Ethic statement

The Animal Experimentation Ethics Committee of Zhejiang Eyong Pharmaceutical Research and Development Centre approved the ethical conduct of animal experiments (SYXK(Zhe) 2023–0027).

Animals

30 male SD rats weighing 200–230 g (6–8 weeks) were obtained from Shanghai Jihui Laboratory Animal Care Co, Ltd. (License number: SCXK (Hu) 2022–0009), and subsequently accommodated in at Zhejiang Eyong Pharmaceutical R&D Co., Ltd (license number: SYXK (Zhe) 2023–0027). The rearing temperature for these rats was maintained at 22–25 °C, with a relative humidity of 55% and exposed to a 12 h light/dark cycle. All rats had access to both food and water at all times. Prior to administration, acclimatization of rats for a week was conducted.

Experiment design

The rats were randomly assigned to five groups with random number table approach (n = 6): Control (Con), IR, IR + ISO Low-dose-treated (ISO-10) (10 mg/kg), IR + ISO Middle-dose-treated (ISO-20) (20 mg/kg), and IR + ISO High-dose-treated (ISO-40) (40 mg/kg) groups on the basis of previous research [19]. All rats were given daily gavage administration for 30 consecutive days and then the establishment of IR model was conducted.

IR rat modeling

The procedure of IR modeling method in our study was carried out by referring to the method previously described [20]. After anesthetizing rats with 1.5% isoflurane, the left anterior descending (LAD) coronary artery was blocked by ligation for 30 min, and then the sutures were loosened for 2 h to establish IR rat model. We observed the presence of significant pallor in the left ventricle, in conjunction with ischemia achieved by ST-segment elevation on electrocardiographic monitoring. In Control group, the same operation was performed on rats without LAD coronary artery ligation. All rats were successfully modeled to complete subsequent experiments, with no death occurred.

Cardiac function measurement

Cardiac function was assayed 24 h after successful IR rat modeling. After rats in each group were anesthetized with 1.5% isoflurane by inhalation, their cardiac function was evaluated by a VEVO 770 High-Resolution Imaging System (Visual Sonics, Canada). The left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), ejection fraction (EF), fractional shortening (FS), left ventricular peak systolic pressure (LVSP), and left ventricular end diastolic pressure (LVEDP) measurements in all rats were recorded using two-dimensional, M-Mode echocardiography.

Sample collection

After detecting cardiac function of all rats and confirming that they were completely under anesthesia, we cut their abdomens open, and approximately collected 3 mL blood from the main abdominal vein. Subsequently, with centrifugation at 3500 rpm for 15 min, the supernatant was collected and utilized to determine creatine kinase-MB (CK-MB), cardiac troponin-I (cTnI), and cardiac troponin-T (cTnT) expression with ELISA kits afterwards. The rats were euthanized following blood sampling by inhalation of compressed CO2 (30% V/min) in cylinders. And then the hearts were rapidly isolated, with apical 1/3 to 2/3 of hearts prepared as paraffin sections for HE staining to observe inflammatory cell infiltration. The apical 1/3 of hearts was frozen at -80 °C for subsequent Western blot assay.

HE staining

The apical 1/3 to 2/3 of rat hearts was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin (H3136, sigma) and eosin (E4009, sigma) to assess histopathological changes in border areas of rat cardiac tissues. The assessment was performed by individuals not involved in experimental process.

Masson trichrome staining

Following deparaffinization of paraffin sections, Weigert iron hematoxylin staining for 10 min and acidic compound red-Lichun red solution (71,019,360, 71,033,761, Sinopharm Chemical Reagent Co.,Ltd) staining for 5–10 min were sequentially performed. Subsequently, phosphomolybdic acid solution (XW514297441, Beijing Wokai Biotech Co.) staining for 3–5 min and aniline blue solution (71,003,644, Sinopharm Chemical Reagent Co.,Ltd) staining for 1–5 min were done. Then, the dehydration, placing in xylene for soaking, sealing and observation were made. Under the microscope, the cell nucleus exhibited blue-black, with collagen fibers and mucus blue, and myofibrils, fibrils and erythrocytes orange-red.

