Rat cardiac H9c2 cells (ATCC, USA) were cultured in Dulbecco's modified Eagle's media (DMEM) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), which is supplemented with 10% fetal bovine serum (FBS; Takara Biotechnology Co., Ltd., Dalian, China), 100 ug/ml streptomycin (Takara Biotechnology Co., Ltd.), and 100 unit/ml penicillin (Takara Biotechnology Co., Ltd.) at 37°C in a humidified atmosphere with 5% CO₂.

Treatment of hypoxia/reoxygenation was performed to mimic the myocardial ischemia/reperfusion (I/R) injury in the body. Cells were cultured in free serum medium overnight and 4 treatment groups were designed for the experiments: i) Normal group, H9c2 cells were cultured with 0.1% DMSO; ii) model group (MG), H9c2 cells were treated with 1 µM Angiotensin II (Ang II) for 48 h; iii) hypoxia/reoxygenation treatment group (H/R), after treated with 1 µM Ang II for 48 h, H9c2 cells were cultured at 37°C with a humidified atmosphere containing 95% N2 and 5% CO₂ for 8 h, following reoxygenation for 4 h; iv) LUTG (Melon, China) pretreatment groups, H9c2 cells were treated with 1 µM Ang II for 48 h prior to cultured in the medium containing LUTG at different doses (10 and 20 µM) for 6 h, then with treatment of 8 h hypoxia/6 h reoxygenation (H/R+10, H/R+20).

H9c2 cells in 96-well plate (1×10⁴ cells/well) were treated as designed. Cells were cultured in serum-free medium overnight before detection. After adding CCK-8 solution the samples were incubated at 37°C for 2 h in the dark. Then cell viability was determined according to the protocols provided by the Cell Counting Kit-8 (CWBio, Beijing, China).

ROS was measured using oxidant-sensitive probe CMH2DCFDA (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). H9c2 cells (1×10⁴ cells/well) in 96-well plate were pretreated as above. The cells were fixed with 16% formaldehyde for 10 to 15 min and then incubated at 37°C for 30 min. Subsequently, the plate was washed with PBS for 3 times before measurement of the fluorescence intensity by flow cytometric analysis (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions.

H9c2 cells (1×10⁴ cells/well) in 96-well plate were pretreated as above. Then, H9c2 cells were harvested and analyzed for cell apoptosis with Annexin V and propidium iodide (PI) staining, using FITC Annexin V apoptosis detection kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Flow cytometric analysis was performed on BD FACS Canto II.

Cardiac troponin-T (cTnT) specific monoclonal antibody (Abcam, Cambridge, UK) was incubated at 4°C overnight after the slides were blocked with 5% BSA for 30 min at room temperature. Then, anti-IgG/FITC (Sigma-Aldrich; Merck KGaA), as secondary antibody, was added at room temperature for 1 h and nuclei were stained with DAPI for 15 min. The slides were observed with a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The cell surface area was analyzed by Image Pro-Plus software.

H9c2 cells (1×10⁴ cells/well) in 96-well plate were collected after treatment as above description. The medium was centrifuged at 3,000 rpm, for 5 min. Then, the supernatant was transferred to a new 96-well plate. The myocardial enzymes spectrum including LDH and CAT were measured by an automatic biochemical analyzer (Hitachi, Tokyo, Japan).

Total RNA was extracted using RNAiso reagent (Takara Biotechnology Co., Ltd.). RNA was reverse transcribed using M-MLV reverse transcriptase (Takara Biotechnology Co., Ltd.). Real-Time reaction was performed using a Real-Time PCR system (ABI 7500; Applied Biosystems, Foster City, CA, USA). The primer sequences used in this study were as follows: β-MHC forward, 5′-CAGACATAGAGACCTACCTTC-3′ and reverse, 5′-CAGCATGTCTAGAAGCTCAGG-3′; Fas forward, 5′-GGATGAACCAGACTGCGTG-3′ and reverse 5′-CTGCATGTTTTCTGTACTTCC-3′; FasL forward, 5′-CTCTGGAATGGGAAGACACC-3′ and reverse 5′-ACCAGAGAGAGCTCAGATACG-3′; PARP forward, 5′-ACGTGTCTGCCTCCACTAAA-3′ and reverse 5′-ACCAGAGTCAGGATCAACGG-3′; Caspase-3 forward, 5′-CTCGCGTTAACAGGAAGGTG-3′ and reverse 5′-GGCAGTGGTAGCGTACAAAG-3′; GAPDH forward, 5′-GGCACAGTCAAGGCTGAGAATG-3′ and reverse: 5′-ATGGTGGTGAAGACGCCAGTA-3′. The relative expression of mRNA was calculated based on 2^−ΔΔCq method (26).

