocardial tissue (Fig. 2K). Further investigation revealed that MI reduced phosphorylated JAK2 levels while increasing SHP1 expression. Although YXS had no significant effect on SHP1 mRNA levels compared to the MI group, it significantly downregulated SHP1 protein expression and restored JAK2 phosphorylation (Fig. 2L–M). Collectively, these findings suggest that the SHP1/JAK2/STAT3 signaling axis plays a pivotal role in mediating the anti-apoptotic effects of YXS. SHP1-mediated dephosphorylation of JAK2 may represent a key pharmacological target of YXS in the treatment of myocardial infarction.Fig. 2YXS promoted STAT3 phosphorylation and suppresses cardiomyocyte apoptosis. A Representative transmission electron microscopy images of heart tissues (scale bar = 5 μm or 1 μm). B Ratio of damaged mitochondrial (n = 6). C, D Immunoblotting to test the levels of proteins in mice hearts (n = 5). E Protein–protein interaction network of common targets of YXS and coronary heart disease. F, G mRNA levels of BCL2 and STAT3 in mice hearts determined with qRT-PCR (n = 3). H Immunoblotting to test the levels of proteins in mice hearts (n = 5). I Protein–protein interaction network of STAT3. J The SHP1/JAK2/STAT3 signaling pathway. K Co-IP experiments in hearts of mice. Lysates were immunoprecipitated with anti-SHP1 and blotted with JAK2 antibody and STAT3antibody. L Immunoblotting to test the levels of proteins in mice hearts (n = 6). M mRNA levels of SHP1 in mice hearts determined with qRT-PCR. Data were shown as mean ± SD. *p < *0.05, *p < 0.01 vs. the sham group; ^#^p < 0.05, ^##^p < 0.01 vs. the MI group
DHT as the active component underlying the anti-apoptotic effect of YXS
An in vitro oxygen–glucose deprivation (OGD) model was established to simulate myocardial ischemia and hypoxia (Fig. S2B). During the early stages of apoptosis, mitochondrial membrane depolarization leads to the release of JC-10 dye from mitochondria, resulting in a fluorescence shift from red-emitting aggregates to green-emitting monomers. JC-10 staining indicated that the cardioprotective effect of YXS was closely associated with its inhibition of mitochondria-mediated apoptosis (Fig. S2C–D). To identify candidate bioactive constituents, we analyzed YXS extracts, blank serum, drug-serum mixtures, and serum samples from mice following oral YXS administration. Ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) was used to profile both the chemical components of YXS and serum metabolites (Fig. 3A-B). A total of 3,810 chemical constituents were identified in the YXS extract based on mass spectrometry databases, with their chemical classes annotated using the NPClassifier system (Table S1). Among these, 324 compounds were detected in the serum post-administration, including 74 terpenoids, 64 alkaloids, 67 shikimates and phenylpropanoids, 23 fatty acids, 23 amino acids and peptides, and 36 polyketides, among others (Fig. 3C). Chromatographic peaks in both positive and negative ionization modes were annotated accordingly (Fig. S2E–F). Subsequent analyses focused on the ten compounds with the highest plasma concentrations (Table 2).Fig. 3DHT as the Active Component of YXS. A BPCs of each sample group under positive ion mode. B BPCs of each sample group under negative ion mode. C Classification and proportion of blood-absorbed compounds from YXS across chemical categories. D–G Danshensu, Dihydrotanshinone-I (DHT), Ginsenoside, Ginsenoside-Rg6 were capable of binding with SHP1. H The binding affinity of SHP1 with DHT was determined using an SPR assay. I Representative Western blot and quantification of STAT3, p-STAT3, p-JAK2, JAK2 and SHP1 (n = 3–5). J Co-IP experiments in Primary neonatal rat cardiomyocytes. Lysates were immunoprecipitated with anti-SHP1 antibody and blotted with ubiquitin antibody and SHP1 antibody. Data were shown as mean ± SD. *p < *0.05, *p < 0.01 vs. sham or control group; ^#^p < 0.05, ^##^p < 0.01 vs. MI or OGD groupTable 2Top ten YXS-derived compounds detected in plasma and their pharmacokinetic profilesNom/zRT minppmcompound nameadductscoreSuperClassInto Blood or None1297.15522.421.84-Hydroxycyclofenil[M + H] + 0.8311None2243.04752.746.45-Bromo-4-hexylpyrimidine[M + H] + 0.9986Tetramate alkaloids + Peptide alkaloidsInto_Blood3341.06526.241.5Lithospermic acid[M + H − C9H10O5] + 0.9723LignansNone4521.10766.621Salvianolic acid b[M + H-C9H10O5] + 0.8114LignansNone5423.36187.071.1(3.beta.,6.alpha.,12.beta.,20Z)-3,12-Dihydroxydammara-20(22),24-dien-6-yl 2-O-(6-deoxy-.alpha.-L-mannopyranosyl)-.beta.-D-glucopyranoside[M + H − C12H24O11] + 0.9716TriterpenoidsNone6285.07567.850.74′,6-Dihydroxy-3′-methoxyaurone[M + H] + 0.9967FlavonoidsNone7423.36189.510.9Panaxoside Rg2[M + H − C12H26O12] + 0.9688TriterpenoidsNone8415.211111.762Schizandrol[M + H − H2O] + 0.9284LignansNone9399.179912.390.8Gomisin A[M + H − H2O] + 0.9394LignansNone10483.237612.710.1Angeloylgomisin H[M + H − H2O] + 0.9419LignansNone11253.158712.880Estra-4,9-diene-3,17-dione[M + H − H2O] + 0.9325SteroidsNone12279.101412.960.1Dihydrotanshinone[M + H] + 0.9872Naphthalenes + DiterpenoidsInto_Blood13401.1956131g-Schizandrin[M + H] + 0.8241LignansNone14431.206213.230.36.beta.-Hydroxyeplerenone[M + H] + 0.7389SteroidsNone15295.132713.5321.5Fatostatin[M + H] + 0.9848Nicotinic acid alkaloidsNone16403.211213.591Schisanhenol[M + H] + 0.9048LignansNone17532.253813.710.7Gomisin B[M + NH4] + 0.9803LignansNone18297.148114.031.3Cryptotanshinone[M + H] + 0.9954Naphthalenes + DiterpenoidsNone19417.226914.550.1Deoxyschizandrin[M + H] + 0.9769LignansNone20401.195614.790.5Deoxygomisin A[M + H] + 0.9918LignansNone21295.132614.871.8Tanshinone IIA[M + H] + 0.9924Naphthalenes + DiterpenoidsNone19417.226914.550.1Deoxyschizandrin[M + H] + 0.9769LignansNone20401.195614.790.5Deoxygomisin A[M + H] + 0.9918LignansNone21295.132614.871.8Tanshinone IIA[M + H] + 0.9924Naphthalenes + DiterpenoidsNone22197.04582.212.9(R)-3-(3,4-Dihydroxyphenyl)lactate[M − H] − 0.942Into_Blood23143.03542.462.6mono-Ethyl fumarate[M − H] − 0.9242Fatty