A total of 28 male SD rats (5-6 weeks old) weighing 165-200 g were used to generate the animal model in the present study (18). The animals were offered access to food and water ad libitum and housed in the animal experimental center of Capital Medical University in a 12: 12 h light: Dark cycle at 22±2˚C. Rats were randomly assigned to 4 groups: Sham; RG3; MI; and RG3 + MI. The rats in the sham and RG3 groups had no ligation of coronary artery, and the rats in the MI and RG3 + MI groups were used to establish the MI model by ligation of coronary arteries.

At 7 days prior to and 28 days after coronary artery ligation, the rats in the Rg3 and Rg3 + MI groups were administered 30 mg/kg/day Rg3 (cat. no., 64139; Sigma-Aldrich; Merck KGaA) intragastrically for 7 days, and an equal amount of saline was used in the sham and MI groups. All animal protocols were approved by the Animal Care and Use Committee in Beijing Anzhen Hospital, Capital Medical University, and conformed with the guidelines of the National Institutes of Health (20).

Coronary artery ligation was used to establish a rat model of MI, as described previously (21). Briefly, the rats were anesthetized, and the depth of anesthesia was evaluated by observing the conjunctival reflex, tail pinch and eyelid reflexes (20). Then, a normal II lead electrocardiogram (ECG) was performed. For the surgical procedure, the skin on the left side of the sternum was prepared, disinfected, and a scalpel incision was made using a 10-0 purse string suture to quickly close the chest following surgery. The intercostal muscles between the 3rd to 4th ribs were separated, revealing the heart. The coronary veins associated with the left coronary artery were identified between the left atrial appendage and the pulmonary artery cone. They were ligated with 6-0 non-invasive sutures at 2-3 mm below the left atrial appendage. The depth of the needle was maintained at 0.1 cm and the width was 0.2-0.3 cm. The heart was placed back into the chest cavity through the intercostal space. The gas in the chest cavity was quickly squeezed to tighten the purse string suture for ligation. Then the incision was sutured. The sham operation and the RG3 groups were only threaded and not ligated. Significant elevation of the S-T segment or widening of the QRS complex was a sign of successful model establishment. Post-surgery intramuscular penicillin was administered continuously for 3 days to prevent infection. The rats were considered to be healthy by observing the skin, activity, food intake, excretion, abdominal respiration, external genitalia, and eyes following surgery. Concomitantly, the cardiac function of the rats was also evaluated by echocardiography and serum myocardial enzymes, and serum inflammatory factors were measured to assess levels of inflammation.

On the 28th day after the MI model was constructed, the experimental rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg), and the depth of anesthesia in the rats was evaluated by observing conjunctival reflex, tail pinch and eyelid reflexes. Following euthanasia by cervical dislocation, the chest cavity was opened to obtain the heart tissue and to collect aneurysm specimens. A portion of the specimen was dehydrated and paraffin-embedded, and the specimen was cut into 5 µm slices using the CUT4062 serial slicer (SLEE medical GmbH). Paraffin sections were routinely dewaxed and washed with H&E staining kits for H&E staining (cat. no., G1120; Beijing Solarbio Science & Technology Co., Ltd.) as follows: The samples were dyed with hematoxylin for 5-10 min at room temperature, washed for 3-5 min, soaked with 0.5-1% hydrochloric acid in alcoholic solution for several seconds at room temperature, washed for 3-5 min, and then colored with alkaline aqueous solution for 1 min, followed by washing with water for 10 min, and examination using an optical microscope (LSM800; Olympus Corporation). Following staining, the slides were washed 1 or 2 times, stained with 0.5% eosin for 1-2 min at room temperature, dehydrated with alcohol (80% ethanol for 30 sec, 95% ethanol for 1 min, 95% ethanol for 1 min, absolute ethanol for 3 min and absolute ethanol for 3 min), washed with xylene, and sealed using a neutral resin. An LSM800 optical microscope (x200 magnification; Olympus Corporation) was used to observe the pathological changes in each slice following H&E staining.

Blood samples were collected from ~1 cm capillaries underneath the eyelids, and the sera were obtained following centrifugation at 1,000 x g for 5 min at room temperature. Creatine kinase myocardial band (CK-MB; cat. no., XF2852b, Shanghai Xinfan Biotechnology Co., Ltd.), lactate dehydrogenase (LDH; cat. no. BC0685; Beijing Solarbio Science & Technology Co., Ltd.), cardiac troponin-1 (cTnI; cat. no., ab246529; Abcam), IL-1β (cat. no., ab100768; Abcam), TNF-α (cat. no., ab100785; Abcam), IL-6 (cat. no., ab100772; Abcam) and IL-10 (cat. no., ab100765; Abcam) ELISA kits were used to detect the CK-MB, LDH, cTnI, IL-1β, TNF-α, IL-6 and IL-10 levels in serum, respectively.

Tissues were fixed with 10% formalin solution at room temperature for 24 h, and slices were prepared. Paraffin-embedded cardiac tissue sections were first dewaxed in xylene for 5-10 min at room temperature. Finally, they were immersed in 90% ethanol for 2 min, 70% ethanol for 2 min and distilled water for 2 min. Then, the one-step TUNEL Apoptosis Detection kit (cat. no., C1088; Beyotime Institute of Biotechnology) was used to detect the levels of apoptosis in the heart tissue following the manufacturer's protocol. Nuclear counterstaining was performed for 5 min using DAPI (5 µg/ml; Sigma-Aldrich; Merck KGaA) at room temperature. An LSM800 optical microscope (x200 magnification; Olympus Corporation) was used to observe apoptotic cells.

Levels of TNF-α, IL-1β, L-6 and IL-10 in the rats were measured as described previously (22) with RT-qPCR using gene-specific TaqMan primer/probe sets in an ABI prism 7000 Sequence Detection System (Applied Biosystems). The total RNA was reverse transcribed into cDNA using a reverse transcription kit (cat. no., RR036B; Takara Bio, Inc.) according to the manufacturer's protocol. Subsequently, a 20-µl RT-qPCR system was prepared using the GoTaq qPCR Master Mix kit according to the manufacturer's protocol (cat. no., 638320; Takara Biotechnology, Co., Ltd.). GAPDH mRNA transcription was used as the internal loading control. The PCR primer sequences were as follows: TNF-α forward (F), 5'-CTGAACTTCGGGGTGATCGG-3'; TNF-α reverse (R): 5'-GGCTTGTCACTCGAATTTTGAGA-3'; IL-1β F, 5'-GAAATGCCACCTTTTGACAGTG-3'; IL-1β R, 5'-TGGATGCTCTCATCAGGACAG-3'; IL-6 F, 5'-TCTATACCACTTCACAAGTCGGA-3'; IL-6 R, 5'-GAATTGCCATTGCACAACTCTTT-3'; IL-10 F, 5'-GCTATCGCCCGGTATAG-3'; and IL-10 R, 5'-GTTTCGCGTCGATATTAGC-3'; GAPDH F, 5'-AGGTCGGTGTGAACGGATTTG-3' and GAPDH R, 5'-GGGGTCGTTGATGGCAACA-3'. The thermocycling conditions for the qPCR were as follows: Initial denaturation (94˚C for 2 min); 40 cycles of denaturation (95˚C for 30 sec), annealing (90˚C for 5 sec) and elongation (65˚C for 30 sec); and a final extension (72˚C for 60 sec). The 2^-ΔΔCq method was used to quantify gene expression levels (23).

