The Role And Mechanism Of DDIT3 In Autophagy And Apoptosis Of Myoblasts Induced By Cyclic Tensile Stress

Objective: Functional correction is an important early treatment for oral and maxillofacial deformities,which is mainly used to modify the tension of the oral and maxillofacial muscles,so as to improve the therapeutic effect.In the present study,the effects and mechanisms of cyclic tensile stress on myoblasts were studied from the microscopic level by the method of in vitro simulated stress stimulation method and combined with molecular biology technology.A preliminary study on the endoplasmic reticulum stress related gene DDIT3 was carried out to further clarify the mechanism of functional orthopedic treatment and to provide a better theoretical support for clinical treatment.Methods: Rats myoblasts cell lines(L6)were cultured in vitro to construct the model of muscle mechanical stimulation in vitro;cells were loaded at a frequency of 10 cycles/min,and tensile strain rate of 15% of the cyclical tensile stress for 2,6,12 and 24 hours,respectively,and 0h group(stress-free)was used as the control under the same experimental conditions except for stress loading time.Hochest 33258 staining was used to observe the effects of different loading time on the apoptosis of myoblasts;Annexin V-FITC/PI flow cytometry was applied to detect the apoptosis rate of each muscle cell,meanwhile,real-time PCR and Western Blot were involved to detect the expression of m RNA and protein expression levels of endoplasmic reticulum stress related gene DDIT3 and apoptosis related gene BBC3,respectively,and also measured the expression of autophagy related genes LC3,P62 and ATG5 in the muscle cells;successful inhibition of autophagy was achieved by 3-MA inhibitor,and then,changes of apoptosis rate were analyzed using flow cytometry after autophagy inhibition;The expression of BBC3 and ATG5 were measured before and after interfering the m RNA expression of DDIT3 with si RNA,Western Blot were used to compare the changes of BBC3 and ATG5 protein after interference with those in tension-force group.The above experimental data were statistically analyzed by SPSS17.0.Results:1.Hochest 33258 staining and flow cytometry detected the apoptosis rate of rat L6 myoblasts,corresponding results indicated that the frequency of 10 cycles/min,and tensile deformation rate of 15% of the cyclical tensile stress could induce myoblast apoptosis,and the apoptosis rate was positively correlated with the duration of stress loading,namely,the apoptosis rate of myoblasts increased with the prolongation of stress loading time,and reached the highest at 24 h.2.RT-PCR results showed that the m RNA expression of DDIT3 and BBC3 increased gradually with the stress loading time,and reached the highest at 24h;meanwhile,Western Blot detection results of DDIT3 and BBC3 protein expression also revealed a positive correlation with the stress loading time(p<0.05).3.Western Blot results implied that the expressions of LC3 and ATG5 were positively correlated with stress loading time,protein expression of p62 increased at 2h firstly,followed by a downward trend(p<0.05).4.After treated with 3-MA autophagy inhibitor,the apoptotic rate of cells was significantly higher by applying tension-force for 24 h than that in the 24 h group applying tension-force but without autophagy inhibitor treatment(p<0.05).5.After successful si RNA interference with DDIT3,m RNA expressions of BBC3 and ATG5 were detected by RT-PCR before and after interference.Relevant results showed that the m RNA expression levels were significantly lower than that in the24 h group applying tension-force(p<0.05).6.The results of Western Blot detection of BBC3 and ATG5 protein expression after DDIT3 interference revealed that expression levels of BBC3 and ATG5 were lower than those of 24 h group,but significantly higher than that of control group and even2 h group(p<0.05).Conclusion:1.The tensile deformation rate of 15% and the tensile stress at the frequency of 10cycles/min could induce the apoptosis of myoblasts;2.The apoptosis of myoblasts was positively correlated with the time of stress loading,that was,the apoptosis rate increased elevated with the increase of the stress loading time;3.The expression of endoplasmic reticulum stress related gene DDIT3 showed that myoblasts could stimulate endoplasmic reticulum stress by cyclic stretch stress;4.The cyclic tensile stress could induce autophagy in myoblasts,and the intensity of autophagy was correlated with the stress loading time,and the intensity of autophagy became stronger with the increase of stress time;5.The apoptosis rate was significantly increased after the inhibition of stress mediated myoblast autophagy.6.DDIT3 might act as a transcription factor to regulate the expression of BBC3 and participate in stress mediated apoptosis induced by endoplasmic reticulum stress,thus involving in stress mediated endoplasmic reticulum stress induced autophagy in myoblasts by regulating the expression of ATG5.