estersNone24173.0092.721.83-(2-Bornyloxycarbonyl)-3-hydroxyglutaric acid[M − H − C10H18O] − 0.9986SesquiterpenoidsNone25111.00942.923.5Citric acid[M − H − CH4O4] − 0.9994Fatty Acids and ConjugatesNone26239.05663.93.2Acetylsyringic acid[M − H] − 0.8304Phenolic acids (C6–C1)None27359.07816.281.9Rosmarinic acid[M − H] − 0.9971Phenylpropanoids (C6–C3)None4717.14846.622.2Salvianolic acid b[M − H] − 0.9587LignansNone28845.49287.081.4Ginsenoside Rf[M + FA − H] − 0.9175TriterpenoidsInto_Blood29283.06197.863Acacetin[M − H] − 0.9963FlavonoidsNone30547.12668.817.8Piscroside A[M − H] − 0.8378MonoterpenoidsNone311107.59710.383Ginsenoside Rb1[M − H] − 0.9988TriterpenoidsInto_Blood32955.492410.631.4Chikusetsusaponin V[M − H] − 0.9971TriterpenoidsInto_Blood33945.544611.66.1Ginsenoside Rd[M − H] − 0.9938TriterpenoidsInto_Blood34239.144812.3319.3IDE2[M − H] − 0.9919Into_Blood35625.244812.6114N-Caffeoyl-O-methyltyramine[2 M − H] − 0.8337Phenylpropanoids (C6–C3)None36389.197912.792.314-Hydroxy-7-O-methylrosmanol[M − H] − 0.8753DiterpenoidsNone37313.145412.892.3Gibberellin A4[M − H − H2O] − 0.8895DiterpenoidsInto_Blood38313.145413.553.2Platyphyllonol[M − H] − 0.969DiarylheptanoidsInto_Blood39315.197414.152.2Kaur-16-en-18-oic acid, 9-hydroxy-15-[[(2Z)-2-methyl-1-oxo-2-butenyl]oxy]-, (4.alpha.,15.alpha.)-[M − H − C5H8O2] − 0.9863DiterpenoidsNone40429.192814.223.7(1-Acetyloxy-3-hydroxy-6,8a-dimethyl-7-oxo-3-propan-2-yl-2,3a,4,8-tetrahydro-1H-azulen-4-yl) 4-hydroxybenzoate[M − H] − 0.9372SesquiterpenoidsNone
To identify the bioactive components of YXS that may interact with SHP1, molecular docking studies were performed using serum-absorbed compounds—Danshensu, Ginsenoside Rd, Ginsenoside Rg6, and Dihydrotanshinone I (DHT) (Fig. 3D–G). Among them, DHT exhibited the strongest binding affinity for SHP1, with a calculated binding energy of − 10.4 kcal·mol^−1^ (Table S2). To further validate this interaction, surface plasmon resonance (SPR) analysis was performed. The results showed that DHT binds directly to SHP1 with a dissociation constant (K_D) of 1.9 × 10^−6^ M (Fig. 3H), indicating a strong binding affinity. These findings suggest that DHT may serve as a key effector molecule in YXS, mediating its downregulation of SHP1 protein. To evaluate its cardioprotective potential, DHT was tested in an in vitro OGD model using primary cardiomyocytes. Cell viability assays revealed that 0.1 μM and 0.2 μM DHT significantly alleviated OGD-induced myocardial injury (Fig. S3A–C). Furthermore, DHT treatment increased the levels of phosphorylated STAT3 (p-STAT3) and phosphorylated JAK2 (p-JAK2), while reducing SHP1 protein expression in OGD-treated cardiomyocytes (Fig. 3I). To determine whether DHT-induced SHP1 downregulation was mediated by ubiquitin-dependent proteasomal degradation, co-immunoprecipitation (Co-IP) and ubiquitination assays were conducted. The results revealed that DHT enhanced SHP1 ubiquitination, suggesting that DHT promotes SHP1 degradation via the ubiquitin–proteasome pathway rather than by suppressing its transcription (Fig. 3J).