Protein levels were analyzed by western blot analysis, as described previously (22). GAPDH protein transcription was used as the internal loading control. RIPA Lysis Buffer (cat. no. P0013K; Beyotime Institute of Biotechnology) was used to lyse fibroblast-like synoviocytes and synovial tissue, and the BCA Protein Assay kit (cat. no. P0010S; Beyotime Institute of Biotechnology) was used to measure lysate protein concentration. A total of 50 µg protein/lane in cell lysates were separated by 10% SDS-PAGE and then transferred to PVDF membranes, which were then blocked with 5% skim milk powder for 1 h at room temperature. The primary antibodies in the present study were as follows for overnight incubation at 4˚C: Anti-p65 phosphorylated (p)-S636 (cat. no. ab86299; 1:5,000; Abcam) and anti-GAPDH (cat. no. ab9484; 1:3,000; Abcam). The following secondary antibodies were used for 1 h incubation at room temperature: Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) H&L (cat. no. ab6721; 1:3,000; Abcam) and HRP-conjugated goat anti-mouse IgG H&L (cat. no. ab6789; 1:3,000; Abcam. ImageJ v1.8.0 (National Institutes of Health) was used to analyze protein gray value.

Data were presented as the mean ± SD. SPSS 20.0 (IBM Corp) software was used to analysis the data in the present study. A Student's t-test was used to compare differences between two groups, and multiple groups were compared using one-way ANOVA followed by a Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

A rat MI model was established by coronary artery ligation, and as shown in Fig. 1A, the S-T segment of the ECG in the rats was significantly increased following coronary artery ligation compared with the sham group. This indicated that the MI model was successfully established. It was also identified that Rg3 could significantly attenuate the increase in rat electrocardiogram S-T values caused by coronary artery ligation. On 28th day post-MI, the serum levels of CK-MB, LDH and cTnI in the RG3 + MI rats were significantly decreased compared with those in the MI group (Fig. 1B-D). In addition, H&E staining was used to detect pathological changes in rat heart tissue 28 days following MI. It was observed that there were a number of pathological changes in the heart tissue of the MI group (Fig. 2C) compared with the sham group (Fig. 2A) and the RG3 group (Fig. 2B), such as myocardial tissue injury, and disordered cell and muscle bundle arrangement. There were a large number of infiltrating inflammatory cells, and the nuclei were dissolved, disappeared or necrotic. However, Rg3 was demonstrated to significantly alter the extent of pathological damage in the heart tissue caused by MI. In the RG3 + MI group (Fig. 2D), the muscle bundles were arranged neatly, the cell morphology was intact, and the level of inflammatory cell infiltration was decreased. It was also observed that the level of cell apoptosis in the RG3 + MI group was significantly decreased compared with that in the MI group (Fig. 2E-H). These data suggested that Rg3 protected cardiac function in rats following MI.

The results of the H&E staining protocol demonstrated that Rg3 attenuated MI-induced inflammatory cell infiltration in the heart tissues of rats. On the 28th day post-MI, the expression of inflammatory factor in the heart tissue in rats was also detected, and it was identified that the mRNA and protein expression levels of TNF-α, IL-1β and IL-6 in the heart tissues of the MI rats were significantly increased compared with that in RG3 + MI group (Fig. 3). The expression of IL-10 was demonstrated to be the opposite. This indicated that Rg3 attenuated inflammation of heart tissue in rats after MI.

On the 28th day post-MI, the levels of TNF-α, IL-1β, IL-6 and IL-10 in the serum of rats were measured, and it was identified that the serum levels of TNF-α, IL-1β and IL-6 in the RG3 + MI group were significantly decreased compared with those in the MI group, and the serum levels of IL-10 were significantly increased compared with the MI group (Fig. 4).

Western blot analysis was used to measure the expression levels of proteins in the heart tissues, and the results are shown in Fig. 5. The expression level of SIRT1 protein was significantly increased following Rg3 treatment in the RG3 group compared with the sham group, and in the RG3 + MI group compared with the MI group, but the expression level of SIRT1 protein in the MI group was significantly compared with that in the sham group. Compared with the MI group, the expression of transcription factor RelB (RelB) was significantly increased, and the expression of p-p65/p65 was significantly decreased.