The Role Of PI3K/Akt Signaling Pathway In Cyclic Stretch-induced Apoptosis Of Myoblast And Its Regulating Mechanism

Objective: Facial jaw muscle tissue plays an important role in the occurrence,development,treatment and maintenance of maxillofacial deformities.Its structure and function can be reconstructed with the change of external stimuli.It is also the basis of orthodontists to regulate its reconstruction with orthopedic forces.In this research,we study the role the possible mechanism of PI3K/Akt signaling pathway in cyclic stretch-induced myoblast apoptosis.This study will further our understanding of the mechanism of orthopedic therapy and provide a theoretical basis for clinical treatment.Methods: The model of L6 rat myoblasts cultured in vitro-mechanical stimulation was successfully constructed.The multichannel cell stress loading system was used to impose the cyclic stress on L6 rat myoblasts for 2h,6h,12 h and 24 h respectively.The loading frequency was 10 cycles/min(each cycle including 3s-stretch and 3s-relaxation),tensile deformation was 15%,non-stressed group as the control group,all groups of cells should be cultured under the same conditions.The morphological changes and apoptosis of myoblasts under different stress loading time were observed by Hoechst33258 staining.The changes of mitochondrial permeability of myoblasts were detected by flow cytometry.We use western blotting to detect the expression of Akt,p-Akt(Ser473),GSK-3β and p-GSK-3β(Ser 9).To clarify the role of PI3K/Akt signaling pathway in stretch-induced apoptosis of myoblasts,we apply PI3 K kinase inhibitor LY294002 to pretreatment myoblasts in each group,and detect the rate of myoblast apoptosis,mitochondrial permeability of myoblasts and the expression of proteins above-mentioned.The experimental data were analyzed by SPSS17.0.Result:1.Hoechst33258 fluorescence staining showed that L6 rat myoblasts were apoptotic under the tensile stress of 15% and 10 cycles/min,the apoptotic rate was increased in a time-dependent manner,and peaked at 24 h.The apoptotic cells in groups treated with LY294002 had a increase in contrast with group loading stress only.2.Mitochondrial permeability transition hole flow detection results: With the extension of the force,the Calcein fluorescence quenching rate increased gradually,and reached the maximum at 24 h;There were statistically significant differences between control group and stretched groups(p <0.01);The fluorescence quenching rate in groups treated with LY294002 was higher than that in the group loading stress only(p <0.05).3.Western blot showed that the protein expression of total Akt and GSK-3β had no statistical difference between control group and stretched groups,while the protein expression of p-Akt(Ser473)and p-GSK-3β(Ser-9)decreases gradually with the extension of time subjected to mechanical stretch,There were statistically significant differences between control group and stretched groups(p <0.01).After adding PI3 K inhibitor,the expression of total Akt and GSK-3β had no statistical difference between each group,but the expression of p-Akt(Ser473)and p-GSK-3β(Ser-9)in inhibitor groups decrease contrast with groups loading stress only(p <0.05).Conclusion:1.Cyclic stretch can induce apoptosis of myoblsts.2.PI3K/Akt signaling pathway is involved in cyclic stretch-induced apoptosis of myoblasts.3.PI3K/Akt/GSK-3β plays an important protective role in stretch-induced myoblasts apoptosis,the mechanism may be that activation of Akt phosphorylates GSK-3β,inactivates GSK-3β,the role of promoting MPTP opening of GSK-3β is weakened,then apoptosis is reduced.