To investigate the anti-apoptotic effects of DHT, we examined both the expression of apoptosis-related proteins and mitochondrial function in primary cardiomyocytes subjected to oxygen–glucose deprivation (OGD). DHT treatment significantly upregulated the anti-apoptotic protein BCL2 while downregulating the apoptotic markers cleaved caspase-3 and BAX (Fig. 4A), thereby effectively suppressing OGD-induced apoptosis in primary cardiomyocytes (Fig. 4B–C). Mitochondrial membrane potential was assessed using JC-10 staining, which revealed that DHT preserved mitochondrial polarization and integrity under OGD conditions (Fig. 4D–E). Co-localization analysis further showed that mitochondrial damage was closely associated with apoptosis in cardiomyocytes, and DHT significantly alleviated this damage (Fig. 4F). To evaluate mitochondrial respiratory function, we conducted a mitochondrial stress test using a Seahorse extracellular flux analyzer. First, oligomycin, an ATP synthase inhibitor, was added to assess ATP-linked respiration. This was followed by the addition of FCCP, a mitochondrial uncoupler, to measure maximal respiration and estimate spare respiratory capacity. Finally, rotenone and antimycin A—specific inhibitors of complexes I and III, respectively—were introduced to determine non-mitochondrial respiration. Compared to the control group, the OGD group exhibited marked reductions in basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity. Notably, DHT treatment significantly restored all of these parameters following OGD insult (Fig. 4G). The anti-apoptotic and mitochondrial protective effects of DHT were further validated in H9C2 cells, showing consistent results with those observed in primary cardiomyocytes (Fig. S3D–F, Fig. 4).Fig. 4DHT inhibited apoptosis and enhanced mitochondrial activity in primary cardiomyocytes. A Representative Western blot and quantification of Cleaved-Caspase3, BAX and BCL2 (n = 3–4). B TUNEL staining (scale bar = 50 μm). C Apoptosis of cardiomyocytes was assessed by flow cytometry (n = 5). D, E Mitochondrial membrane potential was assessed using JC-10 staining (scale bar = 25 μm) (n = 3). F Co-localization of active mitochondria and apoptotic signals (scale bar = 25 μm). G OCR was measured by Seahorse to evaluate mitochondrial bioenergetics. And Quantitative analysis of OCR. n = 3. Data were shown as mean ± SD. *p < *0.05, *p < 0.01 vs. sham or control group; ^#^p < 0.05, ^##^p < 0.01 vs. MI or OGD group
The anti-apoptotic effect of DHT via the JAK2/STAT3 pathway depends on SHP1 downregulation
To confirm the functional significance of SHP1 in DHT-mediated cardioprotection, primary cardiomyocytes were transduced with an SHP1-overexpressing adenovirus (Fig. 5A). Following transduction, both SHP1 mRNA and protein levels were significantly upregulated (Fig. 5B–C). We next assessed the impact of SHP1 overexpression on the DHT-mediated activation of the JAK2/STAT3 signaling axis and cardiomyocyte apoptosis under OGD conditions. Overexpression of SHP1 completely abrogated the DHT-induced phosphorylation of JAK2 and STAT3, as well as the upregulation of the anti-apoptotic protein BCL2 (Fig. 5D). Moreover, SHP1 overexpression reversed the protective effects of DHT on mitochondrial function and apoptosis, as indicated by reduced mitochondrial activity, increased apoptotic cell death, and restored expression of the pro-apoptotic protein BAX (Fig. 5D–G). To further validate these findings in vivo, DHT was administered orally to mice following MI induction. DHT treatment significantly preserved left ventricular systolic function (Fig. 6A), reduced infarct size (Fig. 6B–C), and activated the JAK2/STAT3 pathway, thereby exerting anti-apoptotic effects in cardiac tissue (Fig. 6D). However, intramyocardial injection of the SHP1-overexpressing adenovirus abolished these benefits. In this context, DHT failed to induce JAK2 and STAT3 phosphorylation, and its anti-apoptotic effects were lost (Fig. 6). These findings collectively demonstrate that DHT exerts its cardioprotective and anti-apoptotic effects via activation of the JAK2/STAT3 signaling pathway, and that these effects are critically dependent on the downregulation of SHP1.Fig. 5. The anti-apoptotic effect of DHT is dependent on SHP1 downregulation. A Adenoviral transduction of primary cardiomyocytes. B mRNA levels of SHP1 in primary cardiomyocytes (scale bar = 100 μm) (n = 4). C, D Representative Western blot images and corresponding quantification of protein expression (n = 3–5). E Mitochondrial membrane potential was detected by Mito-Tracker Red CMXRos assay (scale bar = 50 μm) (n = 3). F TUNEL staining (scale bar = 50 μm). G Apoptosis of cardiomyocytes was assessed by flow cytometry (n = 3). Data were shown as mean ± SD. *p < *0.05, *p < 0.01Fig. 6SHP1 overexpression abrogates the cardioprotective effects of DHT. A Representative M-mode echocardiographic images of mice. Quantification of echocardiographic parameters, namely EF, and FS (n = 6). B Representative Masson’s trichrome staining images of heart tissues (scale bar = 500 µm). C Fibrotic area of mice hearts (n = 3). D Representative Western blot images and corresponding quantification of protein expression (n = 3–5). Data were shown as mean ± SD. *p < *0.05, *p < 0.01
Discussion
Modern pharmacological research has demonstrated that traditional Chinese medicine (TCM), characterized by its multi-component, multi-target, and holistic regulatory properties, plays a crucial role in the prevention and treatment of various cardiovascular diseases. Notably, TCM shows distinct advantages in stabilizing disease progression and improving cardiac function. Yixinshu (YXS), a Chinese herbal formula derived from the classical prescription Shengmai San, has long been used as an adjuvant therapy for angina pectoris in coronary artery disease [15]. However, most existing research has focused on clinical outcomes or in vivo efficacy, with limited exploration of the molecular pharmacology underlying its active components—such as dihydrotanshinone I and ginsenosides—and their specific roles in regulating cardiomyocyte apoptosis.
