Effect Of Cordycepin On Skeletal Muscle C2C12 Cell Differentiation In Vitro And Its Mechanism

Objective: Cordycepin,potent active components Cordyceps militaris,is known to modulate skeletal muscle motility.Despite the significant impact of cordycepin on skeletal muscle function,and the cellular-molecular mechanisms underlying its regulation remain poorly studies.Thus,in this study,we used cultured mouse C2C12 cells as a mouse C2C12 cells were cultured as an in vitro model to examine the effects of varying concentrations of cordycepin on the differentiation of skeletal muscle C2C12 cells and explore the potential molecular mechanisms.Methods: 1.Cordycepin was used to determine the safe concentrations on C2C12 cells by MTT assay;2.The effects of cordycepin on the morphology of myotubes during in vitro differentiation process of C2C12 cells was evaluated by observing the tube’s morphology at different concentrations and time;3.Immunofluorescence analysis was conducted to evaluate the effect of different concentrations and time points of cordycepin on the fusion rate of muscle tubes during the in vitro differentiation process of C2C12 cells;4.Fluorescence probes were utilized to assess the impact of different concentrations and time points of cordycepin on energy metabolism and oxidative stress during the in vitro differentiation process of C2C12cells;5.Protein immunoblotting and real-time fluorescent quantitative PCR were employed to investigate the impact of different concentrations and time points of cordycepin on myogenic differentiation genes and protein expression during the in vitro differentiation of skeletal muscle C2C12 cells;6.Protein immunoblotting and real-time fluorescence quantitative PCR were utilized to investigate the impact of different concentrations and time points of cordycepin on myogenic differentiation genes and protein expression during the in vitro differentiation of skeletal muscle C2C12 cells,as well as to assess the changes in ERK1/2 MAPK signaling pathway protein levels.Additionally,the potential mechanism of the impact of cordycepin on the in vitro differentiation of skeletal muscle C2C12 cells was further validated using the ERK1/2 MAPK inhibitor PD98059.Results: 1.The differentiation of skeletal muscle C2C12 cells was significantly inhibited by cordycepin at concentrations of 10 μM and 20 μM,resulting in a notable decrease in myotube diameter and fusion rate;2.Cordycepin led to a reduction in myotube formation and MHC-positive nuclei during the in vitro differentiation of skeletal muscle C2C12 cells;3.Intracellular reactive oxygen content was down-regulated,while mitochondrial membrane potential levels and ATP content were increased by cordycepin during the inhibition of in vitro differentiation of skeletal muscle C2C12 cells;4.Myf5 mRNA expression was up-regulated,while the expression of MyoD and Myogenin mRNA,which are genes related to myogenic differentiation,were down-regulated by cordycepin;5.The expression levels of Myf5 and p130 proteins were up-regulated,while those of MyoD and Myogenin proteins were down-regulated by cordycepin.The reserve cells were characterized by high expression of Myf5 and p130 and low expression of MyoD.6.Cordycepin played a role in regulating the expression levels of genes and proteins related to the ERK1/2MAPK signalling pathway,leading to the inhibition of myogenic differentiation;7.The inhibitory effect of cordycepin on skeletal muscle C2C12 cell differentiation in vitro was significantly blocked by the use of the ERK1/2 MAPK signalling pathway inhibitor PD98059.Moreover,the down-regulation of MyoD and MyoG protein expression and the up-regulation of Myf5 and p130 protein expression induced by cordycepin were simultaneously reversed.Conclusions: 1.The present study demonstrates that cordycepin significantly curbs myogenic cell differentiation while increasing satellite cell reservoirs to regulate skeletal muscle motor function,potentially contributing to the anti-fatigue effects attributed to cordycepin;2.Cordycepin was found to lower oxidative stress and boost energy reserves during myogenic cell differentiation,thereby enhancing skeletal muscle capacity;3.The regulation of the ERK1/2 MAPK signaling pathway by cordycepin affects myogenic cell action,ultimately resulting in the observed effects.The findings of this study will advance our understanding of cordycepin in the field of skeletal muscle exercise physiology,with potential implications for future research and application development.The results of this research are poised to deepen our comprehension of cordycepin in the realm of skeletal muscle exercise physiology,providing opportunities for future research and the development of novel applications.