A total of forty 11–14 week-old male healthy Sprague-Dawley rats were obtained from Experimental Animal Center of Tengzhou Central People's Hospital affiliated to Jining Medical University. They were divided into 3 groups randomly: coarctation of abdominal aorta group (A group, n=20) sham operation group (SH group, n=10) and control group (C group, n=10). Laparotomy was performed after anesthesia by intraperitoneal injection of 3% pentobarbital sodium. Abdominal artery was stripped at approximately 5 mm from the above left renal artery opening, a 6/0 silk suture was tied around and made up to 65–70% constriction of abdominal aorta. RHL treatment rats (A+RHL group) were pretreated with RHL (1.5 g/kg) for 3 days by gavage for 14 additional days. Sham operated animals underwent the same procedure except the ligation. Housing and procedures involving experimental animals were in accordance with the Guide for the Care and Use of Tengzhou Central People's Hospital affiliated to Jining Medical University. All animal experiments were approved by the Animal Care and Studies committee of Tengzhou Central People's Hospital affiliated to Jining Medical University.

RHL was purchased from the Shi-Feng Biological Co., Shanghai, China. The RHL was dissolved in PBS to 10 mg/ml and then diluted with DMEM culture medium containing 10% FBS at different concentrations.

Rats were lightly anesthetized with 1–1.5% isolurane in oxygen until the heart rate stabilized to 400 to 500 beats per minute. Echocardiography was carried out with Vevo 770 and Vevo 2100 (VisualSonics) instruments. Fraction shortening (FS), ejection fraction (EF), let ventricular internal diameter (LVID) during systole, LVID during diastole, end-systolic volume, and end-diastolic volume were calculated with Vevo Analysis software (version 2.2.3). After that, rats were euthanatized by cervical dislocation, and their hearts were collected for further analyses.

Heart tissues were cut into cryosections and subsequently analyzed by H&E staining according to the manufacturer's protocol (Sigma-Aldrich). For the histological analysis, 8 µm sections were incubated with primary antibodies overnight at 4°C. Then, the sections were washed with 0.25% Triton X-100 in PBS and incubated with either fluorescently labeled (Molecular Probes; Invitrogen) or biotinylated secondary (Vector) antibodies for 2 h. Then, the sections were observed using microscopy.

Nuclear fragmentation was detected by TUNEL staining with an apoptosis detection kit (Roche) or by incubating fixed cells using an apoptosis detection kit (R&D Systems) according to the manufacturer's protocol. Cells (500–700) in 10 randomly chosen fields from each dish were counted to determine the percentage of apoptotic nuclei. Each data point indicates results from 1,600 to 2,000 cells from 4 independent experiments.

Neonatal rat ventricular myocytes (NRVMs) were isolated from 1–3-day-old Sprague Dawley rats via combined 0.2% trypsin and 0.1% collagenase type II digestion. The cardiac myocytes were plated at a density of 6.6×10⁴ cells/cm² in DMEM supplemented with 10% FBS supplemented with 0.1 mM 5-bromo-2-deoxyuridine. Fibroblasts were not detected in these cultures as determined by immunocytochemical staining with an anti-fibronectin antibody.

Proteins samples were extracted from cardiomyocytes or myocardial tissue in RIPA buffer (1% TritonX-100, 15 mmol/l NaCl, 5 mmol/l EDTA, and 10 mmol/l Tris-HCl (pH 7.0) (Solarbio, China) supplemented with a protease and phosphatase inhibitor cocktail (Sigma) and then separated by 10% SDS-PAGE, followed by electrophoretic transfer to a PVDF membrane. After soaking with 8% milk in PBST (pH 7.5) for 2 h at room temperature, the membranes were incubated with the following primary antibodies: anti-p-p38, anti-p38, Bcl-2, Bax and anti-GAPDH (Cell signaling). Immunodetection was performed by enhanced chemiluminscence detection system (Millipore) according to the manufacturer's instructions. The house-keeping gene GAPDH was used as the internal control.