Within the pathophysiology of myocardial infarction (MI), the disruption of mitochondrial homeostasis and the activation of programmed cell death (PCD) pathways are recognized as pivotal events contributing to the expansion of the infarcted area [17]. Under hypoxic stress, cardiomyocytes initiate adaptive regulatory mechanisms—through the modulation of mitochondrial activity, fission, and fusion dynamics—in an attempt to maintain function and counteract injury. However, once cellular damage surpasses the reparative threshold, cell death becomes irreversibly activated. Given that cardiomyocytes are terminally differentiated and possess limited regenerative capacity, preventing or attenuating mitochondrial dysfunction during MI has emerged as a crucial cardioprotective strategy.
Based on animal and cellular models of myocardial infarction, this study demonstrates that YXS significantly improves cardiac function and reduces infarct size. Its cardioprotective effects are closely associated with the maintenance of mitochondrial homeostasis. Programmed cardiomyocyte death in MI involves a complex cascade encompassing multiple pathways, including necrosis, apoptosis, ferroptosis, autophagy, and pyroptosis [19–22]. To investigate the underlying mechanisms, we employed network pharmacology to screen for potential molecular targets. The analysis suggests that YXS may exert its effects primarily by regulating the apoptotic pathway.
Apoptosis is a genetically controlled form of programmed cell death, characterized by cell shrinkage, chromatin condensation and margination, nuclear fragmentation, membrane blebbing, and formation of apoptotic bodies. It is considered a vital mechanism for maintaining organismal homeostasis. Apoptosis is initiated via two principal pathways: the extrinsic and intrinsic pathways [23]. The extrinsic pathway (death receptor pathway) is triggered by the binding of extracellular ligands (e.g., Fas, TNF, TRAIL) to death receptors, inducing receptor oligomerization, recruitment of the FADD adaptor protein and caspase-8, and formation of the death-inducing signaling complex (DISC), ultimately leading to the activation of downstream caspases [24]. The intrinsic pathway is mediated by mitochondrial outer membrane permeabilization (MOMP), a process primarily regulated by the BCL2 protein family and often associated with the pathological opening of the mitochondrial permeability transition pore (mPTP). The BCL2 family comprises approximately 25 cytosolic proteins, functionally categorized into three groups: pro-apoptotic effector proteins (e.g., BAX, Bak), BH3-only activator proteins (e.g., tBid, Bad, NOXA, PUMA, Bim), and anti-apoptotic regulatory proteins (e.g., BCL2, Bcl-xL).
Concurrently, the execution phase of apoptosis actively disrupts mitochondrial integrity. Caspase-mediated cleavage of mitochondrial proteins and dysregulation of calcium homeostasis during apoptosis further contribute to the collapse of mitochondrial membrane potential and function [25]. The mitochondrial protective and anti-apoptotic effects of YXS effectively disrupt this vicious cycle between mitochondrial dysfunction and apoptosis, thereby contributing to its cardioprotective action. Building upon this foundation, the present study focuses on the regulation of the STAT3 signaling pathway to investigate the impact of YXS on cardiomyocyte apoptosis following myocardial infarction.
