C57BL/6J male mice (aged 6 weeks and weighing 20–25 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. All mice were placed in a light cycle (half day light and dark) and a temperature of 25 ± 1°C and a humidity of 55 ± 5%, and were allowed free access to water and food. The animals used in the present study were treated in accordance with the Animal Center Guide for the Care and Use of Laboratory Animals. The experimental protocols were approved by the Animal Ethics Committee of the Laboratory Animal Center of Wenzhou Medical University (approval no. wydw2014-0058).

GSPE (purity, 95%) was purchased from Tianjin Jianfeng Natural Product R&D Co., Ltd. GAPDH (5174) was purchased from Cell Signaling Technology, Inc. Primary antibodies against phosphorylated PI3K(C73F8), AKT(C67E7), p-AKT (Ser473), Bcl-2 (D17C4), Bax (2772) and a goat anti‐rabbit secondary antibody (4412) were purchased from Cell Signaling Technology, Inc. Primary antibody against p-PI3K (ab182651) and collagen type Ⅲ polyclonal antibody (ab6310) and collagen type Ⅰ antibody (ab34710) were purchased from Abcam (United Kingdom). Anti-α-SMA (13548-1-AP) antibody were purchased from ProteinTech Group, Inc. Cell Counting Kit-8 (CCK-8), SOD and MDA detection kit were purchased from Nanjing Jiancheng Bioengineering Institute. Annexin V-FITC/PI apoptosis detection kit was purchased from Beyotime Institute of Biotechnology.

Mice were anesthetized with 1.5% isoflurane. Mice were intubated and connected to a ventilator to maintain normal breathing. Their heart was then exposed through a lateral incision along the upper edge of the third or fourth rib. The coronary artery of LAD was ligated with a 7-0 polypropylene suture, ∼2–3 mm from the lower edge of the left auricle. The chest was sutured with surgical suture. The sham operation method was the same, except for the LAD coronary artery, which was not ligated.

The mice were randomly divided into five groups, including 1) a sham-operated group (daily oral gavage by 0.2 ml 0.9% normal saline); 2) a GSPE group (daily oral gavage by 0.2 ml GSPE (200 mg/kg) from sham operation to 14 days after operation) (Shi et al., 2019); 3) an MI group (daily oral gavage by 0.2 ml 0.9% normal saline from operation to 14 days after operation); 4) an MI + GSPE-treated group (daily oral gavage by 0.2 ml GSPE (200 mg/kg) from operation to 14 days after operation); and 5) an MI + GSPE + LY group (daily oral gavage by 0.2 ml GSPE (200 mg/kg) and LY294002 (0.2 mg/mouse) from operation to 14 days after operation).

Echocardiography was performed in the laboratory by a single experienced echocardiologist. Mice were anesthetized with 1.5% isoflurane and connected to a ventilator to maintain normal breathing. Then, an M-mode transducer (Acuson Sequoia 512; Sonos) was used to performed the transthoracic echocardiography. At the papillary level, the Simpson method was used to measure several main cardiac function indexes (Johri et al., 2011), including the left ventricular ejection fraction (LVEF), the ejection fraction (FS), the left ventricular end diastolic diameter (LVIDd, mm) and the left ventricular end systolic diameter (LVIDs, mm).

The 2,3,5-triphenyltetrazolium chloride solution (TTC) staining method was used to determine the myocardial infarct size. 14 days after modeling, euthanasia was done on the mice by intraperitoneal injection of excessive pentobarbital sodium. The hearts of mice were removed quickly and separated from extra connective tissue. The heart was washed with normal saline and frozen at −80°C for 4 h. Five 1–2 mm-thick heart sections were prepared and incubated at 37°C in 2% TTC for 15 min. Next, according to the computer plane measurement, the area of the infarcted tissue was photographed with a digital camera. The infarct area was expressed as the percentage of infarcted area to the risk area x 100%.

