| Objective: As a chronic metabolic disease with high incidence worldwide,diabetes has a complex pathogenesis.Due to the limited ability of islet beta cells to regenerate,beta cell failure and death may be the main driving force for the progressive deterioration of glycemic control in type 2 diabetes(T2DM)and type 1 diabetes(T1DM).The insulin resistance of peripheral tissues caused by abnormal lipid metabolism promotes and aggravates diabetes mellitus to a certain extent and its damage to β-cell itself is inevitable.The mechanism of beta cell damage under abnormal lipid metabolism has not been fully elucidated.In recent years,more and more research evidence has revealed that autophagy plays an important role in regulating the number and function of islet β cells,and oxidative stress is an important factor involved in mediating islet β cell dysfunction and death.Investigation on the relations between abnormal lipid metabolism and the above two,autophagy and oxidative stress,will help us understand the relationship between abnormal lipid metabolism and diabetes mellitus.In this experiment,the mouse insulinoma cell MIN6 was used as a model.Palmitic acid mimics the high-lipid environment in vitro,and explores the molecular mechanism and role of palmitic acid in regulating the autophagy of MIN6 cells through ROS/JNK signaling pathway.Methods: The mouse insulinoma cells MIN6 were cultured in vitro and treated with concentration gradient palmitic acid(PA),Western blot analysis to detect the expression of Beclin-1,LC3.At the optimum concentration of palmitic acid,autophagosome was observed by electron microscopy and the level of reactive oxygen species(ROS)in cells was detected by fluorescence probe.The optimal concentration of palmitic acid and / or oxidative stress inhibitor(NAC)and / or JNK inhibitor(SP600125)treatment,Western blot analysis of intracellular JNK total protein and phosphorylated protein levels and Beclin-1,LC3 expression.The optimal concentration of palmitic acid and/or autophagy inhibitor(3-MA)was used for in vitro intervention.Fluorescence probe was used to detect the level of reactive oxygen species(ROS)in cells,and fluorescence microscopy observed changes in LC3 fluorescence in cytoplasm.Western blot analysis detected the expression of related proteins Beclin-1 and LC3,and apoptosis was detected by Annexin-V/PI staining flow cytometry.Results: 1.Palmitic acid induces autophagy in MIN6 cells.Palmitic acid increased the expression of autophagy-related protein Beclin-1 and the conversion of LC3-I to LC3-II in MIN6 cells and was concentration-dependent within a certain range.2.Palmitic acid promotes ROS production in MIN6 cells.The ROS level in the cells treated with palmitic acid was significantly higher than that in the control group.3.The ROS/JNK signaling pathway is involved in palmitic acid-induced autophagy in MIN6 cells.The ROS/JNK signaling pathway is involved in the development of autophagy in MIN6 cells,which increases the biological effects of autophagy.The levels of p-JNK,Beclin-1 and LC3 were increased in the palmitic acid-treated group and inhibited by oxidative stress inhibitors;JNK inhibitors inhibited the expression of autophagy-related proteins Becine-1 and LC3.4.Autophagy has a potential role in regulating palmitic acid-induced production of ROS in MIN6 cells.The autophagy effect induced by palmitic acid in MIN6 cells can be reversed by the autophagy inhibitor 3-MA,which induces an increase in ROS fluorescence levels,suggesting that autophagy plays an important role in reducing palmitic acid-induced ROS production in MIN6 cells.5.Autophagy regulates palmitic acid-induced apoptosis in MIN6 cells.The results of Annexin-V/PI staining flow cytometry showed that 3-MA induced palmitic acid-induced apoptosis of MIN6 cells.Conclusion: ROS/JNK signaling pathway is involved in palmitic acid-induced autophagy of MIN6 cells,and autophagy regulates intracellular ROS levels and plays a protective role in apoptosis induced by palmitic acid. |
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