Effect of Deer Antler Extract on Muscle Differentiation and 5-Aminoimidazole-4-Carboxamide Ribonucleoside (AICAR)-Induced Muscle Atrophy in C2C12 Cells

Abstract

The effect of deer antler extract on muscle differentiation and muscle atrophy were evaluated to minimize muscle loss following aging. Various deer antler extracts (HWE, hot water extract of deer antler; FE, HWE of fermented deer antler; ET, enzyme-assisted extract of deer antler; UE, extract prepared by ultrasonication of deer antler) were evaluated for their effect on muscle differentiation and inhibition of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced muscle atrophy in C2C12 cells. Morphological changes according to the effect of antler extracts on muscle differentiation were confirmed by Jenner-Giemsa staining. In addition, the expression levels of genes related to muscle differentiation and atrophy were confirmed through qRT-PCR. In the presence of antler extracts, the length and thickness of myotubes and myogenin differentiation 1 (MyoD1) and myogenic factor 5 (Myf5) gene expression were increased compared to those in the control group (CON). Gene expression of AMP-activated protein kinase (AMPK), MyoD1, and myogenin, along with the muscle atrophy factors muscle RING finger-1 (MuRF-1) and forkhead box O3a (FoxO3a) upon addition of deer antler extracts to muscle-atrophied C2C12 cells was determined by qRT-PCR after treatment with AICAR. The expression of MuRF-1 and FoxO3a decreased in the groups treated with antler extracts compared to that in the group treated with AICAR alone. In addition, gene expression of MyoD1 and myogenin in the muscle atrophy cell model was significantly increased compared that into the CON. Therefore, our findings indicate that antler extract can increase the expression of MyoD1, Myf5 and myogenin, inhibit muscle atrophy, and promote muscle differentiation.

Keywords: 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); C2C12 cells; deer antler; muscle atrophy; muscle differentiation.

Fig. 1.. Cell viability of deer antler extracts on C2C12 cells.

Values are presented as the mean±SD for each group. CON, control group; HWE, hot water extract; FE, how water extract of fermented deer antler; ET, enzyme-derived extract; UE, extract prepared by ultrasonication of deer antler.

Fig. 2.. Changes in myotube structure during C2C12 cell differentiation, as observed through Jenner-Giemsa staining.

Values are presented as the mean±SD for each group. CON, control group; HWE, hot water extract; ET, enzyme-derived extract; UE, extract prepared by ultrasonication of deer antler; FE, how water extract of fermented deer antler.

Fig. 3.. Changes in myotube length and diameter during C2C12 cell differentiation.

Values are presented as the mean±SD for each group. The different letters indicate statistically significant (p<0.05) differences among groups, as determined through Tukey’s test. CON, control group; HWE, hot water extract; ET, enzyme-derived extract; UE, extract prepared by ultrasonication of deer antler; FE, how water extract of fermented deer antler.

Fig. 4.. Effects of deer extracts on the relative expression of MyoD1 and Myf5 mRNA in C2C12 cells at two and four days of cell differentiation.

Values are presented as the mean±SD for each group. The different letters indicate statistically significant (p<0.05) differences among groups, as determined by Tukey’s test. CON, normal control; HWE, hot water extract; ET, enzyme-derived extract; UE, extract prepared by ultrasonication of deer antler; FE, how water extract of fermented deer antler; MyoD1, myogenin differentiation 1; Myf5, myogenic factor 5.

Fig. 5.. Effects of AICAR on relative AMPK mRNA expression in C2C12 cells.

Values are presented as the mean±SD for each group. The different letters indicate statistically significant (p<0.05) differences among groups, as determined by Tukey’s test. CON, control group; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; AMPK, AMP-activated protein kinase.

Fig. 6.. Effects of deer antler extract on relative mRNA expression of AMPK, FoxO3a, and MuRF-1 in AICAR-induced muscle-atrophied C2C12 cells.

Values are presented as the mean±SD for each group. Asterisks indicate significant differences: ^*** p<0.001 indicates significance of difference compared with the control, as determined by Student’s t-test. The different letters indicate statistically significant (p<0.05) differences among groups, as determined by Tukey’s test. AMPK, AMP-activated protein kinase; NOR, normal group; CON, control group; HWE, hot water extract; FE, how water extract of fermented deer antler; ET, enzyme-derived extract; UE, extract prepared by ultrasonication of deer antler; FoxO3a, forkhead box O3a; MuRF-1, muscle RING finger-1; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside.

Fig. 7.. Effects of deer antler extract on relative mRNA expression of MyoD1 and Myogenin in AICAR-induced muscle atrophy in C2C12 cells.

Values are presented as the mean±SD for each group. Asterisks indicate significant differences: ^*** p<0.001 indicates significance of difference compared with the control, as determined by Student’s t-test. The different letters indicate statistically significant (p<0.05) differences among groups, as determined by Tukey’s test. NOR, normal group; CON, control group; HWE, hot water extract; FE, how water extract of fermented deer antler; ET, enzyme-derived extract; UE, extract prepared by ultrasonication of deer antler; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside.