Effect of Different Concentrations of Salidroside on Myoblast Differentiation in Vitro and Its Preliminary Mechanism

Objective:This study is aim to explore the effect of different concentrations of salidroside on myoblast differentiation in vitro and its possible mechanism,which can help to find a more effective exogenous regulating substance for myoblast differentiation to promote the myoblast transplantation,and set the foundation for further study of the effect of salidroside on skeletal muscle in vivo.Methods:Part one:The effect of different concentrations of salidroside on myoblast differentiation in vitro:1)observe the morphology of differential myoblast C2C12 which were treated with 0,30,50μg/ml salidroside to induce differentiation by Phase micrographs;2)Detect the cell fusion index affected by salidroside using immunofluorescence;3)Detect the expressions of myogenic differentiation-specific mRNAs and proteins affected by salidroside using Western Blotting and real time-PCR.Part two:The possible mechanism of above study :Detect TGF-β/Smad3 pathway protein expression in differential C2C12 cell treated with 0,30,50μg/ml Sal using Western Blotting.Results:Treated with 50μg/ml salidroside:Part one:1)Salidroside inhibit C2C12 fusion and myotube form in morphological by Phase micrographs;2)Myosin-neonatal Positive area and myogenin Positive Nuclei decreased significantly by immunofluorescence(P<0.05),which inhibited myotube form notably;3)The expressions of MyoD and myogenin in mRNA and protein both decreased notably by real-time PCR and Western blotting(P<0.05),which inhibited cell fusion and myotube form during C2C12 differentiation.Part two:Salidroside inhibit C2C12 differentiation associated with increases in Smad2/Smad3 phosphorylation,especially p-Smad3(P<0.01).The results treated with 30μg/ml salidroside is insignificant(P>0.05).Conclusion:1)Salidroside inhibit myogenic differentiation of C2C12 cell significantly,and promote cell proliferation,to add satellite cells band reserve.2)Salidroside inhibits myogenic differentiation in a dose-dependent manner,50μg/ml Salidroside is the best.3)Salidroside inhibits myogenic differentiation possibly by activating Smad2/Smad3(a key mediator in TGF-β/Smad pathway)phosphorylation.