| Objective:This study is the C2C12 myotube as the research object,to observe the effects of electrical stimulation and pyrroloquinoline quinone(PQQ)supplementation on the expression of genes related to mitochondrial biogenesis and their upstream signaling molecules in the C2C12 myotube.To further understand action of electrical stimulation and pyrroloquinoline quinone supplementation on mitochondrial biogenesis at cellular level,In order to provide experimental basis for the application of pyrroloquinoline quinone in exercise.Methods:1 The C2C12 cells were incubated with different concentrations of PQQ(0,62.5,125,250,500,1000,2000 nmol/L)for 12,24,48 and 72 hours,and examine of cell viability by CCK8,to determination the cell viability of C2C12 cells differentiate for 5days incubated with PQQ at 125 nmol/L for 24 hours by CCK8.2 The C2C12 cells differentiate for 6 days were electrical stimulated by 45 V,20 ms,5 Hz of electrical stimulation at different time(0,15,30,60,90,120,150,180,210,240min),and examine of ROS with reactive oxygen species Kit.and examine of LDH in medium with lactate dehydrogenase Kit in C2C12 myotube were electrical stimulated at60 min and 120 min.3 The C2C12 cells differentiate for 5 days incubated with PQQ at 125 nmol/L for 24 hours,then was electrical stimulated at 60 min and 120 min by 45 V,20 ms,5 Hz.The experiment was divided into six groups:normal control group(NC group)、 PQQ incubating group(NC+PQQ group)、60min electrical stimulation group(E1H group)、60min electrical stimulation group and PQQ incubating group(E1H+PQQ group)、120min electrical stimulation group(E2H group)、120min electrical stimulation group and PQQ incubating group(E2H+PQQ group).Examine of PGC-1a,NRF1,TFAM,SIRT1 mRNA expression in each group by qRT PCR.Examine of PGC-1a,NRF1,AMPK,pAMPK,SIRT1 protein expression in each group by Westren blot;Results:1 Compared with control group,the cell viability of C2C12 cells increasedsignificantly with PQQ incubated at the concentration of 62.5,125,250,500,1000,2000nmol/L,which cell viability of PQQ incubated with 125nmol/L for 24 h was the most significant(P<0.01).Compared with the control group,the cell viability of C2C12 myotube with PQQ incubated 125 nmol/L for 24 hours showed no significant change(P <0.05).2 Compared with the control group,the fluorescence intensity of ROS in the myotube of C2C12 was significantly increased at 15、30、60、90、120、180、210、240min(P<0.05),which showed that the fluorescence intensity of ROS in the muscle tube of C2C12 was increased from the beginning of electrical stimulation to the increase of30 min,the decrease of 30 min to 60 min,the increase of 60 min to 120 min,and the decrease of 60 min to 120 min after 120 minutes of electrical stimulation,which reached the peak value at 120 min.However,there was no significant change in LDH activity in the medium stimulated by electric stimulation for 60 min and 120 min(P < 0.05).3 Compared with NC group,the expression of PGC-1a,NRF1,TFAM mRNA and(or)protein related to mitochondrial biogenesis in C2C12 myotube of NC+PQQ group,E1H+PQQ group,E2 H group and E2H+PQQ group were significantly increased(P<0.05 or P<0.01).Compared with NC+PQQ group,the expression of PGC-1a,NRF1,TFAM mRNA and(or)protein in E1H+QQ group and E2H+PQQ group were significantly increased(P<0.05 or P<0.01).Compared with E1 H group,the expression of PGC-1a,NRF1,TFAM mRNA and(or)protein in E1H+PQQ group and E2 H group were significantly increased(P<0.05 or P<0.01).Compared with E2 H group,The expression of PGC-1a,NRF1,TFAM mRNA and(or)protein in E2H+PQQ group was significantly increased(P<0.05 or P<0.01).4 Compared with NC group,the expressions of pAMPK/AMPK protein and SIRT1 gene and protein in C2C12 myotubes of NC + PQQ group,E1 H + PQQ group,E2 H group and E2 H + PQQ group were significantly increased(P < 0.05 or P <0.01).Compared with NC+PQQ group,the expression of pAMPK/AMPK protein and SIRT1 mRNA and protein in E1H+PQQ group and E2H+PQQ group increased significantly(P<0.05 or P<0.01).Compared with E1 H group,the expression of pAMPK/AMPK protein and SIRT1 mRNA and protein in E1H+PQQ group increasedsignificantly(P < 0.05 or P < 0.01),while the expression of pAMPK/AMPK protein and SIRT1 gene and protein in E2 H group increased,but there was no significant change(P <0.05).Compared with E2 H group,the expression of pAMPK/AMPK protein and SIRT1 mRNA and protein in E2H+PQQ group increased significantly(P < 0.05 or P < 0.01).Conclusions:1 Electrical stimulation can induce mitochondrial biogenesis by up-regulating the expression of PGC-1a-NRF1-TFAM,the key signaling pathway of mitochondrial biogenesis in C2C12 myotubes.The effect of electrical stimulation is significant with the prolongation of electrical stimulation time,which may be related to the increase of reactive oxygen species.2 PQQ supplementation can also induce mitochondrial biogenesis by up-regulating the expression of PGC-1a-NRF1-TFAM,the key signaling pathway of mitochondrial biogenesis in C2C12 myotubes,while electrical stimulation and PQQ supplementation have synergistic effects on mitochondrial biogenesis.3 Mitochondrial biogenesis induced by electrical stimulation and PQQ supplementation may be mediated by AMPK-SIRT1-PGC-1a signaling pathway,and its mechanism needs to be further explored. |
