Low Frequency Pulsed Electromagnetic Field Promotes C2C12 Myoblasts Proliferation Via Activation Of MAPK/ERK Pathway

Background:As a non-invasive,simple,and less side effect method,low frequency pulsed electromagnetic field(PEMF)has been shown to have therapeutic effects on some clinical diseases,such as osteoporosis,fracture nonunion,cartilage injury and so on.Some other works have shown that PEMF can accelerate angiogenesis,peripheral nerve regeneration and repairation of tendon injury.It is not difficult to find that PEMF has a definite role in treating locomotor system disorders.Some clinical and in vivo studies have verified that electromagnetic stimulation could retard denervated muscular atrophy and reduce muscle weakness and girth loss,however,few studies involving effects of PEMF on skeletal muscle have been conducted.Although,PEMF has been shown to affect the activity of various cell types and promote them proliferation,its effect on skeletal muscle cells remains to be determined.Objective:Our study aims to save the following problems on C2C12 myoblasts,an in vitro model of skeletal muscle cells.The first is whether PEMF can promote C2C12 myoblasts proliferation.The second is whether PEMF can activate some MAPK signaling pathway.The third is to find out whether the proliferation of C2C12 cells induced by PEMF results from the activation of some MAPK signaling.Methods:For PEMF exposure,cells were exposed to a sinusoidal 100 Hz PEMF with the density of 1 mT in a cell incubator.To compare the proliferaton of C2C12 cells between PEMF and control groups,we used Cell Counting Kit-8(CCK-8)and 5-Ethynyl-2'-deoxyuridine(EdU)assays.Besides,we tested the cell apoptosis rate between PEMF and control groups by flow cytometry(Annexin V-FITC/PI double staining method).To further study the mechanism of action of PEMF,Western blot was utilized to detect the mitogen-activated protein kinase(MAPK)signaling pathways,we tested the phosphorylation level of extracellular signal-regulated kinase(ERK),p38 MAPK and c-Jun N-terminal kinase(JNK)after exposed to PEMF for 4 h,2 h,1 h,0.5 h,0.25 h,0 h respectively.At last,if we proved that PEMF could promote the proliferation of C2C12 myoblasts and activate some MAPK signaling pathway,we could use an specific MAPK inhibitor to further observe whether it could block PEMF induced activation by CCK-8 and EdU assays.Results:First of all,PEMF promoted the proliferation but did not affact the apoptosis of C2C12 myoblasts.Secondly,MAPK/ERK signaling pathway was activated by PEMF,and the phosphorylation level of ERK1/2 reached its peak in 30 min.Thirdly,C2C12 myoblasts proliferation induced by PEMF required activation of ERK pathway.Conclusion:Our research for the first time demonstrated that PEMF promoted C2C12 myoblasts proliferation via activating MAPK/ERK pathway.