Atorvastatin and lithium chloride (LiCl) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Antibodies targeting GSK-3β, Caspase-9 and B-cell lymphoma 2 (Bcl-2) were provided by BD Biosciences (Franklin Lakes, NJ, USA). Antibody targeting Cytochrome c (Cyt c) was from Abcam (Cambridge, MA, USA). Antibodies targeting α-actin, β-actin and GAPDH were provided by Sigma-Aldrich; Merck KGaA. LY294002 was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The syn-rno-miR-199a-5p miScript miR mimic, anti-rno-miR-199a-5p miScript miR inhibitor, AllStars negative control small interfering (si)RNA, AllStars Mm/Rn cell death control siRNA, and HiPerFect transfection reagent were purchased from Qiagen, Inc. (Valencia, CA, USA) 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from Promega Corporation (Madison, WI, USA).

A total of forty neonatal rat cardiac ventricular cardiomyocytes were prepared from 1–3-day-old neonatal SD rats weighing 8–12 g (20 male, 20 female), bred at 25°C and a humidity of 60% on a 12/12-h dark/light cycle with free access to food and water, and cultured as previously described (20,21). The study was approved by the ethics committee of Wujin People's Hospital (Changzhou, China). The cells were cultured in a humidified incubator (37°C with 5% CO₂). Brdu (0.1 mM) was added to the Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich; Merck KGaA), containing 2 mM L-glutamine, 100,000 IU per L penicillin, 100 mg L21 streptomycin, 5% fetal bovine serum and 200 nM tetradecanoyl phorbol acetate (Sigma-Aldrich; Merck KGaA) for the first 2 days to inhibit fibroblast growth. The neonatal rat myocytes were cultured to 70% confluence and then serum starved in basal medium (DMEM) for 24 h prior to drug treatment. The H9c2 rat myocardial cells (cat. no. CRL 1446; American Type Culture Collection) were cultured in DMEM supplemented with 10% FBS (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), penicillin G (100 U·ml⁻¹), streptomycin (100 µg·ml⁻¹) and glutamine (2 mM). The neonatal rat cardiac ventricular cardiomyocytes and H9c2 cells were treated with oxygen-glucose deprivation/reperfusion (OGD/R) with or without the pretreatment, as described previously with minor modification (22). The pretreatment comprised incubation with 1 µM atorvastatin for 3 h or 10 µM GSK-3β inhibitor (LiCl) for 1 h at a density of 1×10⁹ cells/ml. The cells were then rinsed twice, incubated at a density of 2×10⁵ cells/well in a 6-well plate containing glucose-free DMEM and subsequently placed in an anaerobic chamber containing a mixture of 95% N₂ and 5% CO₂ at 37°C for 6 h. Following OGD, glucose was added to normal levels (final concentration, 4.5 mg·ml⁻¹), and cells were incubated under normal growth conditions (95% air and 5% CO₂) for an additional 18 h as OGD/R, unless otherwise specified.

Following culture for different time periods, the samples were rinsed with PBS, fixed with 4% paraformaldehyde for 20 min, and then permeabilized with glycine/triton solution (PBS/0.1% Triton/10 mM glycine). The samples were incubated with mouse monoclonal anti-α-actin (1:300 dilution; cat. no. A7811; Sigma-Aldrich; Merck KGaA) at 4°C overnight. Following three PBS rinses, the samples were incubated with anti-mouse IgG-FITC (1:300 dilution; cat. no. F2012; Sigma-Aldrich; Merck KGaA) for 60 min at 37°C. The nuclei were counterstained with 100 nM DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min, followed by rinses with PBS. Fluorescent images were captured with a spectral confocal microscope imaging system (Leica TCS SP2; Leica Microsystems, Inc., Buffalo Grove, IL, USA).

Cell viability was assessed using CellTiter vt96 Aqueous One Cell Proliferation assays (Promega Corporation). The cells were seeded at 5×10³ per well in 96-well plates overnight. Following treatment, the cells were incubated with 20 µl/100 µl MTS for 1 h at 37°C. Cell viability was determined by measuring the absorbance at 490 nm using a microplate reader (M200PRO; Tecan Austria GmbH, Grödig, Austria).

