C2C12 myoblast cells were obtained from the Lab of College of Animal Science and Technology of Huazhong Agricultural University. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences,. Logan, UT, USA) supplemented with 1% antibiotics (Gino Biomedical Technology Co., Ltd., Hangzhou, China) and 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences) in a humidified air of 5% CO₂ at 37°C.

Resveratrol was purchased from Sigma-Aldrich (St. Louis, MO, USA). Immediately prior to use, a 0.1 M stock solution stored at −20°C was diluted to the desired concentration with culture medium, and the final concentration of DMSO for all treatments was maintained at 0.2%. SB431542 was purchased from Selleck Chemicals (Houston, TX, USA). NAM was from Beyotime Institute of Biotechnology (Hangzhou, China) and the Cell Counting kit (CCK)-8 was from Dojindo Laboratories (Kumamoto, Japan). The antibodies used were anti-myostatin rabbit polyclonal (Abcam, Cambridge, MA, USA; cat. no. ab98337), anti-P107 rabbit polyclonal (Abcam; cat. no. ab2451), anti-p-P107 rabbit polyclonal (Bioss, Inc., Woburn, MA, USA; cat. no. bs-5696R), anti-β-actin mouse monoclonal (Anbo, Changzhou, China; cat. no. E0012) and anti-rabbit secondary antibody (Abcam; cat. no. ab6721). Other reagents were from Goodbio Technology Co., Ltd. (Wuhan, China) or HyClone.

Exponentially growing C2C12 myoblast cells (8×10³ cells/well; 100 µl) were seeded into 96-well plates 20–24 h prior to replacing fresh serum free medium containing the indicated concentrations of drug. SB431542 was added first, followed by NAM after 30 min in the group of the SB431542 combined with NAM. After incubation for 24 h, culture medium was replaced with drug-free medium (100 µl), and the effect of drug was examined by a CCK-8 assay, according to the manufacturer's protocol. Briefly, 10 µl of CCK-8 was added to each well and the cells were further incubated away from light at 37°C for 1–3 h. Absorbance was measured with excitation at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The cells were cultured in growth media for 12 h, followed by BrdU treatment (BrdU 10 mg/ml stock solution in saline was diluted 1:1,000 in the culture medium) for 1 h prior to harvesting. After washing with phosphate-buffered saline (PBS), cells were then fixed for 20 min in 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 for 10 min. Following blocking with 10% goat serum in PBS for 1 h, the cells were incubated with anti-BrdU antibody (1:200; BD Biosciences, San Jose, CA, USA; cat. no. 552598) at 4°C for 18 h, followed by incubation with and the secondary goat anti-mouse immunoglobulin G antibody conjugated with Alexa Fluor 594 (1:1,000 dilution; Abcam; cat. no. ab150116) for 2 h at room temperature in the dark. The nuclei were visualized by 4′,6-diamidino-2-phenylindole and images were captured using an Olympus microscope (IX71; Olympus, Tokyo, Japan).

After treatment for 6 h, the total RNA from C2C12 myoblast cells was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. First-strand cDNA was synthesized using a PrimeScript™ RT reagent kit (Takara Bio, Inc., Dalian, China; cat. no. RR037A). DNA amplification was performed in a StepOne system (Applied BioSystems; Thermo Fisher Scientific, Inc.) using SYBR Green PCR kit (Takara Bio, Inc.; cat. no. RR420A) for Sirt1, MyoD and β-actin. The primer sequences are listed in Table I.

