IRE-1-JNK Pathway In Apoptosis Myoblasts Stress Mediated Effect And Mechanism

Objective:Orthodontics with functional appliances is an important treatment to correct malocclusion deformity of adolescent and children. Functional appliance can cause a series changes of neuromuscular contraction through occlusal reconstruction. The force can transmit to hard and soft tissues around the oral, which alter the growth potential of jaw via promotting the adaptive changes of tissues and stimulating the growth and reconstruction of the jaw and periodontal tissue. Consequently, the goal of correcting malocclusion deformity can be achieved. Only facial muscles reconstruct coordinated with hard and soft tissues around the oral, functional orthopedic effect can be stable. Therefore, it is significant to explore the mechanism of adaptive reconstruction of facial muscles which contribute to the principle of functional orthopedic. Exploring the underlying mechanism of facial muscles adaptive reconstruction should start with cell culture. Myoblast is the stem cell of muscle tissue. The biological effects of myoblast, such as proliferation, differentiation and apoptosis, play a vital role in adjustment of facial muscles. At present, many researchers pay great attention on the apoptosis of myoblast. There are two classic apoptotic pathways:namely the death receptor-mediated extrinsic pathway and the mitochondria-mediated intrinsic pathway, and endoplasmic reticulum stress (ERS)-mediated pathway, a new apoptotic pathway has been elucidated gradually in recent years. Various stimuli can disturb endoplasmic reticulum homeostasis and result in the excessive accumulation of unfolded and misfolded proteins and calcium dysfunction and pathological consequences, namely endoplasmic reticulum stress. In response to endoplasmic reticulum stress, cells active a series of protective responses collectively termed the unfolded protein response to maintain endoplasmic reticulum homeostasis. However, the activation of unfolded protein response may be insufficient to overcome ERS, ultimately leading to apoptosis. The aim of this study was to investigate the manner of stretch-induced apoptosis and to explore the role of IRE-1-JNK signaling pathways in stretch-induced endoplasmic reticulum stress and the possible mechanisms, which was based on successfully establishing a mechanical stretch model of myoblast in vitro. This study can provide theoretical basis on adaptive changes of facial muscle in the process of functional orthopedic and help orthodontics use force reasonably.Method:L6rat myoblast cells were cultured on collagen-coated six-well Bio flex plates in vitro, and when the cells had reached80%confluence, the medium was switched to DM. Then cells were subjected to15%surface elongation with a frequency of10cycles/min cyclic stretch using multi-channel stress loading system. Cells were harvested after different durations (1h,2h,6h,12h,24h) of cyclic exposure. Control cells were also cultured on same plates and kept in the same incubator without mechanical strain. After cyclic stretch, Hochest33258staining was performed to observe the morphology of the apoptotic cells and flow cytometry detection with Annexin V-FITC/PI staining was used to detect the apoptotic rate of myoblast exposed to different cyclic stretch duration. Real Time PCR was used to detect the mRNA expression of endoplasmic reticulum stress related factors (Chop, TRAF2, ASK1and JNK) and Western blotting was performed to evaluate the protein expression of JNK. Using IRE-1specific inhibitor(STF-083010) to detect the TRAF2, ASK1and JNK mRNA expression and JNK protein expression to investigate the role of IRE-1-JNK signaling pathway in stretch-induced apoptosis and its underlying mechanism. The experimental data were analysed by Statistical software SPSS17.0.Result:1. The results of Hochest33258staining and flow cytometry detection with Annexin V-FITC/PI staining showed that cyclic stretch with15%surface elongationg and at a frequency of10cycles/min can induce myoblast apoptosis in vitro, and the apoptosis rate of myoblast were increased in a time-dependent manner, peaked at24h.2. Real Time PCR results showed that CHOP, TRAF2and ASK1mRNA expression quantity were increased with the extension of stretch time, and reached the maximum at24h; but JNK mRNA expression was decreased followed by an increase range from Oh to2h, and then increased again within24h and peaked at24h. Differences were statistically significant (P<0.05).3. Western blotting result showed that JNK protein increased first and then declined slightly, and peaked at24h as the extension of stretch time. The differences was statistically significant (P<0.05).4. After treated with IRE-1specific inhibitor (STF-083010), the real time PCR results showed that TRAF2, ASK1mRNA expression were significantly decreased compared with the groups without IRE-1inhibitor, and the differences was statistically significant (P<0.05).But there were no significant difference compared the control group (Oh without mechanism stretch and IRE-1inhibitor).The JNK mRNA expression were significantly lower than the groups without IRE-1inhibitor, but still high than the control group, and differences were statistically significant (P<0.05).5. After treated with IRE-1specific inhibitor (STF-083010), the Western blotting results showed that JNK protein expression were significantly lower than the groups without IRE-1inhibitor, but still high than the control group, and differences were statistically significant (P<0.05).Conclusion:1. Cyclic stretch with15%surface elongationg and at a frequency of10cycles/min can induce myoblast apoptosis and within24h the apoptosis rate of myoblast were increased in a time-dependent manner.2. The increase of CHOP、TRAF2、ASK1、JNK mRNA and JNK protein indicate that endoplasmic reticulum stress involve in stretch-induced apoptosis.3. After treated with IRE-1specific inhibitor, the significant decrease of CHOP、TRAF2、 ASK1、JNK mRNA and JNK protein indicate that IRE-1-JNK signaling pathway involve in stretch-induced apoptosis4. The changes of JNK mRNA and protein suggested that JNK can be activated by other pathway except IRE-1, but the underlying mechanism remains to be further study.