All animal experiment procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals established by the National Institutes of Health, as well as with national standards set forth by the People’s Republic of China regarding ethical review for laboratory animal welfare. All protocols related to animal experimentation received approval from the Animal Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine (Number: 2019070). Male Sprague-Dawley (SD) rats weighing approximately 280–300 g (SPF grade) were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China), which were randomly assigned into four groups: sham operation group (Sham), I/R model group (I/R), low-dose DQ group (DQ-L), and high-dose DQ group (DQ-H). Rats in both DQ group received intragastric administration at doses of 0.6 g/kg for DQ-L or 1.2 g/kg for DQ-H daily for 5 days prior to surgery according to the previous study (Ma et al., 2019; Ma et al., 2022). The I/R model was induced through ligation of the left anterior descending (LAD) coronary artery. Following thoracotomy to expose the heart, a 7–0 silk suture was employed to ligate the LAD approximately 2–3 mm below the tip of the left atrial appendage. After a period of 30 minutes, the ligature was loosened to restore the blood supply, after which chest closure occurred once reperfusion had been successfully reinstated. Animals in the Sham group underwent identical surgical procedures without LAD ligation. All samples were collected 24 h after reperfusion.

DQ is composed of 50% Panax notoginseng (Burk.) F. H. Chen [Araliaceae, Notoginseng radix et rhizome] and 50% Salvia miltiorrhiza Bunge [Lamiaceae; Salviae miltiorrhizae radix et rhizoma]. All medicinal materials are sourced from authentic suppliers by China Meheco Great Wall Pharmaceutical Co., Ltd. (Beijing, China) and are identified according to thin-layer chromatography (TLC) as outlined in Appendix VI B of the 2005 edition of the Chinese Pharmacopoeia. Detailed information regarding these two botanical drugs can be found in Supplementary Table 1. The supplementary data provides a comprehensive description of the extraction process and ultra-high performance liquid chromatography (UPLC) analysis of DQ.

The TTC-Evans blue staining technique was utilized to assess myocardial injury induced by I/R. Following a reperfusion period of 24 h, animals were anesthetized, and thoracotomy was performed to expose the heart. After opening the abdominal cavity, a 4% Evans blue staining solution was administered by intravenous injection. Subsequently, the heart was quickly cut off and stored at −20°C. Starting from the apex, the heart was transversely sliced into five to six equal portions of uniformly thick slices. These slices were immersed in a 2% TTC solution dissolved in PBS at 37°C for 15 min before being fixed in paraformaldehyde solution at room temperature. Determine the ischemic risk area (AAR) by the absence of blue dye. The total left ventricular area (LV), AAR, and TTC-negative staining area representing infarcted myocardium were quantified using ImageJ software.

Heart specimens were fixed with paraformaldehyde, embedded in paraffin wax, sectioned into thin slices measuring 5 μm each, and mounted onto glass slides for HE staining procedures. Observations and imaging were conducted under an optical microscope (Olympus, Japan). The pathology score of each heart slices is as follows: (0) nil; (1) minimum (focal myocytes damage); (2) mild (small multifocal degeneration with slight degree of inflammatory process, the order of myocardial fibers is occasionally disordered); (3) moderate (extensive myofibrillar degeneration and/or diffuse inflammatory process, the cells are moderately damaged, Myocardial fibers are deformed in a wave shape, and the nucleus is shed); (4) severe (necrosis with diffuse inflammatory process: The cell is severely damaged, the nucleus shrinks, and myocardial necrosis is accompanied by diffuse inflammation). Four different visual fields were selected for each slide, and the average of the histopathological scores of the four visual fields was calculated to obtain the histopathological score of the slide, the analysis was conducted in a blinded way (Yin et al., 2020).

TUNEL assay kit (G1504, Servicebio) was used to detect the apoptosis of cardiac tissue. Paraffin-embedded heart tissue sections were prepared. After dewaxing and membrane breaking, DAPI staining, incubation and mounting were performed according to the kit instructions. Microscopic images were captured using an Olympus microscope (Japan). The apoptosis rate was calculated by ImageJ software to calculate the total number of apoptotic nuclei and nuclei in the field of view. Cardiomyocyte apoptosis rate = number of apoptotic nuclei/total number of nuclei × 100%.

