Male Sprague-Dawley (SD) rats (4-week old; 250±50 g) were obtained from the Shanghai Laboratory Animal Center (SLAC; Shanghai, China). All experiments were approved by the ethics committee of Jiangxi Provincial People's Hospital (Nanchang, China). MSCs were isolated as previously described, with minor modifications (11). Briefly, 50 SD rats were housed at room temperature with a standard 12-h light/dark cycle and ad libitum access to food and water for at least 7 days, following which the rats were sacrificed by cervical dislocation and soaked in 75% alcohol for 2 min. The bilateral tibial and femoral shafts were isolated and washed twice in cold PBS. The tibia was cut from the middle, and washed in low-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 100 U/ml heparin without serum. According to the Percoll lymphocyte separating method (cat. no. 17-0891-01; Pharmacia; GE Healthcare Life Sciences, Piscataway, NJ, USA), the suspension was centrifuged at a 500 g density gradient for 25 min at room temperature and the mononuclear cell layer was collected. The cells (5×10³/ml) were resuspended in low-glucose DMEM with 15% FBS (GE Healthcare Life Sciences), 100 U/ml penicillin and streptomycin at 37°C and 5% CO₂. The cells were distinguished using flow cytometry with antibodies against CD34 (1:100; cat. no. ab23830, Abcam, Shanghai, China), CD44 (1:100; cat. no. ab46793, Abcam) and CD105 (1:100; cat. no. ab53318; Abcam). The cell growth curve is shown in Fig. 1.

The specific pathogen-free rats (weight, 250±50 g) were purchased from the SLAC and raised in an animal room for at least 7 days prior to experiments. The acute MI model was established by ligating the anterior descending branch of the coronary artery. Following anesthesia via intraperitoneal injection of 40 mg/kg pentobarbital with 0.l mg/kg atropine, the rats were fixed on the bench in the supine position. A non-invasive method was used to insert a tube (18G) into the mouth in order to expose the throat. Positive pressure ventilation was applied using a ventilator to assist with animal breathing. The tidal volume was set at 9–12 ml and the breath rate was set at 1:1.5 with a frequency of 60/min. Following exposure of the heart, the anterior descending branch of the left coronary artery was identified and ligated using 5–0 silk. The ST elevation was determined upon ECG and the tissues downstream of the coronary artery ligation were white or purple in color. Expansion of the left atrial appendage was performed to determine successful modeling of the MI model. The MI rats were then randomly divided into four groups: MSC therapy group, P38MAPK inhibition group, MSC therapy with p38MAPK inhibition group and saline control group. Subsequently, MSCs (1.5×10⁶/100 µ1) and/or SB203580 (0.2 mg/kg) were injected twice each week for a consecutive 4-week period. HE staining and a TUNEL assay were then performed to determine the pathological changes and apoptosis.

The heart tissues were collected for RT-qPCR analysis. Total mRNA was isolated according to the manufacturer's protocol of the kit (cat. no. 74134; Qiagen, Inc., Valencia, CA, USA). The concentration and purity of the total RNA were determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Subsequently, the mRNAs underwent RT-qPCR combined with SYBR staining to amplify the expression (cat. no. RR430A; Takara Biotechnology Co., Ltd., Dalian, China). The primers were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). The primers were as follows: p38MAPK, forward 5′-tcgagaccgtttcagtccat-3′ and reverse 5′-cca cgg acc aaa tatcca ct-3′; transforming growth factor β-activated kinase 1 (TAK1), forward 5′-cca tcc caa tgg cgt atc tta c-3′ and reverse 5′-tca tcc tgg tcc aat tct gca a-3′; GAPDH, forward 5′-act tga agg gtg gag cca aa-3′ and reverse 5′-cca gga aa tga gct tga ca-3′. The Cq value for each gene was detected and the expression levels of target genes were calculated using the 2^−ΔΔCq method (12). RT-qPCR was performed on a real-time PCR system (LightCycler 96; Roche Diagnostics, Basel, Switzerland) with the following procedure: Initial denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 60°C for 13 sec and extension at 72°C for 30 sec, for 37 cycles. The reaction sample consisted of 10 µl 2X SYBR Fast qPCR mix, 0.8 µl forward/reverse primers (10 mM), 0.4 µl 50X ROX Reference Dye II and 2 µl cDNA template.

Myocardial tissues were collected for protein isolation (ReadyPrep; GE Healthcare Life Sciences). The protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). 25 µg proteins from each group were obtained for sodium dodecyl sulfate polyacrylamide gel electrophoresis (12%) and transferred onto nitrocellulose membranes for western blot analysis. The membranes were incubated with the following primary antibodies: p38MAPK (1:2,000; cat. no. sc-17852-R, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), TAK1 (1:2,000; cat. no. sc-7967, Santa Cruz Biotechnology, Inc.) and actin (1:3,000; cat. no. sc-58673, Santa Cruz Biotechnology, Inc.). Following incubation with the primary antibodies at 4°C overnight, the nitrocellulose membranes were washed three times and incubated with secondary antibody (HRP-labeled goat anti-rabbit IgG; cat. no. A16104SAMPLE, Thermo Fisher Scientific, Inc.) at 4°C for 2 h. The staining of the blots was enhanced using an ECL kit (Thermo Fisher Scientific, Inc.). The densities of the blots were quantified using Quantity One software (v4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Data are presented as the mean ± standard error of the mean. Statistical analyses were performed using one-way analysis of variance with GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

An acute MI model was produced using rats by ligating the anterior descending branch of the coronary artery. The modeling process is shown in Fig. 2A. During the present study, four rats died due to infection-induced heart failure. The ECG examination showed that the model rats exhibited acute anterior ST elevation (Fig. 2B and C). At 4 weeks post-treatment, the heart tissues were collected for HE staining. As shown in Fig. 3A, the cells in the model control group were loosely arranged with numerous bubbles, inflammatory cell infiltration and tissue injury. Following treatment with MSCs or SB203580, the cells were tightly arranged with few bubbles (Fig. 3B and C). The combined application of MSCs with SB203580 exhibited optimal effects (Fig. 3D). A TUNEL assay was also performed to detect apoptosis. Apoptotic cells in the control group were apparent (Fig. 4A). Following treatment with MSCs or SB203580, the number of apoptotic cells was significantly decreased (Fig. 4B and C). Consistent with the HE staining, the combined application of MSCs with SB203580 (Fig. 4D) showed optimal effects in decreasing apoptosis.

mRNA and proteins were isolated to examine the underlying mechanisms. The expression levels of p38MAPK and TAK1 were determined. Compared with the control group, MSC therapy or SB203580 injection significantly decreased the expression of p38MAPK at the mRNA (Fig. 5A) and protein (Fig. 5B) levels. In addition, the combined application of MSC therapy with SB203580 led to a further decrease in the expression of p38MAPK. The expression of TAK1 was also detected, which is upstream of the p38MAPK signaling pathway in the different groups. A consistent trend was observed in the groups as shown for p38MAPK (Fig. 5A and B).