The suppliers and the catalog numbers of the reagents are as follows. Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA, United States ; 22,400–089), fetal bovine serum (Life Technologies; 10,099), antibiotic-antimycotic (Life Technologies; 15,240–112), phosphate-buffered saline (PBS) (Life Technologies; 10010-049, pH7.4), trypsin-EDTA (Life Technologies; 25300-054, 0.05%), bovine serum albumin (Life Technologies; 15560012); D-Hanks solution (Beyotime, Jiangsu, China; C0218); type II collagenase (Sigma, St. Louis, MO, United States ; C6885); 5-bromo-2'-deoxyuridine (5-BrdU) (Sigma; B5002); Cell Counting Kit-8 (CCK-8; DOJINDO, Kumamoto, Japan; CK04), IRDye 680CW (Licor, Lincoln, NE, United States ), protein molecular weight markers (Beyotime; P0066), BCA protein concentration determination kit (Solarbio, Beijing, China; PC0020). Antibodies: anti-glycogen synthase kinase beta (GSK3β) (Cell Signaling Technology (CST), Danvers, MA, United States ; 12,456), anti-AKT serine/threonine kinase 1 (AKT1) (CST; 2,967), anti-AKT serine/threonine kinase 2 (AKT2) (CST; 2,962), anti-β-catenin (Abcam, Cambridge, MA, United States ; ab24925), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abbkine, Wuhan, China; Abp57259).

The purity of ginkgo flavonol glycosides in GBE50 (24%) was increased to more than 80% by modified macroporous resin (LSA-12S) column chromatography, and the purity of ginkgo ginkgolides was improved from 6% to over 80% by normal phase silica gel column chromatography combined with recrystallization, according to previously published methods (Zheng et al., 2018a; Zheng et al., 2018b)_. Then, ginkgo flavonol glycosides and ginkgolides with purity higher than 80% were mixed in a ratio of 4:1 (mass ratio), which was consistent with the yield ratio of ginkgo flavonol glycosides and ginkgolides in GBE50. GBE80 (2.5 mg/ml) was dissolved in 10% dimethyl sulfoxide (DMSO) + 30% polyethylene glycol (PEG) aqueous solution, and 1.6 ml was diluted in 50 ml water to obtain semi-finished GBE80 injection product. The product was preliminarily filtered by precision filtration, and was immediately canned and sealed after passing the qualified filtration test. The GBE80 injection was steam sterilized at 100°C, and leak detection was performed after sterilization.

The isolation and primary culture of rat neonatal cardiomyocytes were performed in accordance with previously published methods (Bergmann et al., 2004; Duan et al., 2015). The cell suspension was collected and mixed into the DMEM containing 10% (V/V) fetal bovine serum (FBS), passed through a 200-µm mesh sieve, and centrifuged at 1,000 rpm for 10 min. The cell pellets were resuspended and cultured in DMEM containing 10% FBS for 2 h to allow fibroblasts to adhere preferentially. Non-adherent cells were transferred into a new flask and cultured in DMEM containing 10% FBS and 0.1 μM of 5-BrdU, which was used to inhibit the growth of fibroblasts. Cells were passaged when they reached 80–90% confluence and cultured in DMEM containing 10% FBS without 5-BrdU. Cardiomyocytes at passage 3 to 5 were used in this study.

The cells were seeded into a 96-well plate at 2000 cells/well. In the safety assay, serial dosages of GBE80 were used to treat the cardiomyocytes. In the viability assay, serial dosages (10 μg/ml, 30 μg/ml, and 100 μg/ml) of GBE80, GBE50, or captopril were added into each well together with 0.03% H₂O₂. After 48 h, 10 µl of CCK-8 reagent was added into each well and incubated for 4 h. The absorption at 450 nm was measured using a microplate reader.

Healthy aged male Sprague-Dawley rats (aged 22–24 months, weight: 330 ± 530 g) were purchased from B&K universal Group Co. Ltd. (Shanghai, China). All rats were housed at a controlled temperature (22 ± 2°C), relative humidity (55 ± 5%), and 12-h light/dark cycle, and were allowed food and water ad libitum. All animal procedures were performed following the “Guidelines for the Care and Use of Laboratory Animals” of Shanghai University of Traditional Chinese medicine.

Coronary artery ligation was conducted to establish the AMI rat model according to the procedures of Chen et al. (2019). The aged rats were anesthetized intramuscularly using 10:1 tiletamine/zolazepam and xylazine. After left-sided thoracotomy in the fourth rib interspace, the left anterior descending coronary artery was ligated using a 6–0 silk thread, which was confirmed by the color change of the myocardial tissue. The chest and skin were closed using 2–0 sutures. For the sham (control) operation, a similar procedure was performed, but without ligation.

After ligation, the rats were treated daily for seven consecutive days, followed by euthanasia after another month of follow-up. Seventy rats were randomly divided into seven groups (n = 10 per group): The sham group 1) and model group 2) were intravenously injected with 10% DMSO +30% PEG +60% saline solution (0.9%); 100 mg/kg GBE80 3), 30 mg/kg GBE80 4), 10 mg/kg GBE80 5) and 30 mg/kg captopril 7) were injected intravenously for 7 days; 30 mg/kg GBE50 6) was administered intragastrically with GBE80 for 7 days.