The rat heart was fixed with electron microscope solution, and samples measuring approximately 1 mm³ were taken within 1–3 min. The myocardial tissue was immersed in the electron microscopy solution for fixation and stored at 4°C. Following a series of procedures including rinsing, fixing, dehydration, embedding, polymerization, positioning, and solidification, ultra-thin sections with a thickness of 60–80 nm were prepared. These sections were stained with a saturated alcohol solution of 2% uranium acetate for 8 min in darkness. After washing three times with 70% alcohol and ultrapure water, the sections were treated with a light-protected solution of 2.6% lead citrate for another 8 min. They were then washed three times with ultrapure water before being blotted dry on filter paper. Finally, the copper mesh slices were placed into a copper box and allowed to air-dry at room temperature overnight. The mitochondrial ultrastructure was observed using a Hitachi transmission electron microscope (Japan), and images were collected. The total number of mitochondria was counted by ImageJ software, and further divided it into three intervals according to the mitochondrial area: <0.5 μm², 0.5–1 μm², >1 μm². The proportion of mitochondria in these three intervals was calculated, and the number of mitochondria in these three area intervals was statistically analyzed (Sun et al., 2022).

H9c2 cardiomyocytes, obtained from Meisen Chinese Tissue Culture Collections (China), were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. The cells were maintained in a humidity cell incubator at 37°C with 5% CO₂. The culture medium was replaced every two to 3 days. The cardiomyocytes were divided into four experimental groups: control group (Con), H₂O₂ group (H₂O₂), low-dose DQ group (DQ-L) and high-dose DQ group (DQ-H). For cell experiments, the water-soluble components of DQ were utilized. Prior to modeling, the cardiomyocytes underwent pretreatment with DQ for a duration of 2.5 h.

Cell viability was assessed using the methyl thiazolyl tetrazolium (MTT) assay. Cells were seeded into 96-well plates at a density of 5 × 10³ cells per well and cultured at 37°C. Following various treatments as described above, the cells were washed twice with phosphate-buffered saline (PBS) before adding MTT (Sigma Aldrich,Unites States) to each well at a final concentration of 0.5 mg/mL for 4 h incubation. Subsequently, 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals at room temperature for 10 min. The optical density (OD) value of each well was recorded at a wavelength of 570 nm using a microplate reader (Thermo Scientific, Unites States).

H9c2 cells were cultured in 6-well plates at a density of 2.5 × 10⁵ cells per well. Following treatment, the apoptosis rate was analyzed using the Annexin V-FITC apoptosis detection kit (Beyotime, China) according to the manufacturer’s instruction. The mitochondrial membrane potential was measured with JC-1 (Beyotime, China), which serves as a specific probe following the manufacturer’s protocol. The opening degree of the mitochondrial permeability transition pore (mPTP) was evaluated using a mPTP detection kit (Beyotime, China), employing Calcein AM as a fluorescent precursor compound according to manufacturer guidelines. Additionally, intracellular reactive oxygen species (ROS) levels were determined utilizing a ROS detection kit (Beyotime, China), adhering strictly to the manufacturer’s instructions. All fluorescence intensities were quantified with a flow cytometer (Agilent, Unites States).

The activity levels of lactate dehydrogenase (LDH; Sigma-Aldrich, Unites States) and cardiac troponin T (cTnT; Elabscience, China) in serum were evaluated according to the protocols provided by their respective manufacturers. Malondialdehyde (MDA; Beyotime, China), total glutathione (GSH; Beyotime, China), and glutathione peroxidase activity levels (GPx; Beyotime, China) were measured according to provided instructions. Furthermore, Caspase-3 activity, Caspase-8 activity and Caspase-9 activity assays were conducted based on manufacturers’ guidelines from Beyotime Biotechnology Co., Ltd., China.

According to the Mito-Tracker Deep Red FM (Beyotime, China) manual, cells were inoculated in a cell culture plate. Following treatment with drugs, the cell medium was removed. The prepared working solution of Mito-Tracker Deep Red FM was added and incubated at 37°C for 20 min. After staining, the cells were washed with PBS and fixed using fresh 3.7% formaldehyde for 15 min. Post-fixation, the cells underwent two washes with PBS before being incubated in 0.2% Triton X-100 in PBS at room temperature for 5 min, followed by another wash with PBS. A small volume of Hoechst 33342 staining solution was added and allowed to place at room temperature for 5 min. Subsequently, the slides were washed three times with PBS prior to sealing and observation under laser confocal microscopy.

Mitochondrial respiration is assessed by oxygen consumption rate, which is conducted using a Seahorse XFe24 Analyzer (Agilent, Unites States) following the manufacturer’s instructions. The final results are analyzed utilizing Seahorse software.