In order to evaluate the morphological changes and degree of myocardial fibrosis 14 days after MI, the heart tissues of each group were washed with PBS and fixed with 4% paraformaldehyde solution overnight at 4°C. After the heart tissue was paraffin embedded, the heart was sliced to 5 μm-thick sections. After dehydration and dewaxing of slices, HE staining as well as Masson’s trichrome stain was performed according to the staining kit instructions, followed by observation under a fluorescence microscope (Leica Microsystems GmbH).

Frozen tissue sections were placed at room temperature for >30 min. After washing three times with PBS, the antigen was blocked with 2% BSA. The tissues were then incubated with anti-α-SMA antibody (1:200) at 4°C for 24 h. The next day, the tissues were incubated at 37°C for 60 min in the dark with a goat secondary antibody (1,500). After three washes with PBS, the tissues were re-stained with DAPI by incubation in the dark for 5 min. Images were obtained with a fluorescence microscope (Leica Microsystems GmbH) and analyzed with Image Proplus 6.0 software (Media Control Silver Spring).

After 14 days of MI, 400 µl blood was collected from the abdominal aorta of mice. The blood was centrifuged at 3,000 rpm at 4°C for 20 min, and then the supernatant was carefully collected. The SOD and MDA test kits pursed from Nanjing Jiancheng Bioengineering Institute were wsed to determine the SOD activity and MDA levels.

H9C2 cells were cultured in glucose- and serum-free DMEM without penicillin/streptomycin in a hypoxic atmosphere (95% N₂, 5% CO₂) at 37°C (An et al., 2019). The cells were then inoculated into 96-well plates, at a density of 5,000 cells per well. The activity of the cells under different periods of hypoxia and GSPE concentration were measured. The cell activity was measured with a CCK-8 detection kit.

H9C2 cells in a 6-well plate (1 × 10⁵ cells/well) were treated as described before and collected with 0.25% trypsin. The H9C2 cells (about 100 μl)) were incubated with 5 μl Annexin V-fluorescein isothiocyanate for 5 min, followed by addition of 3 μl disodium propionate iodide and incubation at room temperature in the dark for 15 min. Finally, the apoptosis rate was measured with a flow cytometer (BD AriaIII; BD Biosciences).

The animal proteins extracted from the left ventricular myocardium and the proteins in the cells were cleaved and quantified, and then quantified using the Bradford protein assay (Bio-Rad Laboratories). Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% fat-free milk at room temperature for 2 h, the membranes were incubated overnight with the corresponding primary antibody at 4°C, including anti-p-PI3K (1, 1,000 dilution), anti-AKT (1, 2,000 dilution), anti-p-AKT (1, 1,000 dilution), anti-Bcl-2 (1, 1,000 dilution), anti-Bax (1, 2,000 dilution), anti-Col-1 (1, 1,000 dilution), anti-Col-3 (1, 1,000 dilution), anti-PI3K (1, 1,000 dilution), anti-α-SMA (1, 3,000 dilution), anti-tubulin (1:5,000 dilution) and anti-GAPDH (1:10,000 dilution). After being washed with TBST, the membranes were incubated with secondary antibodies (1:5,000) for 2 h at room temperature and washed again. ChemiDoc™ XRS + System with Image Lab™ Software purchased from Bio-Rad was used to visualize the signals.

All statistical data were analyzed using GraphPad Prism 5 (GraphPad Software, Inc.). Values were expressed as the mean ± SEM. Differences between two groups were analyzed using Student’s t-test. The significance of the difference between groups were analyzed by one-way analysis of variance (ANOVA) and followed by LSD post hoc least significant difference test. A value of p <0.05 was considered statistically significant difference for all analyses.

At 14 days post-MI, all mice in the sham group survived, while the survival rate was 61% in the MI group and 74% in the GSPE group (Figure 1A). Compared with that of the MI group, the heart weight/body weight ratio of the GSPE treatment group was significantly lower (Figure 1B). Echocardiography showed that, compared with those in the MI group, the LVEF and FS of the GSPE treatment group were significantly increased, while LVIDd and LVIDs were significantly decreased (Figure 1C).