TTC staining

The perfusion was performed for collection of heart weighed and sectioned in 6 segments (1–2 mm thick). The samples were stained using 1% TTC solution (F603BA0025, Shenggong Biotechnology Engineering Co.) at 37 °C for 15 min. Photography was taken following 4% paraformaldehyde fixation for 30 min. The areas of non-infarcted area (red) and infarcted area (white) were analyzed and calculated. Infarcted areas of cardiac tissues (%) = infarcted areas of the heart/total areas of the heart × 100%

ELISA

The supernatant was taken and we followed instruction steps of the rat CK-MB ELISA kit (CB10461-Ra, COIBO), rat cTn-I ELISA kit (RX302234R, RuiXin), rat cTnT ELISA kit (RX301216R, RuiXin), Total-superoxide dismutase (T-SOD) kit (A001-1, NanJing JianCheng Bioengineering Institute), malondialdehyde (MDA) kit (A003-1, NanJing JianCheng Bioengineering Institute), and glutathione (GSH) kit (A006-2–1, NanJing JianCheng Bioengineering Institute) to estimate contents of CK-MB, cTnI, cTnT, T-SOD, MDA, and GSH levels. The absorbance values were calculated at 450 nm.

Cell culture, treatment and grouping

Rat cardiomyocytes H9c2 cells (iCell-r012, iCell Company) were cultured with reference to method previously described [21]. The logarithmically grown H9c2 cells were randomly classified into five groups: Control (Con), IR model (IR), IR + 1 μmol/L ISO (IR + ISO-1), IR + 10 μmol/L ISO (IR + ISO-10), and IR + 100 μmol/L ISO (IR + ISO-100) groups on the basis of previous research [11]. Cells in Control group were cultured in normal conditions (21% O2, 5% CO2, 37 °C) without any treatment. Following 8 h incubation in serum-free medium containing corresponding concentration of ISO, H9c2 cells of IR + ISO-1, IR + ISO-10, and IR + ISO-100 groups underwent modeling processes.

We followed previously described protocols to establish myocardial IR model in H9c2 cells [21]. To simulate ischemia, after rinsing cells three times with PBS, they were cultured in glucose-free DMEM at 37 ℃ under light protection and hypoxic conditions (94% N2, 5% CO2, and 1% O2) for 4 h. Subsequently, reperfusion was simulated by replacing the glucose-free DMEM with complete DMEM containing 4.5 mg/mL glucose). The cells were incubated in a 37 °C incubator with 95% air and 5% CO2. Cell samples were obtained after reperfusion period of 24 h.

Cell counting kit-8 (CCK-8)

H9c2 cells were seeded into 96-well plates and cultured for 48 h, followed by adding CCK8 solution (10 μL) to each well and incubating at 37 °C for 4 h. The absorbance was calculated by a microplate spectrophotometer at 450 nm.

Flow cytometry (FC)

To determine the apoptosis rate of H9c2 cells, FC assay was conducted in accordance with instructions of Apoptosis Kit (556,547, BD). The collection of H9c2 cells was done at a concentration of 1 × 106 cells/mL, and addition of 500 μL of binding buffer, centrifugation, and subsequent addition 100 μL of binding buffer was performed. The reaction was carried out at room temperature, avoiding light for 15 min, following addition of 5 μL Annexin V-FITC and 10 μL PI. The final 400 μL of binding buffer was added, and detection of apoptosis rate was made by FC assay within 1 h.

Transmission electron microscopy (TEM)

The collected cells were fixed in 2.5% glutaraldehyde solution for 4 h, rinsed, and then fixed in 1% osmium acid solution for 1–2 h. Following dehydration treatment with gradient concentrations of ethanol solution, H9c2 cells were embedded, sectioned (50–70 nm thickness), and subsequently completed with uranyl acetate and lead citrate staining. Samples were placed under transmission electron microscope to view ultra-structural changes.

Immunofluorescence

The H9c2 cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with serum. Cells were incubated overnight at 4 °C with primary antibody (AF5402, Affinity), and then Goat-Anti-Rabbit lgG (Alexa FLIOR 488) (ab150077, Abcam) was added with incubation at room temperature for 1 h. After counter-staining of cell nuclei with DAPI, observation was performed with a fluorescence microscope (Ts2-FC, Nikon).

Western blot

After protein extraction of rat myocardial tissues in infarct and non-infarct areas and H9c2 cells, quantification was accomplished with a BCA kit (pc0020, Beyotime). With separation by 10% SDS-PAGE gel electrophoresis, protein transferring to PVDF membranes (10,600,023, GE Healthcare Life) was completed. Primary antibodies (Table 1) were incubated at 4 °C overnight, followed by block with 5% milk. Subsequent secondary antibodies (Table 1) incubation was performed. Protein bands were detected by chemiluminescence (610,020-9Q, Clinx) and band analysis was done by ImageJ.

Table 1 Antibody informationStatistical analysis

SPSS 20.0 statistical software was used to analyze these data. The one-way-ANOVA was employed when measurement information between multiple groups met the normal distribution and Chi-square test, and further two-by-two comparisons between groups were performed by Turkey test. The analysis of data that did not conform to normal distribution was done by Kruskal–Wallis H-test. All data were expressed as mean ± standard deviation. The level of significance was α = 0.05, with P < 0.05 considered statistically significant.