Following treatment for 24 h, the C2C12 myoblast cells were harvested in phosphate-buffered saline, centrifuged at 5,000 g for 10 min at 4°C and the supernatant was then removed. The samples were homogenized in ice-cold radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology) with 1% protease inhibitor cocktail (Sangon Biotech, Shanghai, China) and 1% phosphatase inhibitors (Goodbio Technology Co., Ltd.), and were incubated on ice for 20–30 min. The samples were centrifuged at 10,000 g for 10 min at 4°C. The protein concentration of the supernatant was measured using the bicinchoninic acid Protein Quantification kit (BestBio, Shanghai, China). Supernatant fractions of equal protein concentration (50 µg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene membranes (EMD Millipore, Bedford, MA, USA). Blocking was performed in Tris-buffered saline with 5% (w/v) non-fat dry milk for 2 h at room temperature. Following blocking, the membranes were probed with primary antibodies at 4°C overnight. The antibodies used were anti-p-P107 (1:500), anti-P107 (1:250), anti-myostatin (1:500) and anti-β-actin (1:5,000). The membranes were washed (6×5 min) with Tris-buffered saline containing Tween-20, and incubated with the appropriate secondary antibody (1:5,000) for 2 h at room temperature. The membranes were washed as before and horseradish peroxidase activity was detected using Chemiluminescence reagent (Beyotime Institute of Biotechnology) and exposure to G:BOX Chemi xT⁴ Gel imaging system instrument (Syngene UK, Cambridge, UK). The blots were quantified by densitometric analysis using the ImageJ software (version 1.46; imagej.nih.gov/ij/).

The data are presented as the mean ± standard error of the mean for at least three individual experiments. All analyses were performed using SPSS 19.0 software (IBM SPSS, Chicago, IL, USA). The results were analyzed by analysis of variance, least significant differences test for comparisons between two groups, and correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

To elucidate the role of Sirt1 in the control of C2C12 myoblast cell growth, C2C12 cells were treated with resveratrol and NAM (Fig. 1). The present study administered different concentrations of resveratrol on C2C12 myoblast cells and measured the cells growth after 24 h. The results showed that following treatment of C2C12 cells with the resveratrol at different concentrations of 10, 20, 50, 100 or 200 µM, the cell proliferation index was significantly higher compared with that of the untreated control group (P<0.05; Fig. 1A). Additionally, the results demonstrated that the cell proliferation index of C2C12 myoblast cells increased with the growth following treatment with resveratrol in a dose-dependent manner when the resveratrol concentration ranged between 0 and 100 µM (Pearson correlation coefficient, 0.993). However, when the concentration of resveratrol reached >100 µM, the cell proliferation index decreased and the proliferation of cells was inhibited. C2C12 myoblast cell proliferation stabilized at increased doses of resveratrol to 165±7.2% (Fig. 1A) of the control group at 100 µM and the BrdU positive rate significantly increased by 18.75% (Fig. 1E).

Subsequently, the effect of different concentrations of NAM on cell growth of C2C12 myoblast cells was detected. It was revealed that following treatment of C2C12 myoblast cells with NAM at different concentrations of 10, 20, 40, 50 and 60 mM, the cell proliferation index was significantly lower compared with that of the untreated control group (P<0.05; Fig. 1B). In addition, the results revealed that the survival rate of C2C12 myoblast cells decreased with the increase of NAM concentration in a dose-dependent manner in the NAM concentration range of 0–60 mM. C2C12 myoblast cell viability reached 34±8.8% of the untreated cells (Fig. 1B) at 50 mM and the BrdU positive rate was 18.75% (Fig. 1E). However, when the concentration of NAM reached >20 mM, the cell proliferation rate was rather lower and the cell condition steadily deteriorated (Fig. 1B).

It has been demonstrated that SB431542 stimulates proliferation and differentiation, and inhibits the activity of TGF-β1 and activin receptor-like kinases (ALKs). It is a selective and potent inhibitor of the phylogenetically related subset of ALK-4 (activin type I receptor) and ALK-5 (TGFβ type I receptor). The results showed that following treatment with SB431542 at different concentrations of 2, 4, 5 and 10 µM, the cell proliferation index was significantly higher compared with that of the control group (P<0.05; Fig. 2A). The total number of cells (255±10.27; Fig. 1D) and the BrdU positive rate of 47.73±1.26% were both significant compared with the control group (Fig. 1E).

