Effects Of High Expression Of INOS On Myogenesis Of Satellite Cells Of Levator Anal Muscle In Rats And Its Study

Objective:To investigate the effect of high expression of inducible nitric oxide synthase(iNOS)on myogenesis of rat levator anorectus satellite cells(satellite cell,SC),and to reveal the effect of iNOS-p38-MAPK-HGF signaling pathway on SC myogenesis.Methods:High expression of iNOS satellite cells and normal muscle satellite cells were cultured in vitro after cryopreservation of liquid nitrogen.After culture,some high expression of iNOS satellite cells were added into Smethylisothiourea(SMT),a drug with specific inhibitory effect on iNOS,and were divided into three groups:normal muscle satellite cell group,high expression of iNOS satellite cell group and high expression of iNOS satellite cell+SMT group.At the same time,apoptosis and necrosis were detected by flow cytometry,proliferation ability was measured by MTS and cell cycle was detected by flow cytometry.Then we analyzed the effect of high expression of iNOS on the myogenesis of satellite cells of levator anal muscle.The expression of iNOS,HGF,Pax7,Myod,P38MAPK,Desmin,?-SCA at the protein and mRNA levels was detected by Western Blot(WB)and Real-time quantitative PCR(QPCR),respectively.Results:Apoptosis and necrosis:the ratio of apoptosis and necrosis in high expression of iNOS satellite cells+SMT group was significantly higher than that in normal SC cells and high expression of iNOS satellite cells group.The ratio of apoptosis and necrosis in high expression of iNOS group was lower than that in SC cell group.The ability of cell proliferation:the ability of growth and proliferation of SC cells and high expression of iNOS satellite cells in normal culture were similar to those of high expression of iNOS satellite cells at 7 days,but the proliferation ability of high expression of iNOS satellite cells in SMT group was significantly lower than that in the above two groups.Cell cycle change:G1 phase:the percentage of cells in high expression of iNOS satellite cell group was significantly higher than that in normal SC cell group and high expression of iNOS satellite cell+ SMT group.There was no difference between the normal SC cells and the high expression of iNOS satellite cells+ SMT in this phase.S phase:the cells in the three groups changed significantly in this phase,and the percentage of cells in the high expression of iNOS satellite cells group was significantly lower than that in the normal SC cells during this phase.The percentage of high expression of iNOS satellite+SMT cells in this phase was significantly higher than that of normal SC cells and significantly higher than that of high expression of iNOS satellite cells.G2 phase:G2 phase cells showed no significant change compared with the three groups of cells in G2 phase.The percentage of normal SC cells in this phase was slightly higher than that of high expression of iNOS satellite cells,and there was no significant difference among other groups.Western Blot and Real-time quantitative PCR in detecting the expression of related protein molecules in levator anal muscle of rats:compared with normal SC cells and SMT transfected with iNOS satellite cells,iNOS,HGF,Myod,P38MAPK,Desmin,was transformed into iNOS satellite cells.The expression level of a-SCA was increased and the expression of Pax7 was decreased.Conclusion:The results showed that iNOS promoted the myogenesis of SC,and iNOS mediated SC activation,thus promoting the differentiation of satellite cells,and confirmed the effect of iNOS-p38-MAPK-HGF signaling pathway on the myogenesis of satellite cells.