Tissue or cells was lysed using RIPA buffer supplemented with a protease inhibitor cocktail (Roche, Unites States). Protein concentration was determined employing the Pierce BCA Protein Assay Kit (Beyotime, China). Low molecular weight markers (CST, Unites States) and protein samples were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto a polyvinylidene fluoride membrane and blocked at room temperature for 1.5 h in Tris buffer containing 5% BSA. Membranes were incubated overnight at 4°C with antibodies against Bcl-2 (Abcam, UK; dilution: 1:1000), Bax (CST, Unites States; dilution: 1:1000), Caspase-3 (Affinity, China; dilution: 1:1000), Cleaved-Caspase-3 (CST, Unites States; dilution: 1:1000), Phospho-Drp-1(Ser616) (Affinity, China; dilution: 1:1000), CaMKII (Abmart, China; dilution: 1:1000), Phospho-CaMKⅡ Antibody (CST, Unites States; dilution: 1:1000), Cytochrome c (Biovision, Unites States; dilution: 1:200), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (CST, Unites States; dilution: 1:5000). The secondary antibodies (CST, Unites States) utilized in this study are labeled with horseradish peroxidase and diluted at a ratio of 1:3000, followed by incubation at room temperature for 1.5 h. The immunoblot results were visualized using E-blot Plus. The relative band density of proteins in the protein immunoblot hybridization was normalized to GAPDH as an internal reference. By normalizing the data to the control value, the final results are expressed as fold changes.

Statistical analysis and plotting were conducted using GraphPad Prism version 8.0.2 (263) (GraphPad Software, Unites States) and SPSS version 23.0 (IBM Corporation, Unites States). Data are presented as mean ± SEM. One-way ANOVA and post hoc Dunnett’s tests were applied in the independent variable with more than two groups. A difference was deemed statistically significant when p < 0.05.

To assess the cardioprotective effect of DQ on I/R injury, we evaluated the infarct area through TTC-Evans blue staining (Figure 1A). Compared with I/R group, the ratios of infarct area to the area at risk (AAR) were significantly reduced in DQ-L and DQ-H groups (Figure 1B). The ratios of AAR to the total ventricular area (LV) in all groups had no significant difference (Figure 1C). Additionally, myocardial enzyme levels of cTnT and LDH in both DQ-L and DQ-H groups showed significant decreases compared with those observed in I/R rats (Figures 1D, E). We further examined alterations in heart tissue pathology via HE staining; results indicated that cardiomyocyte membranes appeared clear and intact within the Sham group, with muscle fibers arranged neatly without signs of inflammatory cell infiltration. Conversely, the myocardial fibers were irregularly arranged, the structure was disordered, extensive cardiomyocyte lysis, necrosis, and neutrophil infiltration were evident within the I/R group tissues; however, pretreatment with DQ significantly reversed these pathological changes (Figure 1F). DQ showed a significant improvement in myocardial histopathological lesions (Figure 1G). Compared with sham group, the histopathological scores of I/R group was significantly increased. Compared with the I/R group, the histopathological scores of DQ-L group, DQ-H group were significantly reduced. Collectively, these findings suggest that DQ effectively mitigates myocardial I/R injury in vivo.

To investigate the role of DQ in mitigating cardiomyocyte apoptosis during myocardial I/R injury, we performed TUNEL staining to evaluate cardiomyocyte apoptosis in a rat model (Figure 2A). The I/R group exhibited a significant increase in the number of apoptotic cells compared to the Sham group (Figure 2B). Western blot analysis demonstrated that Bcl-2 protein levels were significantly reduced, while Bax protein levels were markedly elevated in the I/R group relative to those observed in the Sham group (Figures 2C–E). Treatment with DQ led to an upregulation of Bcl-2 expression and a downregulation of Bax expression (Figures 2C–E). Additionally, Cleaved-Caspase three levels were significantly higher in the I/R group compared to those found in the Sham group; however, these levels decreased following treatment with DQ (Figure 2F). Collectively, these findings suggest that DQ effectively reduces cardiomyocyte apoptosis associated with myocardial I/R injury.

To investigate the protective mechanism by which DQ exerts its effects against I/R injury, we focused on mitochondrial dynamics. We examined morphological changes of mitochondria using transmission electron microscopy. As illustrated in Figure 3A, myofilaments within the Sham group exhibited neat and regular arrangements and mitochondrial morphology was normal. Conversely, within the I/R group, myofilaments broke, dissolved and disappeared. The mitochondrial cristae appeared irregularly shaped or fragmented. The cristae space was enlarged. In contrast, both DQ-L and DQ-H pretreatment groups demonstrated more regular myofilaments and cristae structure, accompanied by an increase in the number of larger mitochondria (Figure 3B). To further investigate the mechanism by which DQ regulates mitochondrial fission, we assessed the levels of fission-related proteins Drp-1 and its upstream regulator CaMKII using Western blotting (Figure 3C). The expression levels of p-Drp-1 and p-CaMKII in the I/R group were found to be elevated compared to those in the Sham group, while these levels significantly decreased following DQ treatment (Figures 3D, E). These findings suggest that DQ may mitigate mitochondrial damage by inhibiting Drp-1 phosphorylation and thereby reducing CaMKII activation to protect against myocardial I/R injury.

