OBJECTIVE: To investigate the effect of constant compressive stress induced by imitating Tuina stimulation with various durations on the cell cycle,cellular secretion,apoptosis,and expression of myogenic regulatory factors (MRFs),myogenic factor 5 (Myf5) and myogenic differentiation (MyoD) of rat skeletal muscle cells (RSkMCs) in vitro.METHODS: Third passage RSkMCs were subjected to constant compressive stresses with various durations at 2000 μstrain for 15,30,60,90,and 120 min via a four-point bending system.The control group (CG) was cultured in the absence of mechanical loading.Alterations of the cell cycle and apoptosis rate were detected by flow cytometry (FCM).The concentrations of interleukin 6 (IL-6)/prostaglandin E2 (PGE2) and nitric oxide (NO) in supernatants were determined by enzyme-linked immunosorbent assays and the nitrate reductase method,respectively.Expression of Myf5 and MyoD was detected by immunohistochemistry.RESULTS: Compared with the CG,a significant alteration was observed in the synthesis phase fraction (SPF) (P ＜ 0.01).The SPF and proliferation index (PI) were reduced from 15 to 90 min,but reached levels similar to those at 120 min.Apoptosis was increased significantly at 30 min (P ＜ 0.05) and especially at 90 and 120 min (P ＜ 0.01).Expression of MyoD and Myf5 was increased significantly at 15,30,and 90 min (P ＜0.01).Compared with 15 and 30 min,MyoD and Myf5 expression at 60 and 120 min was decreased significantly (P ＜ 0.01).Compared with 60 min,MyoD expression at 90 min was increased significantly (P ＜ 0.05),whereas MyoD and Myf5 expression at 120 min was significantly lower (P ＜ 0.05).The IL-6 concentration was increased at 60 min compared with the CG and 15 min (P ＜ 0.05),whereas the concentrations of PGE2 and NO were the highest at 15 and 30 min,respectively,compared with the CG and other time points (P ＜ 0.05).CONCLUSION: The cell cycle,secretion,apoptosis,and Myf5 and MyoD expression of RSkMCs were regulated by compressive stress in a time-dependent manner.SPF and PI were inhibited at short durations (＜ 90 min),but NO and PGE2 secretion was the highest at shorter durations (＜ 30 min).With the prolongation of stimulation time,SPF,PI,and apoptosis were increased,but Myf5 and MyoD expression was decreased gradually at 15-30 min.