NRVMs (5,000 cells/well) were plated in 24-well plates and pretreated with RHL for 1 h and then treated with the indicated concentrations of H₂O₂ for 24 h. All assays were carried out in triplicate. The cells were incubated with 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-di-phenyl-tetrazolium bromide for 4 h. And the relative fluorescence was determined at 490 nm as previously described (13). The MTT kit was purchased from Roche Applied Science (Indianapolis, IN).

The living NRVMs were stained with 10 µmol/l DHE (Sigma) for 30 min in a dark humidified chamber at 37°C. ROS generation was indicated by red fluorescence and visualized with fluorescence microscopy. The fluorescence intensity was analyzed as previously described (13).

Data were presented as mean ± SD from 3 independent experiments or 5 rats. Statistical analysis was carried out with Student's t test. Multiple comparisons were evaluated by ANOVA followed by Turkey's multiple-comparison test. P<0.05 was considered as statistically significant difference.

Firstly, primary cardiomyocytes were treated with 10 µM H₂O₂ for 24 h. Then, the cells were incubated with 0.1, 0.5, 1, 3, 5 µM RHL for 24 h. Treatment with 10 µM H₂O₂ decreased cell viability by more than 55%. In contrast, primary cardiomyocytes viability was increased by 19, 25, 32, 42, 48% with 0.1, 0.5, 1, 3, 5 µM RHL incubation by in a dose- dependent manner (Fig. 1A). Meanwhile, the cells were incubated with 1 µM RHL for 12, 24, 48, 72 h. As shown in Fig. 1B, H₂O₂ treatment decreased cardiomyocyte viability by 16, 28, 39, 46% at 12, 24, 48, 72 h, repectively. However, RHL could improve H₂O₂-reduced cardiomyocyte viability by 8, 15, 24, 28% in a time- dependent manner (Fig. 1B).

DHE staining demonstrated that H₂O₂ induced the production of ROS by approximately 3.6 fold. In comparison, incubation with 1 µM RHL decreased ROS production by about 2.1 fold than that of H₂O₂ (Fig. 2A). Next, we further evaluated the role of RHL in H₂O₂-induced cardiomyocytes apoptosis. TUNEL staining indicated that H₂O₂ treatment significantly increased apoptotic cells by 42% than that of normal control (0.1%) (Fig. 2B). In comparison, RHL incubation significantly decreased H₂O₂-induced cardiomyocytes apoptosis by 30% (Fig. 2B).

Next, we explored the potential molecular mechanism in which RHL protects cardiomyocytes from apoptosis. We found that H₂O₂ treatment with markedly activated p38MAPK signaling by ~1.12 fold (Fig. 3). Furthermore, the pro-apoptotic protein, Bax, was significantly increased by about ~1.54 fold, while an anti-apoptotic protein, Bcl-2, was decreased by 57% (Fig. 3). In comparison, RHL markedly reduced p38MAPK signaling activation by 80%. Furthermore, RHL treatment reduced the expression of Bax by 98%, but increased the protein level of Bcl-2 by approximately ~1 fold (Fig. 3). These data indicated that RHL protected primary cardiomyocytes from H₂O₂-induced apoptosis mainly by suppressing p38MAPK activation.

To further explore the effect of RHL on heart function, echo analysis was carried out. Compared with control and sham group, coarctation of abdominal aorta group demonstrated reduced ejection fraction (EF)% and fraction shortening index (FS)% by 28 and 16%, respectively (Fig. 4A and B). But pre-treatment with RHL markedly increased heart function by 21 and 11% than that of the operation group, respectively (Fig. 4A and B). Furthermore, we also found that RHL treatment markedly inactivated p38MAPK signaling in coarctation of abdominal aorta group. Moreover, the protein level of Bcl-2 was increased by ~1.73 fold after RHL treatment, while the expression of Bax was decreased by 35% after RHL incubation in NRVMs (Fig. 4C). These data demonstrated that RHL protects heart failure mainly by suppressing p38MAPK in vivo.