TTC staining showed that the infarct size of the GSPE group was significantly smaller than that of the MI group (Figure 2A). The MDA level in the MI group increased, while GSPE treatment could significantly reduce the increase in MDA. On the contrary, the serum SOD level in the MI group decreased, and GSPE treatment could significantly increase the SOD value (Figure 2B). Masson’s trichrome stain showed that the collagen deposition area of myocardial fibrosis of the MI group was significantly higher than that of MI + GSPE group (Figure 2C). H&E staining showed that myocardial necrosis, inflammatory infiltration and interstitial edema were decreased in the MI + GSPE group compared with those in the MI group (Figure 2C).

The Bcl-2 protein family is associated with the regulation of cell apoptosis. In this family, Bcl-2 plays an anti-apoptotic role, while Bax plays a pro-apoptotic role (Bogner et al., 2020). GSPE significantly increased the expression of Bcl-2 and decreased the expression of Bax in myocardial tissue after MI (Figure 3A). In addition, GSPE reduced the expression of collagen Ⅰ, collagen Ⅲ and a-SMA (Figure 3B). Furthermore, immunofluorescence showed that the integral optical density of a-SMA in the GSPE treatment group was significantly lower than that in the MI group (Figure 3C).

The PI3K/AKT pathway is one of the important signaling pathways in cells, which participates in the regulation of multiple signaling molecules (Fajgenbaum et al., 2019). Western blotting revealed that p-PI3K and p-AKT expression increasing was detected in the MI group, and GSPE treatment enhanced this activation (Figure 3D).

Echocardiography showed that, compared with those in the MI + GSPE group, the LVEF and FS of the LY294002 treatment group were significantly decreased, while LVIDd and LVIDs were significantly increased (Figure 4A). Masson’s trichrome stain also showed that the collagen deposition area of myocardial fibrosis of the MI + GSPE + LY294002 group was significantly higher than that of MI + GSPE group (Figure 4B). H&E staining showed that myocardial necrosis, inflammatory infiltration and interstitial edema were increased in the MI + GSPE + LY294002 group compared with those in the MI + GSPE group (Figure 4B).

After administration of the PI3K inhibitor LY294002, the p-PI3K and p-AKT levels in the LY294002-treated group were lower than those in the GSPE group after MI (Figure 5A). Compared with those in the GSPE group, LY294002 increased the level of Bax protein, decreased Bcl-2 levels (Figure 5B), and increased collagen Ⅰ, collagen Ⅲ and α-SMA levels (Figure 5C). In other words, LY294002 significantly increased apoptosis and fibrosis compared with the results of the MI + GSPE group. Furthermore, immunofluorescence showed that the integral optical density of α-SMA in the LY294002 treatment group was higher than that of the MI + GSPE group (Figure 5D).

The time-cell activity experiment showed that the most suitable hypoxia time for H9C2 cell apoptosis was ∼24 h (Figure 6A). The dose response experiment showed that the optimal concentration of GSPE was 40 μg/ml (Figure 5A). Western blotting showed that the increasing of Bax under hypoxia, and GSPE treatment significantly decreased the level of Bax (Figure 6B). Furthermore, GSPE treatment increased the expression of Bcl-2 (Figure 6B). In addition, flow cytometry also showed that GSPE could reduce the apoptosis of H9C2 cells under glucose and oxygen deprivation (Figure 6C).

In vitro, western blotting showed that the levels of p-PI3K and p-AKT in the MI + GSPE group were higher than those in the hypoxia group, indicating that GSPE can enhance the activation of the PI3K/AKT pathway in H9c2 cells under hypoxia (Figure 6D).

After administration of the PI3K inhibitor LY294002, the p-PI3K and p-AKT levels in the LY294002 treatment group were lower than those in the GSPE group under OGD (Figure 7A). Compared with those in the GSPE treatment group, the level of Bax increased and the level of Bcl-2 decreased in the LY294002 group (Figure 7B). In addition, flow cytometry showed the same results (Figure 7C). This suggests that GSPE plays an anti-apoptotic role via the PI3K/AKT pathway in vitro, which was corroborated by flow cytometry.