The proteins were extracted using total protein extraction kit (CWBio). Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membrane. Primary antibody directly against PARP, Fas, Fasl, casepaes-3 and GAPDH were from Cell Signaling Technology, Inc. (Danvers, MA, USA). The primary antibodies against p-ERK1/2, p-JNK, p-P38 were puchased from Sigma-Aldrich; Merck KGaA, while p-ERK5, ERK5, ERK1/2, JNK, P38MAPK were from Cell Signaling Technology, Inc. HRP-conjugated secondary antibodies are from CWBio. And the signals were visualized with BeyoECL Plus (EMD Millipore, Billerica, MA, USA).

Data was expressed as the mean ± standard deviation. Comparisons of differences between groups were made by one-way ANOVA or t-test. P<0.05 was considered to indicate a statistically significant difference..

Immunofluorescence staining assay (IFA) was performed to identify the cardiac phenotype of the cells (Fig. 1A). Thereafter, Angiotensin II (Ang II) was adopted to induce cardiomyocyte hypertrophy in this study. The images showed that the relative cell surface area was enlarged (Fig. 1B and C) following the treatment of Ang II. Meanwhile, RT-PCR and Western blot assays showed that the expression of β-MHC was upregulated significantly under the Ang II stimulation (Fig. 1D-F).

Subsequently, the effect of LUTG (2, 5, 10, 20, 40 and 60 µM) on the viability of H9c2 cells was tested with CCK-8 assay. The results showed that LUTG at each concentration did not cause cytotoxicity significantly (Fig. 2A). Moverover, the potential effect of LUTG was also determined in the H/R treated cardiomyocytes. It was shown that LUTG improved the cell survival in a dose dependent manner. The pretreatment with 2 µM LUTG could not suppress the H/R induced damage significantly (Fig. 2B). Thus, in this study, we adopted 10 nd 20 µM to explore the effects of LUTG on the cardiomyocytes, which was correspond to the previous studies (3,27,28). The balance between ROS production and elimination is vital to cell survival through maintaining the cellular redox homeostasis (29). By flow cytometry analysis, it was displayed that ROS levels were elevated obviously both in the model group and in the H/R group. By contrast, the ROS production was inhibited significantly in the LUTG pretreatment groups (Fig. 2C and D). Moreover, the amount of released lactate dehydrogenase (LDH), as a common indicator to determine membrane leakage and cellular damage (30), was decreased in the LUTG pretreatment group compared to the H/R group (Fig. 2E). Furthermore, the activity of catlase (CAT), one of the main extracellular antioxidant enzymes (31), was evaluated substantially in the LUTG pretreatment groups compared to that of the H/R group (Fig. 2F).

Oxidative stress is involved in the apoptosis of hypertrophic myocardial cells (32). The flow cytometry analysis revealed that percentage of cell apoptosis induced by hypoxia/re oxygenation were significantly reduced by LUTG compared to the H/R group (Fig. 3A and B). In addition, the mRNA expression of apoptosis-related genes including PARP, Fas, Fasl and Caspase-3 were lower than that in the H/R group. And the Western blot analysis presented that PARP, Fas, Fasl and Caspase-3 were downregulated in the LUTG pretreatment groups (Fig. 3C-E).

To determine the mechanisms of the myocardial protection mediated by LUTG, the expression of p-ERK1/2, p-JNK, p-P38 and p-ERK5 was measured by Western blot analysis. It was presented that the expression of p-ERK1/2, p-JNK and p-P38 were increased in the model group. Moreover, the phosphorylation levels of ERK1/2 JNK and P38 were boosted after hypoxia/reoxygenation treatment in the H/R group. By contrast, the expression of p-ERK1/2, p-JNK and p-P38 were inhibited, while the level of p-ERK5 was elevated in the LUTG pretreatment groups compared to those of the H/R group (Fig. 4A-F).