Supernatant was obtained from the culture medium following centrifugation for 1 min (15,550 × g) at 37°C. The activity of LDH in the supernatant was detected using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing China).

Total RNA was extracted with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cDNA was synthesized from 0.5 µg of total RNA using a reverse transcription system (Promega Corporation) according to the manufacturer's protocol. Subsequently, the cDNA was subjected to PCR using GoTaq qPCR master mix (Promega Corporation). Reactions were performed on a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Included in the 25 µl reaction system was 300 nmol/l primers. Thermal cycling conditions consisted of 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. The primers used for RT-qPCR were as follows: GSK-3β, forward 5′-TCCATTCCTTTGGGATCTGCC-3′ and reverse 5′-ATCAGCTCTGGTGCCCTGTAGTAC-3′; β-actin, forward 5′-CAGGGTGTGATGGTGGG-3′ and reverse 5′-GGAAGAGGATGCGGCAG-3′. The fold change in expression of each gene was calculated using the 2^−ΔΔCq method (23).

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RT-qPCR was performed using the All-in-One miRNA qRT-PCR detection kit (GeneCopoeia, Inc., Rockville, MD, USA). The reactions were performed on a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers used for RT-qPCR were as follows: the specific primer for rno-miR-199a-5p was 5′-CCCAGTGTTCAGACTACCTGTTC-3′ and 5′-GACTATCATATGCTTACCGT-3′ for U6 (GeneCopoeia, Inc.). The thermal cycling conditions were the same as described above. The fold change in the expression of each gene was calculated using the 2^−ΔΔCq method.

The H9c2 cells at a density of 2×10⁵ cells/well were seeded in 6-well plates for 12 h. Prior to transfection, the medium was replaced with DMEM without serum and antibiotics. The AllStars Mm/Rn Cell Death Control siRNA (Qiagen, Inc.) at 10, 20 and 50 nM were added and incubated for 24, 48, and 72 h to determine the optimal conditions. Transfection efficiency was observed and evaluated under a light microscope (Olympus Corporation, Tokyo, Japan). Transfection with 20 nM was selected due to the highest efficiency, which was >70%. HiPerFect transfection reagent (Qiagen, Inc.) was used as a transfection reagent. Syn-rno-miR-199a-5p miScript miR mimic and anti-rno-miR-199a-5p miScript miR inhibitor were used (20 nM) to respectively overexpress and inhibit the expression of miR-199a-5p. AllStars negative control siRNA was used as a negative control.

Total cell lysates were obtained by incubation of cells in radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA and 0.1% SDS) supplemented with 50 µg/ml aprotinin and leupeptin. Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein extracts (30 µg) were separated by 10% SDS-PAGE, and then transferred onto PVDF membranes. The membrane was blocked with 5% milk at 37°C for 1 h. The membranes were blotted with specific antibodies against GSK-3β (1:2,000), Caspase-9 (1:2,000), B-cell lymphoma 2 (BCL-2; 1:2,000), cytochrome c (1:5,000), GAPDH (1:1,000) and β-actin (1:1,000) at 4°C overnight, and then incubated with the horseradish peroxidase-conjugated secondary antibody (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-2020) at 37°C for 1 h. The membranes were then processed for chemiluminescent detection according to the manufacturer's protocol (Santa Cruz Biotechnology, Inc.). All bands were evaluated using densitometry with Quantity One V4.6.2 software (Bio-Rad Laboratories, Inc.). Bands of interest were normalized against GAPDH and β-actin. Data are presented as relative density ratios.

The cells were fixed with 4% paraformaldehyde for 20 min, and then permeated with glycine/triton solution (PBS/0.1% Triton/10 mM glycine). The samples were incubated with mouse monoclonal anti-GSK-3β (1:100 dilution; BD Biosciences) at 4°C overnight. Following rinses, the samples were then incubated with anti-mouse IgG-FITC (1:300 dilution; BD Biosciences) for 40 min at 37°C. The nuclei were counterstained with 100 nM DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min followed by rinses with PBS. Fluorescent images were captured using a spectral confocal microscope imaging system (Leica TCS SP2).