The JAK/STAT pathway is an evolutionarily conserved transmembrane signaling cascade composed of cell surface receptors, receptor-associated tyrosine kinases, and downstream transcription factors. The JAK family comprises JAK1, JAK2, JAK3, and TYK2 [26], while the STAT family includes STAT1 through STAT6 [27]. Upon ligand binding, JAKs phosphorylate specific tyrosine residues on the receptor, creating docking sites for STAT proteins. Subsequently, STATs undergo phosphorylation, dimerization, and nuclear translocation to regulate gene transcription.
STAT3, encoded on chromosome 17q21, is an approximately 89 kDa protein [28]
Its activation depends on phosphorylation at two critical sites: tyrosine 705 (Y705) and serine 727 (S727). Phosphorylation at Y705 promotes STAT3 nuclear translocation and subsequent activation of anti-apoptotic gene transcription. In contrast, phosphorylation at S727 enables mitochondrial localization of STAT3, allowing direct regulation of mitochondrial function [29, 30]. STAT3 exerts its anti-apoptotic effects through a dual mechanism: (1) Nuclear STAT3 upregulates the expression of anti-apoptotic genes (e.g., BCL2) while suppressing pro-apoptotic genes (e.g., BAX), thereby blocking apoptosis signaling [31–33]. (2) Mitochondrial STAT3 stabilizes the mitochondrial membrane potential (ΔΨm), prevents the opening of the mitochondrial permeability transition pore (mPTP), and restricts excessive reactive oxygen species (ROS) production, ultimately inhibiting caspase activation and DNA fragmentation [34, 35]. Our Western blot analysis demonstrated that YXS significantly increased BCL2 protein levels while reducing the expression of BAX and cleaved caspase-3, suggesting that its anti-apoptotic effects are mediated through the STAT3 signaling pathway. Notably, YXS treatment also markedly elevated the level of phosphorylated STAT3 (p-STAT3) in myocardial tissue.
To identify the active constituents of YXS, we employed liquid chromatography-mass spectrometry (LC–MS) and detected 10 components that entered the bloodstream, including dihydrotanshinone I (DHT), salvianolic acids, ginsenoside Rg6, and ginsenoside Rd, among others. Molecular docking simulations suggested that DHT is likely the key component responsible for modulating SHP1 expression. As a major tanshinone derivative derived from Salvia miltiorrhiza, DHT has been reported to exert significant cardioprotective effects, primarily through its antioxidant mechanisms [36]. Although previous studies have suggested that its action involves the inhibition of arachidonic acid metabolism, its upstream signaling targets have not been fully elucidated [37]. It is noteworthy that the anti-cancer properties of DHT are also associated with mitochondrial regulation and STAT3 phosphorylation, further supporting its potential involvement in cardioprotection via this pathway [38].
In both animal models and primary rat cardiomyocyte experiments, DHT treatment significantly suppressed SHP1 protein expression, restored JAK2/STAT3 signaling transduction, preserved mitochondrial function, reduced cellular apoptosis, diminished infarct size, and improved cardiac function. Mechanistically, we discovered that DHT promotes the degradation of SHP1 protein via the ubiquitin–proteasome pathway without affecting its transcription (Fig. 7). Importantly, overexpression of SHP1 completely abolished the beneficial effects of DHT, confirming that SHP1 downregulation is a central event in its cardioprotective mechanism.Fig. 7. The potential mechanism of YXS by which it regulates myocardial apoptosis and mitochondrial homeostasis
It is noteworthy that SHP1 overexpression in normal cardiomyocytes did not induce apoptosis, suggesting its function may be context-dependent and differ between physiological and pathological states. Furthermore, while this study did not investigate other forms of cell death, we cannot exclude the possibility that other bioactive components of YXS may regulate alternative cell death modalities through different signaling molecules. Concurrently, the precise mechanism by which DHT promotes SHP1 degradation via the ubiquitin–proteasome pathway warrants further investigation. We hypothesize that it may involve alterations in SHP1 conformation, facilitating its interaction with E3 ubiquitin ligases.
