Mimicking L6 Myoblast Quiescence In Vitro And Stretch-induced Crosstalk Between P38 And JNK To Regulate Cell Cycle

Objective: In this study,firstly we generate homogeneous populations of quiescent myoblasts by manipulating culture conditions and cells in G0 phase which can still be able to re-enter the cell cycle.The aim of establishing G0 model is to ensure a homogeneous and novel condition for molecular analysis of quiescence and reactivation of L6 myoblasts.Then,expose L6 myoblasts to appropriate cyclic stretch to clarify the effect of stress on cell cycle induced by the interaction of p38 MAPK and JNK pathway which could activate downstream cell-cycle related proteins.The purpose of this study is to further investigate the mechanism of the adaptive remodeling of the masticatory muscles,and to provide a more perfect theoretical support for the functional orthopedic treatment.Method: L6 myoblasts,as the experimental objects,were induced to G0 phase.The cell cycle and Ki67 protein expression were detected to confirm cells arresting in the G0 phase.Then,reactive quiescent myoblasts by exposing them to proliferation promoting medium containing 10% fetal bovine serum.Samples were extracted to detect the changes of cell cycle,the expression of Ki67 and other cell cycle-related proteins at 6 h,12 h,18 h and 24 h to confirm cells' reactivation.The cells reactivated for 12 hours were used to construct cyclic stretch stimulated model.We applied the cyclic stretch of 15% amplitude and 10 cycles/min frequency on L6 in order to simulate the biological orthodontical force.As well as observing the cell morphology and arrangement direction under stress,we also investigate the difference of cell cycle distribution and whether p38 MAPK(p-p38MAPK)and JNK(p-JNK)pathways are activated(or inhibited).The expression changes of CyclinD1,p21,p57,which are related to cycle regulation downstream are also be included in Western Blot test.Finally,p38 specific inhibitor(SB203580)was used to further clarify the interaction between p38 and JNK and its effect on cell cycle.Results:1.The G0-phase model of rat myoblasts was successfully constructed showing less than2% S-phase cells and complete inhibition of Ki67 expression.2.Quiescent cells re-entered S phase,and the proliferation rate peaked at 12 h.3.The cells re-activated at 12 h were observed that the shape and arrangement direction of cells would adjust with the stress stimulating.4.The stretched cells group showed decreased proportion of S-phase especially at 3 h and6 h.5.The expression of p-p38 MAPK was significantly increased after stretching.On the contrary,the expression of p-JNK in the experimental group was appropriately decreased,and the total JNK content was basically the same.At the same time,the expression of Cyclin D1 was lower in the stressed group than in the control group,while the expression of p21 and p57 were higher in the stress group.6.The p38 inhibitor(SB203580)slightly increased cell proliferation rate(the difference was not statistically significant).While expression of p-p38 was sharply decreased,the expression of JNK was dramatically increased,with increasing of CyclinD1;Interestingly,the expression of p21 was not significantly decreased after using p38 inhibitor.Conclusion: Cyclic stress induced p38-inactivated CyclinD1 pathway to prevent the infinite proliferation of myoblasts.There is a crosstalk between p38 MAPK and JNK,seeing when blocking p38 pathway,JNK is obviously activated.Interestingly,the inactivation of the p38 pathway did not induce a sharp increase in the proportion of S-phase cells.It is speculated that there are other pathways and proteins involved in the regulation of cell cycle in this process.