Astragaloside was purchased from Chengdu Mansite Biological Technology Co., Ltd. (Chengdu, China). Polyclonal anti-HIF-1α (cat. no. #4426) and monoclonal anti-Notch1 (cat. no. #3608) primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), and monoclonal anti-Jagged1 (cat. no. ab124524) and anti-GAPDH (cat. no. ab181602) antibodies were purchased from Abcam (Cambridge, UK). A biotinylated horseradish peroxidase (HRP) -conjugated secondary antibody (cat. no. A0216) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). TRIzol^® reagent was obtained from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Reverse transcription (RT), bicinchoninic acid protein (BCA) assay and SYBR^®-Green Real-Time Polymerase Chain Reaction (PCR) kits were purchased from Thermo Fisher Scientific, Inc. Radioimmunoprecipitation assay (RIPA) lysis buffer was obtained from JRDUN Biotechnology (Shanghai, China).

Male Wistar rats (mean weight, 250±30 g; age, 2 to 3 months; n=45) were supplied by the Changchun Yisi Experimental Animal Technology Co. (Changchun, China). They were housed at 22±2°C with 40–50% humidity, a 12 h/12 h light/dark cycle and free access to food and water. The study protocol was approved by the Ethical Committee of the Harbin Medical University (Harbin, China) and performed in accordance with the guidelines of Laboratory Animals published by the US National Institutes of Health.

Rats were randomly divided into sham control or acute MI groups. Animals were subjected to coronary artery ligation to induce acute MI as described previously (19). Briefly, rats were anesthetized with an intraperitoneal injection of 40 mg/kg 1% sodium pentobarbital (Merck KGaA, Darmstadt, Germany). Following tracheal intubation, all rats received positive pressure aerobic ventilation with a small animal ventilator (55–705B; Harvard University, Cambridge, MA, USA), and the chest was opened at the left fourth intercostal space. The heart was exteriorized following incision of the pericardium. A 6–0 silk suture was used to ligate the left anterior descending branch ~3–5 mm distal to the aortic root. Successful generation of the acute MI model was verified by regional cyanosis of the infarct areas. Sham control animals were subjected to an identical surgical procedure without coronary artery ligation.

A total of 18 rats (35% of rats died) surviving for 24 h were randomly assigned to three groups: MI model, 2.5 mg/kg/day Astragaloside-treated, 10 mg/kg/day Astragaloside-treated or control (n=6/group). Following surgery, all animals were kept in an air conditioned-controlled room at 22±2°C with 40–50% humidity in a 12 h light/dark cycle with free access to water and standard rat feed. Astragaloside-treated rats were administered with the designated doses of Astragaloside by intraperitoneal injection for 28 days, whereas rats in the MI model and sham control groups received equal amounts of saline.

After 28 days of Astragaloside or saline treatment, rats were anesthetized with an intraperitoneal injection of 40 mg/kg 1% sodium pentobarbital. The heart was rapidly dissected and rinsed with saline, following which the left intraventricular pressure was maintained at 20 mmHg by using water-filled balloon. The aortic arch, right ventricle, atrium and auricle were removed, and horizontally sectioned into five 1-mm thick sections. Infracted areas of the extracted left ventricle were fixed in 4% paraformaldehyde for 24 h. The fixed tissue specimens were cut into 6-µm sections for hematoxylin-eosin (H&E) staining. Morphological alterations of the stained tissues were assessed under a light microscope (CH20BIMF200; Olympus Corporation, Tokyo, Japan) at ×400 magnification.

Reverse transcription-quantitative PCR (RT-qPCR) analysis was performed to measure HIF-1α, Notch1 and Jagged1 mRNA expression. Briefly, total RNA was extracted from 100 mg samples of ischemic myocardium tissue with TRIzol reagent according to the manufacturer's protocol. The purity of RNA was measured according to the A₂₆₀/A₂₈₀ ratio spectrophotometrically. Total RNA (2 µg) from each sample was used to synthesize cDNA with Moloney Murine Leukemia Virus Reverse Transcriptase (Thermo Fisher Scientific, Inc.). The following primer sequences were synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China): Forward, 5′-CAGCGATGACACGGAAAC-3′ and reverse, 5′-AGTGACTCTGGGCTTGAC-3′ for HIF-1α; forward, 5′-CTTCGTGCTCCTGTTCTTTG-3′ and reverse, 5′-GCCTCTGACACTTTGAAACC-3′ for Notch1; and forward, 5′-CACCCGAACTGGACAAAC-3′ and reverse, 5′-AGCCTCAGACTGGGATAC-3′ for Jagged1. These primer pairs resulted in 210, 105, and 170 bp amplified products, respectively. Rat GAPDH was used as an internal control with the following primers (182 bp): Forward, 5′-GTCGGTGTGAACGGATTTG-3′ and reverse, 5′-TCCCATTCTCAGCCTTGAC-3′. qPCR reactions were prepared using a SYBR Real-Time PCR kit. The PCR products were quantified by detecting the SYBR-Green fluorescence signal intensity and analyzed by Applied Biosystems Prism 7300 SDS software (Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative mRNA expression levels of HIF-1α, Notch1 and Jagged1 were quantified by the 2^−∆∆Cq method, normalized to the levels of GAPDH.

