Study On The Mechanism Of Mitochondrial Biorhythm In Skeletal Muscle Myogenic Differentiation Damaged By Oxidative Stress

Objectives: In this study,C2C12 myoblasts were used as the research object to prepare oxidative stress model cells by exogenous tert-butyl hydroperoxide,and the dynamics of mitochondrial homeostasis and the expression of circadian clock-related proteins during myogenesis were observed.To explore the mechanism of oxidative stress on mitochondrial biological rhythm in the process of myogenic differentiation.Intervene in mitochondrial homeostasis to verify whether mitochondria can reversibly regulate the clock.Research methods:(1)Exogenous tert-butyl hydroperoxide(t-BHP)grope for C2C12 myoblasts in different concentration gradients(0,20,40,60,80,100,and 120 μM),MDA Assays evaluate cellular oxidative stress levels to determine the concentration of t-BHP that causes oxidative stress.(2)Exogenous t-BHP regulates mitochondrial homeostasis during myogenic differentiation: C2C12 myoblasts were divided into control group and t-BHP treated group,horse serum was added to induce myogenic differentiation,t-BHP treated group At the beginning of differentiation,cells were added with 100 μM t-BHP,and 48 h after the initiation of differentiation was defined as ZT0.The relevant indicators for cell detection were collected on ZT0,ZT4,ZT8,ZT12,ZT16,ZT20 and ZT24,respectively.Western-blotting was used to detect the expression of Mfn2,OPA1,Drp1,COXIV,PGC-1α and Bmal1,and the expression of Myo D m RNA was detected by fluorescent quantitative PCR.(3)Mitochondrial homeostasis intervention experiments: C2C12 myoblasts were divided into 4 groups: empty plasmid control group,OPA1 si RNA group,DMSO control group,Drp1 inhibitor group.Horse serum was added to initiate myogenic differentiation.C2C12 myoblasts were differentiated and incubated with tert-butyl hydroperoxide.ZT0 was defined at 48 h after the initiation of differentiation.The cells were harvested every 4 hours.They were ZT0,ZT4,and ZT8,respectively.ZT12,ZT16,ZT20,ZT24.Experimental group: control group and t-BHPtreated group;Western-blotting wasused to detect the expression of Mfn2,OPA1,Drp1,COXIV,PGC-1α and Bmal1,and the expression of Myo D in myogenic differentiation by fluorescence quantitative PCR.At the beginning of differentiation,the blank plasmid control group was added with the blank interference fragment,the OPA1 si RNA group was added with the OPA1 si RNA interference fragment,the DMSO control group was added with DMSO solution,the Drpl inhibitor group was added with 50 μM of Mdivi1.48 hours after differentiation,the relevant indicators for cell detection were collected.Western-blotting was used to detect the expression of OPA1,Drp1 and Bmal1 protein.Real-time fluorescence quantitative PCR was used to detect Myo D and PGC-1α.Mitochondrial force was used to detect reactive oxygen species and membrane potential.Results:(1)In the mitochondrial rhythm experiment in the regulation of myogenic differentiation by exogenous t-BHP,JTK-CYCLE algorithm analysis showed that the control group(Mn SOD,Mfn2,OPA1,L-OPA1,S-OPA1,Drp1 The expression of COXIV,PGC-1α,OMA1 and Bmal1)protein and the expression of Myo D gene had significant biological rhythms.The expression levels of Mn SOD,OPA1,COXIV,PGC-1α,OMA1,S-OPA1,protein and Myo D gene were all significant.The wave peaks were significantly higher than the mean(p<0.05).The trough values of Drp1,L-OPA1,OMA1,Drp1,and Bmal1 were significantly lower than the mean(p<0.05),and the gene expression of Myo D was correlated with Mn SOD.The expression of Mfn2,OPA1,L-OPA1,S-OPA1,OMA1,Drp1,COXIV,PGC-1α and Bmal1 all showed a significantly lower trough than the peak(p<0.05).The t-BHP treated group(Myo D gene expression,Mn SOD,Mfn2,OPA1,L-OPA1,S-OPA1,OMA1,Drp1,COXIV,PGC-1α,and Bmal1 protein expression)did not show a significant biological rhythm,Myo D gene The expression of Mn SOD,Mfn2,OPA1,L-OPA1,S-OPA1,OMA1,Drp1,COXIV,PGC-1α and Bmal1 showed no significant difference(p>0.05)in the expression of peaks,troughs and mean values.Compared with the control group,the total expression level of the gene expression of Myo D,Mn SOD,Mfn2,OPA1,L-OPA1,S-OPA1,OMA1,Drp1,COXIV,PGC-1α and Bmal1 was significantly increased in the t-BHP treated group.Decreased(p<0.05-0.01)(2)In the mitochondrial homeostasis experiment,compared with the empty plasmid group,the ROS in the OPA1 si RNA group was significantly higher(p<0.05),and the membrane potential(p<0.05-0.01).The expression levels of PGC-1α and Myo D and protein expression of Bmal1,OPA1 and Drp1 were significantly decreased(p<0.05),and the membrane potential of Drp1 inhibitor group was significantly higher than that of DMSO control group(p<0.05-0.01).There was no significant change in the gene expression of Myo D and the expression of protein Bmal1(p>0.05),and the gene level and reactive oxygen species of PGC-1α were significantly decreased(p<0.05).Conclusions:1.During the process of myogenic differentiation of C2C12 cells,the mitochondrial homeostasis(biosynthesis,fusion/division)and myogenic differentiation-related factor expression showed a significant biological rhythm.In this study,exogenous tert-butyl hydroperoxide was used to establish C2C12 cell oxidative stress model,and it was proved that oxidative stress leads to the abnormal or disappearance of mitochondrial steady-state biological rhythm in the process of myogenesis.In this study,OPA1 si RNA and Drp1 inhibitors were used to establish a mitochondrial homeostasis model for C2C12 cells,demonstrating that mitochondrial homeostasis inhibits the expression of circadian clock genes,and the specific mechanism needs further study.