HSYA powder (95-99.0% purity, determined by high performance liquid chromatography) was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Sodium pentobarbital was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Cardiac troponin I (cTnI; cat. no. ml059498) and interleukin-6 (IL-6; cat. no. ml064292) ELISA kits were obtained from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). The superoxide dismutase (SOD; cat. no. S0101), lactate dehydrogenase (LDH; cat. no. C0016) and malondialdehyde (MDA; cat. no. S0131) kits were purchased from Beyotime Institute of Biotechnology (Haimen, China). Dulbecco's modified Eagle's medium (DMEM) and other cell culture reagents were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) were obtained from Sigma-Aldrich; Merck KGaA. The specific inhibitors of JAK2 and STAT1 (named AG490 and S1491, respectively) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). The antibodies used were purchased from Abcam (Cambridge, MA, USA) and Cell Signaling Technology, Inc. (CST, Inc.; Danvers, MA, USA), and were as follows: Anti-cleaved caspase (cat. no. ab49822; dilution, 1:500; Abcam), anti-Fas (cat. no. ab82419; dilution, 1:1,000; Abcam), anti-FasL (cat. no. ab15285; dilution, 1:500; Abcam), anti-Bcl-2 (cat. no. ab59348; dilution, 1:1,000; Abcam), anti-Bcl-2-associated X protein (Bax; cat. no. ab32503; 1:3,000; Abcam), anti- phosphorylated (p)-JAK2 (cat. no. 4406; dilution, 1:1,000; CST, Inc.), anti-p-STAT1 (cat. no. 7649; dilution, 1:1,000; CST, Inc.), anti-β-actin (cat. no. ab8226; dilution, 1:5,000; Abcam), anti-GAPDH (cat. no. ab9385; dilution, 1:1,000; Abcam) and horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. no. ab205718; dilution, 1:5,000; Abcam).

The present study was performed, according to the Guide for the Care and Use of Laboratory Animals, 8th Edition (27) and approved by the Animal Subjects Committee of the Affiliated Hospital of Hangzhou Normal University (Hangzhou, China). The experiments were performed on 40 healthy adult male Sprague-Dawley rats (weighing 250±20 g; 2 months old) obtained from Guangdong Medical Laboratory Animal Center (Foshan, China). The animals were housed in a pathogen-free environment and maintained following standard laboratory animal feeding protocols. The animals had ad libitum access to food and water in a light/dark cycle (12/12 h). The animals were given 2 weeks to acclimate prior to the experiments. The local myocardial I/R model was constructed as follows: The animals were anesthetized by intraperitoneal administration of sodium pentobarbital (60 mg/kg); ligation of the anterior descending thoracic branch of the coronary artery was performed for 30 min prior to 2 h of perfusion. The animals were randomly divided into four groups, which were: Control (control), which comprised rats without I/R treatment; the Sham operated group (sham), in which open heart surgery was performed but the anterior descending branch of the coronary artery was not ligated; the I/R group (I/R), which included rats that underwent I/R treatment; and the I/R+HSYA group (I/R+H), which included rats that underwent I/R treatment, but 5 mg/kg HSYA was intraperitoneally injected 30 min prior to ischemia. As previously described and following our preliminary experiments, concentration of HSYA was determined (28,29). Following perfusion, the anesthetized animals were prepared for subsequent experiments. Finally, the animals were sacrificed by intraperitoneal injection of sodium pentobarbital (200 mg/kg). Mortality was verified by lack of visible signs of respiration and measurable heartbeat.

H9c2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM medium containing fetal bovine serum (10%) and streptomycin/penicillin (1%) in an incubator with 5% CO₂. The cells were seeded at a density of 2.5×10³ cell/well. The cells were randomly grouped as follows: i) Control group (control); ii) the hypoxia/reoxygenation group (H/R); iii) the H/R +20 µM HSYA group (H/R + H); iv) the H/R +10 µM AG490 group (H/R + A); v) the H/R +10 µM AG490 +20 µM HSYA (H/R + H + A) group; and vi) the H/R +5 µM S1491 +20 µM HSYA (H/R + H + S1491) group. As previously described and following our preliminary experiments, the doses of each agent were selected (29-32). The cells were collected for the following experiments.