To investigate the protective effect of DQ on cardiomyocyte damage, we focused on the cellular apoptosis in a cell model of oxidative stress injury induced by H₂O₂ (Figure 4A). The MTT assay was employed to assay the survival rate of cardiomyocytes, revealing a gradual decrease in cell viability with increasing concentrations of H₂O₂ (Figure 4B). We selected 400 μM as the modeling concentration for subsequent experiments (Figure 4B). Furthermore, we treated H9c2 cells with different concentrations of DQ, and found that DQ (150–600 mg/mL) exhibited no cytotoxicity towards H9c2 cells (Figure 4C). Subsequently, we exposed H9c2 cells injured by H₂O₂ to different concentrations of DQ; results indicated that DQ significantly enhanced the viability of these injured cells (Figure 4D). The optimal concentrations were designated as the DQ-L group (300 mg/mL) and the DQ-H group (600 mg/mL, Figure 4D). Flow cytometry analysis revealed a significant increase in apoptosis rate within the H₂O₂ group compared to controls; conversely, both the DQ-L and DQ-H groups demonstrated markedly reduced apoptosis rates relative to the H₂O₂ group (Figures 4E, F). Additionally, Western blotting results indicated that exposure to H₂O₂ led to decreased expression levels of Bcl-2 and increased expression levels of Bax, Cleaved-Caspase-3, and Cytochrome C in cardiomyocytes when compared with control groups (Figures 4G–K). In contrast, treatment with DQ resulted in elevated Bcl-2 expression while decreasing Bax, Cleaved-Caspase-3, and Cytochrome C levels (Figures 4G–K). We also assessed enzyme activities for Caspase-3, Caspase-9, and Caspase-8 within these cells (Figures 4L–N). Compared to control groups, activities for all three caspases were significantly elevated in the H₂O₂-treated group; however, treatment with DQ notably reduced these enzymatic activities. In summary, our findings demonstrate that DQ effectively mitigates cardiomyocyte apoptosis induced by H₂O₂.

To assess the protective effect of DQ on mitochondrial damage in cardiomyocytes caused by H₂O₂, we conducted mito-tracker immunofluorescent staining, JC-1 assays, mPTP analysis and ROS detection. The immunofluorescence staining revealed a distinct and rod-shaped mitochondrial grid structure was obvious and in the Con group (Figure 5A). In contrast, the H₂O₂ group exhibited significant mitochondrial damage characterized by the loss of grid structure and fragmentation. However, both the DQ-L and DQ-H groups effectively reversed these alterations; following DQ treatment, there was a notable reduction in punctate mitochondria alongside a significant increase in rod-shaped mitochondria (Figure 5B). Compared to the Con group, the membrane potential was significantly reduced in the H₂O₂ group; however, treatment with DQ-L and DQ-H effectively restored this parameter (Figures 5C, D). The fluorescence intensity of mitochondrial calcein AM was markedly decreased in the H₂O₂ group relative to the Con group, indicating an enhancement in mPTP opening following H₂O₂ treatment. Notably, pretreatment with DQ resulted in a significant increase in fluorescence intensity compared to the H₂O₂ group, suggesting that DQ inhibited H₂O₂-induced mPTP opening in H9c2 cells (Figure 5E). Furthermore, we measured intracellular ROS production and found that ROS release was markedly elevated in the H₂O₂ group compared to controls. Notably, ROS generation after treatment with either DQ-L or DQ-H was significantly lower than that observed in the H₂O₂ group (Figure 5F). Collectively, these data indicate that DQ can mitigate mitochondrial damage inflicted upon cardiomyocytes by H₂O₂.

The oxygen consumption rates (OCRs) of H9c2 cells were assessed to evaluate mitochondrial function. A visual representation of real-time OCR measurements for evaluating mitochondrial function was obtained using the Seahorse XFe system (Figure 6A). Compared to the H₂O₂ group, both basal respiration OCR and maximal OCR were significantly enhanced in the DQ-treated groups, indicating that DQ treatment promotes higher respiratory efficiency under normal physiological conditions as well as stress conditions (Figures 6B, C). Additionally, mitochondrial ATP production within the DQ-treated groups also demonstrated a significant increase (Figure 6D).

Compared to the Con group, the MDA level in the H₂O₂ group was significantly elevated, and the GSH and GPx levels were markedly decreased. The DQ treatment effectively reversed these alterations (Figures 7A–C). These finding indicate that DQ can alleviate oxidative stress damage in cardiomyocytes caused by H₂O₂.

The expression levels of the mitochondrial fission protein Drp-1 and its upstream regulator CaMKII were assessed using Western blotting (Figure 8A). The results demonstrated that compared with the Con group, both p-Drp-1 (ser616) and p-CaMKⅡ levels were significantly increased in the H₂O₂ group; however, treatment with DQ group led to a reduction in the expression of p-Drp-1 and p-CaMKⅡ (Figures 8B, C). Based on these experiments, we conclude that DQ diminishes mitochondrial fission in cardiomyocytes caused by H₂O₂, thereby providing protective effects for these cells.