In order to investigate the role of the myostatin signaling pathway in Sirt1-mediated regulation of C2C12 myoblast cell proliferation, the present study divided C2C12 myoblast cells into four groups and treated with SB431542 (10 µM), resveratrol (100 µM), NAM (50 mM) and a combination of SB431542 (10 µM) and NAM (50 mM). The cell growth of C2C12 myoblast cells was then assessed after 24 h treatment. Consistent with previous studies, the present results showed that following treatment with SB431542, the cell proliferation index (Fig. 2A) and BrdU positive rate (Fig. 1E) were significantly higher compared with that of the control group (P<0.05). Following treatment with SB431542 and NAM, the BrdU positive rate and cell viability were significantly higher compared with that of NAM alone (P<0.05; Figs. 1E and 2B, respectively). This indicated that Srit1 inhibited the expression of myostatin. By contrast, the inhibition of Sirt1 with NAM may promote the expression of myostatin, therefore, the inhibition of myostatin by SB431542 may release the effect of Sirt1 inhibition.

To confirm the role of Sirt1 in C2C12 cell proliferation, the present study measured the mRNA expression of Sirt1. As shown in Fig. 2C, the mRNA expression of Sirt1 was upregulated by resveratrol treatment (P<0.05), and was significantly downregulated by NAM treatment (P<0.05). Notably, the mRNA expression of Sirt1 was significantly higher in cells treated with SB431542 and NAM compared with in cells treated with NAM alone (P<0.05); however, no significant difference was observed in the mRNA expression of Sirt1 between control cells and cells treated with SB431542 and NAM (P<0.05). These data demonstrated that resveratrol, NAM and SB431542 combined with NAM induced cell proliferation by the activation or inhibition of Sirt1 expression.

MyoD is a myogenic regulatory factor critical for myogenic specification. Previous studies (19,20) reported that myostatin downregulates the expression of MyoD, which indicates that MyoD is the downstream target of myostatin. The members of the retinoblastoma (RB) family include RB1/P105, RB2/P130 and RBL1/P107 (21–23), and these genes encode retinoblastoma protein (pRb). P107 belongs to a subline of pRb and has numerous common functions with pRb (24). Additionally, certain research has shown that myostatin may also be involved in the inhibition of myoblast by the RB1 gene, which can be regarded as an indicator of Sirt1 improving C2C12 proliferation via myostatin. Myostatin can inhibit the proliferation of myoblast cells by controlling the transition of G1 to S via the phosphorylation and inactivation of pRb (17). To determine whether Sirt1 mediated the regulation of C2C12 myoblast cell proliferation via the myostatin signaling pathway, the present study measured the mRNA expression levels of MyoD and myostatin following treatment with resveratrol, NAM or the combination of SB431542 and NAM. Treatment with resveratrol significantly upregulated the mRNA expression of MyoD (P<0.05), while the treatment of NAM significantly downregulated the expression of MyoD (P<0.05), and the expression of MyoD of C2C12 cells treated with SB431542 and NAM was higher compared with those treated with NAM alone (P<0.05) (Fig. 2C). However, the mRNA expression of MyoD in the C2C12 cells treated with SB431542 and NAM showed no notable change compared with the NAM group. Treatment with resveratrol significantly downregulated the mRNA and protein expression levels of myostatin (P<0.05; Figs. 2C, 3A and B). Treatment with NAM significantly downregulated the protein expression of p-P107 (P<0.05; Fig. 3A and C), and significantly upregulated the protein expression levels of myostatin and P107 (Fig. 3B and D). The expression of p-P107 was significantly higher in cells treated with SB431542 and NAM compared with those treated with NAM alone. By contrast, the expression of P107 was significantly lower in cells treated with SB431542 and NAM compared with those treated with NAM alone (P<0.05).