Radioimmunoprecipitation assay (RIPA) lysis buffer was used to extract total proteins from the cultured cardiomyocytes. A BCA protein concentration determination kit was used for protein quantification. Equivalent amounts of protein (20 μg) were separated using 10% SDS-PAGE gels and transferred electrically onto a polyvinylidene fluoride (PVDF) membrane. After blocking using 5% fat milk powder, the PVDF membrane was incubated overnight in the primary antibody solution (4°C) with the anti-GAPDH antibody as the internal protein control. Then, the membrane was incubated with IRDye-680CW-conjugated secondary antibody and developed using the enhanced chemiluminescent (ECL) reagent at room temperature. The optical density of the immunoreactive protein bands was quantified using ImageJ software (NIH, Bethesda, MD, United States ) and normalized to the signal of GAPDH.

Measurement of the infarction size was performed according to the procedure by van Rooij et al. (2002) and Yang et al. (2018). The left ventricle was cut transversely into 2–3 mm slices starting from the apex. The slices were incubated with 1% TTC at 37°C in the dark and then fixed using 4% paraformaldehyde solution for 8 h. The TTC unstained (white) part was the infarcted heart region, which was regarded as the infarction area (INF), while the stained (red) part was the normal heart tissue. The myocardial infarct size was expressed as the weight ratio: The total weight of INF/the total weight of the left ventricle.

The left ventricle was cut into slices of 5-µm thickness using a microtome (CM 1900; Leica, Wetzlar, Germany) and then fixed overnight in 4% polyoxymethylene solution. A commercial TUNEL kit (Beyotime) was used to detect the apoptotic cells in the frozen myocardium. The ratio of the number of TUNEL positive cells to the total number of cells was analyzed using ImageJ.

The left ventricle was fixed with 10% formaldehyde for 3 h at 4°C, and then paraffin slices were prepared by making the tissue transparent, embedding, and slicing. The paraffin slices were treated with citrate buffer for 20 min at 98°C to repair the antigens. After 15 min of blocking using 10% goat serum, the slices were incubated with the primary antibody overnight at 4°C. After washing three times with PBS-Tween 20 (PBST), the myocardium was incubated with the biotin-labeled secondary antibody at 37°C for 30 min. Then, the positive cells in the myocardium were stained using with 3,3′-diaminobenzidine (DAB) solution (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China). The quantitative analysis for positively stained cells was performed by Image-Pro Plus software (v6.0) to express results as average optical density (AOD) at a magnification of ×100.

Continuous variables were expressed as the mean ± standard deviation (SD). GraphPad Prism software 5.0 (GraphPad Inc., La Jolla, CA, United States ) was used to analyze the data using one-way analysis of variance (ANOVA). A p value ≤0.05 indicated that the difference was statistically significant.

When the concentration of GBE80 was lower than 1,000 μg/ml, the viability of cardiomyocytes was not affected, while GBE80 at 1,000 μg/ml decreased the cardiomyocyte viability to less than 80% (48 h, p < 0.05, Figure 1A). Therefore, in this study, 0–100 μg/ml GBE80 was used as the safety concentration. H₂O₂ at 0.03% induced significant oxidative damage in cardiomyocytes. GBE80, GBE50, and captopril reversed the cytotoxicity induced by H₂O₂, and at the same concentration, GBE80 had a better effect on cardiomyocyte viability than GBE50 (Figure 1B).

The levels of phosphorylated (p) AKT, pGSK3β, and β-catenin decreased when the cardiomyocytes were treated with H₂O₂. Western blotting confirmed that GBE80 restored the decreased levels of pAKT, pGSK3β, and β-catenin caused by oxidative damage (Figure 2). In addition, GBE80 significantly increased pAKT, pGSK3β, and β-catenin proteins levels in a dose-dependent manner. These results revealed that GBE80 activated the AKT/GSK3β/β-catenin signaling pathway, and played an antioxidant role in cardiomyocytes in vitro.

The TTC staining results shown in Figure 3A were representative slices that delineated the heart infarction size. Obviously, the myocardial slice of the sham group did not present a pale infarct area, whereas the pale infarction area was distinct in the model group slice. The middle and high doses of GBE80 effectively reduced the area of the myocardial infarction dose-dependently. Quantitative analysis further confirmed that the weight ratio of the myocardial infarction in the GBE80 treatment group was significantly smaller than that of the model group (p < 0.05, Figure 3B), which indicated that GBE80 could protect the myocardial tissue of the AMI model rats.

The TUNEL staining results showed that AMI caused cardiomyocyte apoptosis, and the TUNEL-positive nuclei ratio in myocardial tissue was suppressed significantly after intravenous injection of GBE80 for 7 days (Figure 4A). With the increase in GBE80 dose, the ratio of TUNEL-positive nuclei decreased significantly (Figure 4B), which indicated that GBE80 treatment had a protective effect on the myocardial tissue of aged rats.

The molecular mechanism of GBE80 myocardial protection was studied by detecting the levels of AKT/GSK3β/β-catenin pathway-related proteins. The levels of GSK3β, AKT, and β-catenin in the model group were reduced significantly compared with those in the sham group, which confirmed that AKT/GSK3β/β-catenin signaling was affected by AMI injury (Figure 5A). GBE80 treatment resulted in a dose-dependent increase of GSK3β, AKT, and β-catenin levels in aged AMI rats. These results suggested that GBE80 might protect myocardial tissue in AMI by activating the AKT/GSK3β/β-catenin pathway.