Effects Of EPA And DHA On Proliferation, Differentiation And Apoptosis In C2C12 Myoblasts

This study was conducted to elucidate the biological effects of n-3 polyunsaturated fatty acids(n-3 PUFA)on the proliferation,differentiation,and apoptosis of C2C12 myoblasts,and explore its mechanism by studying the global gene expression patterns and the mitogen activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK)1/2 and phosphatidyl inositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathways in C2C12 myoblasts.1.This experiment was conducted to investigate the effects of eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA)on the proliferation,differentiation,and apoptosis of C2C12 myoblasts.After C2C12 myoblasts were treated with EPA or DHA at different concentrations(0,6.25,12.5,25,50 or 100 ?M)for 12,24,48 or 72 h,cell proliferation was detected by CCK-8 kit.Inducing differentiation of myoblasts and treating C2C12 myoblasts at different concentrations(0,6.25,12.5,25,50,or 100 ?M)with EPA or DHA for 48 or 72 h.The expression of myogenic marker genes were assessed at the transcriptional and translational levels by using qRT-PCR,immunoblotting and immunofluorescence.Apoptosis was detected by TUNEL after C2C12 myoblasts treated with EPA or DHA at different concentrations(0,50,or 100 ?M)for 48 h.The results showed that: Cell growth was significantly inhibited after incubation with EPA(50 or 100 ?M)for 48 h,or with DHA(100 ?M)for 72 h.EPA and DHA inhibited the expression of differentiation marker genes MHC,Myogenin,and skeletal ?-actin in C2C12 myoblasts in a dose-dependent manner.And the induced inhibition of myoblast differentiation was not accompanied by a transdifferentiation process in the early stage of EPA or DHA treatment.In addition,high concentration of EPA could promote apoptosis,but the effect of DHA on apoptosis was not obvious.These results indicate that EPA and DHA can inhibit the proliferation and differentiation and promote apoptosis of C2C12 myoblasts.2.This experiment was conducted to investigate the effects of EPA and DHA on the gene expression profile and the MAPK/ERK1/2 and PI3K/Akt signaling pathways in C2C12 myoblasts in order to explore the mechanisms.After C2C12 myoblasts were treated with 50 ?M EPA or DHA for 48 h,the cells were harvested for RNA-sequencing and data analysis.The mRNA expression levels of the selected genes were determined.Besides,after C2C12 myoblasts were treated with 50 ?M EPA or DHA for 1 or 6 h,the levels of ERK1/2 and Akt phosphorylation were assessed by immunoblotting.The results showed that: 50 ?M of EPA or DHA treated C2C12 myoblasts for 48 h resulted in a large number of mRNA changes,and EPA treatment had a more significant effect on the gene expression in C2C12 myoblasts.By using qRT-PCR to verify six significantly down-regulated genes screened by RNA-sequencing,we found that their mRNA expressions were all significantly reduced,which was consistent with the RNA-sequencing results.Furthermore,the down-regulation of EPA was more obvious.In addition,after C2C12 myoblasts were treated with 50 ?M EPA or DHA for 1 or 6 h,the phosphorylated protein levels of ERK1/2 and Akt were significantly reduced.These results indicate that EPA and DHA significantly down-regulate the expression of muscle-specific genes in C2C12 myoblasts,which may be related to their inhibitory effects on differentiation.Meanwhile,EPA and DHA may inhibit myoblast proliferation,and differentiation,promote apoptosis through the MAPK/ERK1/2 and PI3K/Akt signaling pathways.