In summary, the traditional Chinese medicine formula Yixinshu (YXS) confers cardioprotective effects in the setting of myocardial infarction by downregulating SHP1 protein expression, thereby relieving its inhibitory effect on the JAK2/STAT3 signaling pathway. This activation of JAK2 and STAT3 leads to modulation of apoptotic regulators, including upregulation of the anti-apoptotic protein BCL2 and suppression of the pro-apoptotic protein BAX, ultimately attenuating cardiomyocyte apoptosis and preserving mitochondrial homeostasis. Dihydrotanshinone I (DHT), a key bioactive constituent of YXS, was identified to directly bind SHP1 and promote its ubiquitin-dependent degradation, highlighting a critical molecular mechanism through which YXS exerts its anti-apoptotic and cardioprotective actions.
Supplementary Information
Supplementary Material 1. Fig.S1. Establishment of the Myocardial Infarction (MI) Model and Identification of Therapeutic Targets. A Representative electrocardiogram (ECG) of the sham group. B ECG showing characteristic changes following left anterior descending (LAD) coronary artery ligation. C Representative M-mode echocardiographic images from each group. D, E Quantification of left ventricular systolic function by ejection fraction (EF) and fractional shortening (FS) (n = 10). F Creatine kinase-MB (CK-MB) and G lactate dehydrogenase 1 (LDH1) levels were significantly elevated in the MI group compared to the sham group, indicating successful establishment of the myocardial infarction model (n = 10). H Herb–ingredient–target interaction network of YXS, constructed based on the intersection of YXS-associated targets and coronary heart disease-related genes. Data were shown as mean ± SD. *p < 0.05, **p < 0.01 vs. the sham group.Supplementary Material 2. Fig.S2. YXS protected myocardial mitochondrial integrity and function. Stock solutions of YXS (2 g in 4 mL DMSO) or CDDP (4 g in 4 mL DMSO) were prepared with ice-bath sonication for 5 min, filtered through a 0.22-μm filter, and applied to cells. A Density of mitochondrial cristae and maximum width of mitochondrial cristae (n = 3). B Viability of primary cardiomyocytes (n = 5). C, D Assessment of mitochondrial membrane potential by JC-10 staining (scale bar = 25 μm) (n = 3). E Base peak chromatograms (BPCs) under positive ion mode. F BPCs under negative ion mode. Data were shown as mean ± SD. *p < 0.05, **p < 0.01 vs. sham or control group; ^#^p < 0.05, ^##^p < 0.01 vs. MI or OGD groupSupplementary Material 3. Fig.S3. DHT attenuated OGD-induced injury in cardiomyocytes. A, B Cardiomyocyte metabolic activity (n = 4–6). C LDH levels in the cell culture supernatant (n = 3). D Mitochondrial membrane potential was detected by Mito-Tracker Red CMXRos assay (n = 4). E Cellular reactive oxygen species (ROS) levels (scale bar = 50 μm) (n = 3). Data were shown as mean ± SD. *p < 0.05, **p < 0.01 vs. sham or control group; ^#^p < 0.05, ^##^p < 0.01 vs. MI or OGD groupSupplementary Material 4. Fig.S4. The protective effects of DHT on H9C2. A The effect of different concentrations of SalB on OGD-induced H9C2 cardiomyocyte viability (n = 9). B The effect of different concentrations of DHT on OGD-induced H9C2 cardiomyocyte viability (n = 9). C The effect of DHT on the expression of apoptosis-related proteins BAX and Bcl-2 (n = 3–4). Data were shown as mean ± SD. *p < 0.05, **p < 0.01 vs. sham or control group; ^#^p < 0.05, ^##^p < 0.01 vs. MI or OGD groupSupplementary Material 5.