As described previously, the risk area and infarct size was calculated using a double-staining method with TCC and Evans blue stains (33). In brief, following perfusion, the heart was perfused with 3% Evans blue to indicate the area at risk (AAR). Then, the tissues were incubated with 2% TTC solution in the dark at 37°C for 15 min, and then were then stored in 4% paraformaldehyde overnight at 4°C to demarcate the infarct area. Areas with non-perfusion and blue staining were considered AAR, while areas with non-perfusion with TTC staining were labeled as the infarct area. The AAR and infarct area were calculated using Image-Pro Plus software V6.0 (Media Cybernetics, Inc., Rockville, MD, USA). The infarct size was measured as a percentage of the infarct area over the AAR.

Following reperfusion, blood samples in rats and cells in the culture medium were harvested. The blood and cells were then centrifuged at 3,000 × g for 15 min at room temperature to obtain sera and cell supernatant. The levels of cTnI, IL-6 and LDH release were examined to estimate myocardial damage. The levels were measured using commercially available ELISA kits according to the manufacturer's protocol. The absorbance was read on a microplate reader (Thermo Fisher Scientific, Inc.). The serum and the cell culture supernatant were also used to estimate oxidative stress markers, including the content of MDA and the activity of SOD, which were detected with commercial assay kits using fluorescence spectrophotometry (F-7100; Hitachi, Ltd., Tokyo, Japan) following the manufacturer's protocols.

A terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and flow cytometry analysis were performed to detect the apoptosis levels in heart tissue and cardiomyocytes, respectively. The heart tissue was fixed in 4% paraformaldehyde for 10 min at room temperature and embedded in paraffin. The tissue was subsequently cut into 4-5 µm sections, and the slides were incubated with TUNEL mixture reagent (Sigma-Aldrich; Merck KGaA) at 37°C for 1 h. The cells were also incubated with DAPI (1 µg/ml) for 30 min at room temperature to stain the nuclei. ProLong™ Gold Antifade Mountant (cat. no. P36930; Thermo Fisher Scientific, Inc.) was used to prevent photo-bleaching. The apoptotic cells in ≥6 fields were randomly selected. The percentage of apop-tosis was calculated as the number of TUNEL-positive cells within the total populations of cardiomyocytes. The images and the ratios were obtained using an Olympus fluorescence microscope (BX51; Olympus Corporation, Tokyo, Japan) and ImageJ software (version 2.0.0; National Institutes of Health, Bethesda, MD, USA), respectively. An Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Biosciences, San Jose, CA, USA) was used to assess cell apoptosis in vitro according to the manufacturer's protocols. The collected cells were stained with 5 µl Annexin V-FITC and 10 µl PI in the dark at room temperature for 10-15 min. Apoptosis was determined by a FACSCalibur flow cytometer (BD Biosciences) and analysis software BD CellQuest™ Pro Software version 1.2 (BD Biosciences).

The cells (5×10³ per well) were seeded in a 96-well plate and treated as described. Next, the cells were incubated with Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) reagent (10 µl) at 37°C in an incubator for 3 h. The absorbance was determined on a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm. Caspase-3 activity (cat. no. 69-21755; MSK Laboratory, Wuhan, China) was determined in the cell lysate by conducting an ELISA assay. The treated cells were collected and plated in a 96-well plate. Cell lysates were subject to protein quantitation using a Bicinchoninic Acid Protein Assay kit (Bio-Rad Laboratories, Inc.) prior to detection. Each well was incubated with substrate solution. Then, the 96-well plate was maintained at 37°C for 30 min. Finally, the reaction was terminated with a stop solution. A microplate reader was used to read the absorbance at a wavelength of 405 nm.

