Effect of constant compressive stress induced by imitating tuina stimulation with various durations on the cell cycle,cellular secretion,apoptosis,and expression of myogenic differentiation and myogenic factor 5 of rat skeletal muscle cells in vitro

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 2000μstrain 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,M yoD 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.

Gyejibokryeong-Hwan improves recovery of injured muscles in mouse model of muscle contusion

OBJECTIVE: To investigate the effects of Gyejibokryeong-Hwan(Guizhifuling-wan, GBH) on muscle injury in a mouse model of muscle contusion.METHODS: C57/BL6 mouse biceps femoris muscles were injured using the drop-mass method and injured animals were treated orally with GBH(50,100, or 500 mg/kg) once a day for 7 d. Open field and treadmill running tests were performed to assess functional recovery from muscle injury. The production of pro-inflammatory cytokines was examined by enzyme-linked immunosorbent assay and Western blotting analysis. Expression of the muscle regeneration biomarkers, myoblast determination(Myo D), myogenic factor 5(Myf5), and smooth muscle actin(α-SMA), in the biceps femoris muscle was investigated at the protein and m RNA level by Western blotting and real time-PCR, respectively. Histological analysis was performed using hematoxylin and eosin staining. Finally, myosin heavy chain production was investigated in differentiated C2C12 myoblasts in the presence of GBH.RESULTS: GBH treatment markedly improved locomotion and running behavior. GBH significantly inhibited the secretion of monocyte chemoattractant protein-1 into the bloodstream in muscle-contused animals. The levels of Myo D, Myf5, and α-SMA protein and m RNA were significantly up-regulated by GBH in injured muscle tissue. Histological studies suggested that GBH facilitated recovery from muscle damage. However, GBH did not induce the production of myosin heavy chain in vitro.CONCLUSION: Overall, the present study suggested that GBH improves the recovery of the injured muscles in the mouse model of muscle contusion.