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目的:通过第三丁基过氧化氢(t-BHP)诱导小鼠胰岛β细胞凋亡,研究氧化损伤对内质网应激JNK凋亡通路相关分子表达的影响。方法:将不同浓度t-BHP(0~400μmol/L)分别加入胰岛β细胞株M IN6细胞中,于不同时间(0~8 h)收集细胞悬液进行相关检测。CCK-8法检测细胞增殖活力;An-nexin-V-PI流式细胞技术(FCM)检测细胞凋亡率及坏死率;Caspase-3检测试剂盒测定caspase-3活性;W estern b lot检测内质网应激相关分子IRE1,αJNK,P-JNK,Caspase-3蛋白的表达。结果:随t-BHP浓度的增高,①M IN6细胞的存活率降低;②t-BHP以浓度≥25μmol/L作用≥1 h时,Caspase-3活性有明显变化(P<0.05);③随t-BHP浓度增大、作用时间延长,内质网应激跨膜蛋白IRE1α表达逐渐减少,P-JNK、活性caspase-3表达明显增多。结论:①t-BHP引发细胞凋亡坏死呈一定的时效和量效关系;②持续t-BHP氧化损伤可诱导M IN6细胞发生内质网应激并发生凋亡;③内质网应激凋亡通路相关分子表达在细胞凋亡过程中有浓度和时间依赖性。
OBJECTIVE: To study the effect of oxidative damage on the expression of JNK apoptosis-related molecules in endoplasmic reticulum (ER) induced by t-BHP in mouse pancreatic β-cell. Methods: Different concentrations of t-BHP (0-400μmol / L) were added into the pancreatic β cell line M IN6 cells, and cell suspensions were harvested at different times (0-8 h). Cell viability was detected by CCK-8 assay; apoptosis rate and necrosis rate were detected by An-nexin-V-PI flow cytometry (FCM); caspase-3 activity was detected by Caspase-3 assay kit; Expression of stress-related molecules IRE1, αJNK, P-JNK and Caspase-3 in the cytoplasm. Results: The survival rate of M IN6 cells decreased with the increase of t-BHP concentration. ② The activity of Caspase-3 significantly changed when t-BHP was ≥1 h at the concentration of ≥25 μmol / L (P <0.05) The concentration of BHP increased and the action time prolonged. The expression of IRE1α in the endoplasmic reticulum stress transmembrane protein gradually decreased, and the expression of P-JNK and active caspase-3 increased obviously. Conclusion: ①T-BHP induces apoptosis and necrosis in a time-dependent and dose-dependent manner. ② Oxidative damage induced by t-BHP induces endoplasmic reticulum stress and apoptosis in M IN6 cells. ③ Endoplasmic reticulum stress-induced apoptosis Pathway-related molecules expressed in a concentration and time-dependent manner during apoptosis.