The alteration of MMP was measured by Rhodamine-123 (Rho-123) dye (Sigma-Aldrich; Merck KGaA). Following reperfusion, the cells were incubated with 10 µg/ml Rho-123 working buffer at 37°C for 30 min. The cells were then washed, and analyzed using a flow cytometer and BD Accuri™ C6 software (BD Biosciences). Subsequently, 2,7-dichlorofluorescein diacetate (DCFH-DA) dye (Sigma-Aldrich; Merck KGaA) was used to detect the ROS level. Briefly, prior to fixation with 4% paraformaldehyde at room temperature for 15 min, the cells were stained with 20 µM DCFH-DA at 37°C for 30 min. ROS generation was then determined at wavelength 488 nm using a fluorescence plate reader (BioTek Instruments, Inc., Winooski, VT, USA).

Cells were pretreated as described. The total RNA was isolated with TRIzol^® reagent (Thermo Fisher Scientific, Inc.). DNaseI was used to digest the genomic DNA. Reverse transcription was completed with random hexamer primers using a Moloney-Murine Leukemia Virus Enzyme (Promega Corporation, Madison, WI, USA). RT-qPCR was performed on ABI Prism 7500 using the SYBR Premix Ex Taq II (Takara Bio, Inc., Otsu, Japan). The qPCR conditions were set as follows: 10 min pretreatment at 95°C, 96°C for 15 sec, 63°C for 45 sec (35 cycles) and a final extension at 75°C for 10 min. The specific PCR primers used were as follows: Fas forward, 5′-CCAGCCACAAAGAGAGGAGA-3′; Fas reverse, 5′-AACGGTTCCTCTCAACACCT-3′; FasL forward, 5′-TGCTGTGTGACAATGCAGAG-3′, FasL reverse, 5′-GAGCCTCCTTTCTCACCCTT-3′; Bcl-2 forward, 5′-CCTGGCATCTTCTCCTTCCA-3′; Bcl-2 reverse, 5′-GGACATCTCTGCAAAGTCGC-3′; Bax forward, 5′-TGGCCTCCTTTCCTACTTCG-3′; Bax reverse, 5′-AAAATGCCTTTCCCCGTTCC-3′; β-actin forward, 5′-TCTATGAGGGTTACGCGCTC-3′; β-actin reverse, 5′-GCTGTGGTGGTGAAGCTGTA-3′. The 2^−ΔΔCq method was used to quantify gene expression levels (34).

The harvested cells were treated with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). A Bicinchoninic Acid Protein Assay kit (Bio-Rad Laboratories, Inc.) was used to assess protein concentration. Following denaturation in boiling water for 5 min, the proteins (20 µg/lane) were separated by electrophoresis on 10% SDS-PAGE gels. Then, the cells were transferred onto a polyvinylidene fluoride membrane. Subsequently, 5% skimmed milk was incubated with the membrane to block the non-specific antigens at room temperature for 2 h. The primary antibodies were then added, to interact with the target proteins, at 4°C overnight. Next, the membrane was incubated with HRP-coupled secondary antibodies at 4°C for 1 h. Blots were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) An enhanced chemiluminescence system (GE Healthcare, Chicago, IL, USA) was adopted to detect the bands. The protein gray intensity was calculated using Quantity one software (4.6.2; Bio-Rad Laboratories, Inc.).

GraphPad Prism Software 6 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. Statistical analysis was conducted by one-way analysis of variance with Dunnett's post-test comparison. Data are presented as the mean ± standard deviation from three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

Infarct size and the release of cTnI and IL-6 were measured to estimate the effect of HSYA on I/R injury. In contrast to the Sham group, animals in the I/R group exhibited a significant increase in myocardial infarct size. The infarct size was markedly decreased in the HSYA group in comparison with that in the I/R group (Fig. 1A and B). In addition, animals in the I/R group exhibited a marked increase in serum cTnI and IL-6 levels, compared with those rats in the Sham group. Notably, the treatment with HSYA was associated with a minor increase in cTnI and IL-6 levels compared with the I/R group (Fig. 2A and B). Furthermore, as a marker of tissue damage, the increased activity of LDH induced by I/R injury was decreased by the treatment with HSYA (Fig. 2C). In addition, the animals in the I/R group exhibited a noticeable increase in the content of MDA and a significant decrease in SOD activity compared with the Sham group. The administration of HSYA in animals with I/R injury exhibited cardio-protective effects by alleviating oxidative stress (Fig. 2D and E).