HIF-1α, Notch1 and Jagged1 protein expression levels were measured by western blot analysis. Briefly, 20 mg samples of frozen tissues were powdered and homogenized in 150–250 µl RIPA lysis buffer. Protein levels were determined by performing a BCA assay according to the manufacturer's protocol. Prepared protein samples were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with milk powder in TBS for 60 min at room temperature, and subsequently incubated with anti-HIF-1α (1:1,000 dilution), anti-Notch1 (1:1,000 dilution), anti-Jagged1 (1:500 dilution) and anti-GAPDH (1:1500 dilution) primary antibodies. Following overnight incubation at 4°C, the membranes were washed three times with TBST (TBS with 0.1% Tween-20) and subsequently incubated with HRP-conjugated secondary antibodies (1:2,000 dilution) in TBS with 0.1% Tween-20 for 30 min at 37°C. The bands were visualized using Enhanced Chemiluminescence reagents (EMD Millipore, Billerica, MA, USA), according to the manufacturer's protocol. The relative protein expression levels of HIF-1α, Notch1 and Jagged1 were normalized to the intensity of GAPDH bands from the same sample. All experiments were repeated in triplicate, and mean values were calculated.

Data are presented as mean ± standard deviation. One-way analysis of variance followed by the Student-Newman-Keuls method was applied for multiple comparisons. All statistical analyses were performed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Heart structures were healthy in sham control rats (Fig. 1A), whereas untreated MI rats exhibited dilation of the heart, pale lesion sites, thinner ventricular walls and total ventricular wall involvement (Fig. 1B). In contrast, treatment with 2.5 (Fig. 1C) or 10 mg/kg/day (Fig. 1D) Astragaloside attenuated the dilation of heart and reduced the infarction size of MI rats.

Histopathological analysis of myocardial tissues was observed by H&E staining. Myocardial tissues in control rats demonstrated apparent integrity of the myocardial cell membrane with no inflammatory cell infiltration (Fig. 2A). Myocardial tissues in infarct rats exhibited dissolved cardiomyocytes, distorted cardiac muscles, myocardial necrosis, a large number of fibroblasts or collagen fibers, and a small amount of inflammatory cell infiltration (Fig. 2B). In contrast, treatment with 2.5 (Fig. 2C) or 10 mg/kg/day (Fig. 2D) Astragaloside attenuated the above histopathological abnormalities, and the boundary between the infarct area and non-infarct area was obvious.

As presented in Fig. 3A, no significant differences were observed in HIF-1α mRNA expression levels in the myocardium of untreated MI rats compared with sham control rats (1.7006±0.9756 and 1.1495±0.6861, respectively; P>0.05). Treatment with Astragaloside significantly increased HIF-1α mRNA expression to 15.3790±4.2757 at a dose of 2.5 mg/kg/day and 20.8603±6.8249 at 10 mg/kg/day (both P<0.01). Furthermore, HIF-1α mRNA expression levels were significantly increased following treatment with 10 mg/kg/day Astragaloside compared with 2.5 mg/kg/day (P<0.05).

As presented in Fig. 3B, no significant differences were observed in Notch1 mRNA expression levels in the myocardium of untreated MI rats compared with sham control rats (2.3227±0.6524 and 1.8110±2.6816, respectively; P>0.05). Treatment with Astragaloside significantly increased Notch1 mRNA expression to 22.4513±5.0885 at 2.5 mg/kg/day and 26.3550±10.4480 at 10 mg/kg/day compared with the expression level in MI model rats (both P<0.01). However, no significant differences in Notch1 mRNA expression were observed between the different doses of Astragaloside (P>0.05).

As presented in Fig. 3C, no significant differences were observed in Jagged1 mRNA levels in the myocardium of untreated MI rats compared with sham control rats (1.5589±2.0397 and 1.4880±1.5084, respectively; P>0.05). Treatment with Astragaloside significantly increased Jagged1 mRNA expression to 8.4783±6.4066 at 2.5 mg/kg/day and 12.1670±2.0818 at 10 mg/kg/day (both P<0.01). However, no significant differences were observed in Jagged1 mRNA expression between the different doses of Astragaloside (P>0.05).

Representative HIF-1α protein bands with a molecular weight of 120 kDa are presented in Fig. 4A. Myocardial HIF-1α protein levels were significantly increased in MI model rats compared with sham control rats (0.5668±0.1855 and 0.3295±0.1553, respectively; P<0.05). HIF-1α protein expression levels following treatment with 2.5 and 10 mg/kg/day Astragaloside were 0.7259±0.2112 and 0.8009±0.2167, respectively. Treatment with 10 mg/kg/day Astragaloside resulted in significantly increased HIF-1α expression levels compared with MI model rats (P<0.05). HIF-1α protein expression levels did not differ significantly between rats treated with different doses of Astragaloside (P>0.05).

Representative Notch1 protein bands with a molecular weight of 120 kDa are presented in Fig. 4B. Myocardial Notch1 protein expression levels were significantly increased in MI model rats compared with sham control rats (0.7873±0.2207 and 0.4781±0.1393, respectively; P<0.05). Notch1 protein expression levels increased to 0.9567±0.2099 and 1.1336±0.2014 following treatment with 2.5 and with 10 mg/kg/day Astragaloside, respectively. Treatment with 10 mg/kg/day Astragaloside led to significantly upregulated Notch1 compared with the MI model rats (P<0.01). No significant differences in Notch1 protein expression levels were observed between the different doses of Astragaloside (P>0.05).

Representative Jagged1 protein bands with a molecular weight of 134 kDa are presented in Fig. 4C. Myocardial Jagged1 protein expression levels were significantly increased between MI model and sham control rats (0.5244±0.0915 and 0.3900±0.5689, respectively; P<0.05). Treatment with 2.5 and 10 mg/kg/day Astragaloside significantly increased Jagged1 protein expression levels in rats (0.7380±0.1091 and 1.0580±0.1211, respectively), compared with untreated MI rats (P<0.01). Furthermore, Jagged1 protein expression levels were significantly increased following treatment with 10 mg/kg/day Astragaloside compared with 2.5 mg/kg/day (P<0.01).