Abstract:

Objective To discuss the regulatory effect of velvet antler polypeptide on mitochondrial energy metabolism efficiency during myoblast differentiation， and to clarify its related mechanism. Methods The C2C12 myoblasts were maintained in growth medium （GM） and then induced to differentiate in differentiation medium（DM） for 1， 3， and 5 d. The differentiated C2C12 myoblasts were divided into control group and velvet antler polypeptide group. The VAP group was treated with 80 mg·L^-1 velvet antler polypeptide， while the control group was given an equal volume of phosphate buffer saline （PBS）. Cell counting kit-8 （CCK-8） method was used to detect the proliferation rate of the undifferentiated cells in various groups after treated with 0， 5， 10， 20， 40， 60， 80， 120， 160， and 200 mg·L^-1 velvet antler polypeptide； immunofluorescence staining was used to detect the fusion indexes of the cells in various groups； Western blotting method was used to detect the expression levels of myosin， myogenin， myogenin：ubiquinone oxidoreductase subunit B8 （NDUFB8）， mitochondrially encoded cytochrome c oxidase Ⅰ （MT-CO1）， and succinate dehydrogenase complex flavoprotein subunit A （SDHA） proteins in the cells in various groups； 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） fluorescence probe assay was used to detect the reactive oxygen species （ROS） level in the cells in various groups； JC-1 mitochondrial membrane potential assay kit was used to detect the mitochondrial membrane potential of the cells in various groups； commercial kits were used to detect the adenosine triphosphate （ATP） level， glucose uptake， and lactate level in the cells in various groups. Results The CCK-8 results showed that compared with 0 mg·L^-1 velvet antler polypeptide group， the proliferation rates of undifferentiated C2C12 myoblasts in 60， 80， 120， 160， and 200 mg·L^-1 velvet antler polypeptide groups were significantly increased （P<0.05）. The immunofluorescence results showed that compared with control group at the same differentiation stage， the fusion indices of the C2C12 myoblasts in velvet antler polypeptide group at 3 and 5 d of differentiation were significantly decreased （P<0.05）， and the number of multinucleated myotubes was significantly decreased. The Western blotting results showed that compared with control group at the same differentiation stage， the expression levels of myosin in the cells in velvet antler polypeptide group at 1， 3， and 5 d of differentiation were significantly decreased （P<0.05）； the expression levels of myogenin protein in the cells in velvet antler polypeptide group at 3 and 5 d of differentiation were significantly decreased （P<0.05 or P<0.01）. Compared with control group at the same differentiation stage， the expression levels of NDUFB8 and MT-CO1 proteins in the cells in velvet antler polypeptide group at 5 d of differentiation were significantly decreased （P<0.05 or P<0.01）； the expression levels of SDHA protein in the cells in velvet antler polypeptide group at 1， 3， and 5 d of differentiation were significantly decreased （P<0.05 or P<0.01）. The DCFH-DA results showed that compared with control group at the same differentiation stage， the ROS level in velvet antler polypeptide group at 5 d of differentiation was significantly decreased （P<0.01）. The JC-1 assay results showed that compared with control group at the same differentiation stage， the mitochondrial membrane potentials in the cells in velvet antler polypeptide group at 3 and 5 d of differentiation were significantly increased （P<0.05）. Compared with control group at the same differentiation stage， the glucose uptake in the cells in velvet antler polypeptide group at 1 d of differentiation was significantly decreased （P<0.01）， and the glucose uptake at 5 d of differentiation was significantly increased （P<0.05）； there were no significant differences in lactate levels in the cells in velvet antler polypeptide group at 1， 3， and 5 d of differentiation （P>0.05）； the ATP levels in velvet antler polypeptide group at 1 and 3 d of differentiation were significantly increased （P<0.05）. Conclusion Velvet antler polypeptide inhibits the differentiation of the C2C12 myoblasts， and its mechanism may be related to the down-regulation of mitochondrial oxidative phosphorylation complex subunit expression and the improvement of mitochondrial oxidative phosphorylation efficiency.

Key words: Velvet antler polypeptide, Myoblast differentiation, Mitochondria, Aging, Muscular degenerative diseases