IBM SPSS 17.0 software (IBM SPSS, Armonk, NY, USA) was used to perform statistical analyses. The data are expressed as the mean ± standard deviation. Differences between groups were analyzed using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference. The 2^−ΔΔCq method was used for the RT-qPCR analysis.

To evaluate whether atorvastatin protects H9c2 myocardial cells from OGD/R, the present study examined the direct cytotoxic effect of OGD/R on H9c2 cells with and without atorvastatin pretreatment. The data shown in Fig. 1A indicates that atorvastatin protected against OGD/R. The cell viability was significantly decreased following OGD/R, compared with that in the control group (P<0.05). As shown in Fig. 1, pretreatment with atorvastatin increased cell viability and reduced the OGD/R-induced LDH release and expression of cell damage-associated proteins, including Caspase-9, Bcl-2 and Cyto C.

The present study examined the effect of OGD/R on the expression of GSK-3β and miR-199a-5p in H9c2 cells with and without atorvastatin pretreatment. As shown in Fig. 2A-C, the mRNA and protein expression levels of GSK-3β were significantly decreased following OGD/R, compared with those in the control group and normoxia group (P<0.05). Pretreatment with atorvastatin increased the mRNA and protein expression levels of GSK-3β. As shown in Fig. 2D, the mRNA level of miR-199a-5p was significantly increased following OGD/R (P<0.05), whereas pretreatment with atorvastatin significantly reversed this effect (P<0.05).

To analyze the exact mechanism of atorvastatin protecting H9c2 cardiomyoblast cells from OGD/R, the present study examined the effect of GSK-3β inhibitor (LiCl) on H9c2 cells with and without atorvastatin pretreatment. As shown in Fig. 3A, the cell viability was significantly decreased in the LiCl+atorvastatin+OGD/R group, compared with that in the control group (P<0.05). As shown in Fig. 3B-D, pretreatment with LiCl significantly increased the atorvastatin-inhibited release of LDH and expression of cell damage-associated proteins Caspase-9, Bcl-2 and Cyto C, compared with the control group (P<0.05).

To evaluate the effect of miR-199a-5p on the expression of GSK-3β, the present study examined the effects of miR-199a-5p mimic and inhibitor transfection on the expression of GSK-3β. As shown in Fig. 4A-C, miR-199a-5p mimic transfection significantly decreased the expression of GSK-3β at the mRNA and protein levels, whereas transfection with the miR-199a-5p inhibitor increased their expression (P<0.01). The mRNA levels of miR-199a-5p in the mimic and inhibitor groups are shown in Fig. 4D.

To evaluate whether the miR-199a-5p inhibitor protected the H9c2 cardiomyoblast cells from OGD/R, the present study examined the direct cytotoxic effect of OGD/R on H9c2 cells with and without miR-199a-5p inhibitor pretreatment. As shown in Fig. 5, pretreatment with miR-199a-5p inhibitor increased cell viability and reduced the OGD/R-induced release of LDH and expression of cell damage-associated proteins and mRNA levels of Caspase-9, Bcl-2 and Cyto C.

To evaluate whether atorvastatin protected neonatal rat cardiac ventricular cardiomyocytes from OGD/R as was shown for H9c2 myocardial cells, the present study first verified neonatal rat cardiac ventricular cardiomyocytes (Fig. 6A). The direct cytotoxic effects of OGD/R on neonatal rat cardiac ventricular cardiomyocytes with and without atorvastatin pretreatment were then examined. The data, as shown in Fig. 6B indicated that atorvastatin protected against OGD/R. The cell viability was significantly decreased following OGD/R, compared with that in the control group (P<0.05). As shown in Fig. 6B and C, pretreatment with atorvastatin increased cell viability and reduced the expression of OGD/R-induced cell damage-associated proteins Caspase-9, Bcl-2 and Cyto C. As was observed in the H9c2 myocardial cells, the protective effects of atorvastatin acted through the miR-199a-5p/GSK-3β pathway (Fig. 6D-G) The expression levels of Caspase-9 and Bcl-2 in the OGD/R group were significantly upregulated compared with those in the control group, as illustrated in Fig. 6H.