To estimate the effect of HSYA on apoptosis, the TUNEL-positive cardiomyocytes and the expression levels of apoptosis-associated proteins were determined in vivo. The percentage of TUNEL-positive cells was elevated in the I/R group in comparison with the Sham group. Compared with the I/R group, HSYA significantly decreased the number of TUNEL-positive cardiomyocytes (Fig. 3A and B). In addition, the expression levels of pro-apoptotic proteins, including cleaved caspase-3, Fas, FasL and Bax, were markedly induced by I/R. However, the administration of HSYA decreased their expression levels. Concomitantly, the level of anti-apoptotic protein Bcl-2 was decreased in the I/R group, but it was recovered in the I/R + HSYA group (Fig. 3C and D).

The expression levels of p-JAK2 and p-STAT1 were markedly increased in animals with I/R injury. No significant difference in the expression levels of total JAK2 and STAT1 between Sham and I/R group was identified. The administration of HSYA decreased the levels of p-JAK2 and p-STAT1 (Fig. 4A-D).

CCK-8 data indicated that compared with the control group, the cell viability was significantly inhibited following I/R injury, while pretreatment with HSYA or inhibitors of JAK2/STAT1 (AG490 or S1491) significantly rescued the cell viability (Fig. 5A). In addition, the release of cTnI and IL-6 was decreased in the H/R group compared with the control group, while such a release was decreased by the pretreatment of HSYA, AG490 or S1491 (Fig. 5B and C). The protective effect appeared more apparent in the HR + A + H or H/R + S + H group compared with the H/R + H group.

Data from the assays for MDA content and SOD activity demonstrated that I/R injury accelerated the rate of oxidative stress compared with the control. The MDA content and SOD activity were markedly decreased and recovered, respectively, in the group pretreated with HSYA. Although no significant difference was identified in the MDA content and SOD activity in H/R + S + H group when compared with those in the H/R + H group, the protective effects of HSYA were more significantly enhanced by the combined use with AG490 or S1491 (Fig. 6A and B). Loss of MMP may be associated with the disruption of redox status, which then leads to ROS-triggered oxidative stress. The results indicated that the loss of MMP was all partially alleviated in H/R + A + H, H/R + H, H/R + A and H/R + S + H groups, compared with the control group. Consistently, the ROS contents in these groups were decreased compared with those in the control group. The ROS level was significantly decreased in the H/R + A + H and H/R + S + H groups, compared with the H/R + H group. Although no significant difference was identified in the H/R + S + H group in comparison with that in the H/R + H group, the MMP level was also decreased in the H/R + A + H and H/R + S + H groups, compared with the H/R + H group (Fig. 6C-E).

To confirm the effect of HSYA on apoptosis, the levels of apoptosis were detected by flow cytometry. The results revealed that the apoptosis rate was accelerated in the H/R group, while decreased cell apoptosis was identified in the H/R + A + H, H/R + H, HR + A and H/R + S + H groups compared with the H/R group (Fig. 7A and B). Furthermore, the caspase-3 activity was decreased in the H/R + A + H, H/R + H, HR + A and H/R + S + H groups compared with the H/R group (Fig. 8A). Additionally, the expression levels of apoptosis-associated factors were evaluated. The data from the in vitro analyses were compatible with those from the in vivo experiments, which demonstrated that the expression levels of pro-apoptotic proteins (cleaved caspase-3, Fas, FasL and Bax) were decreased and anti-apoptotic protein Bcl-2 was increased in the H/R + A + H, H/R + H, H/R + A and H/R + S + H groups compared with that in the H/R group (Fig. 8B and C). Similar results were obtained with regards to the mRNA expression levels of these apoptosis-associated factors (Fig. 8D). Therefore, the treatment with AG490/S1491 enhanced the protective effect of HSYA.