Studies On The Mechanisms Of Apoptosis/Autophagy Induced By Xiaokeping Tablet In Pancreatic Beta Cell

Diabetes mellitus is a global,chronic metabolic disease which is charactered by high blood glucose concentration.With high morbidity,diverse complication,complicated pathogenesis,and with no chance to be completely cured,diabetes mellitus has become one of the most difficult diseases all over the world.Chronic hyperglycemia affects various of tissues and cells,including pancreatic ? cells.As insulin producer,pancreatic ? cell plays an important role in the pathogenesis and therapy of diabetes.Xiaokeping tablet(XKP)a Chinese medicine which has hypoglycemic effect,has been used to cure diabetes patients for many years.However its molecular mechanism is unclear yet.In this study,a apoptotic model of pancreatic ? cell under high glucose condition has been established.The ? cells were treated with high glucose concentration and/or XKP.The status of apoptosis and autophagy of ? cells under different treatments are detected by Flow cytometry test,Immunofluorescence test,and western blotting test.The results are listed below:1.There are no difference between contol and high glucose treatments(concentration of glucose: 6.0 g/L,7.5 g/L,9.0 g/L)within 12 h and 24 h in pancreatic ? cell(cell line: MIN-6);After treated for 36 h and 48 h,the apoptosis rate and necrosis rate of cells under high glucose treatments were significantly higher than control group.With the glucose concentration increased,the apoptosis rate of ? cell was increased first and then decreased.While the necrosis rate of ? cell had obviously positive correlation with glucose concentration.Considering both the apoptosis rate and necrosis rate,the best conditions for the apoptotic cell model are glucose concentration of 6.0 g/L,and treatment for 36 h.2.After treated for 36 h,the autophagy level of MIN-6 cells under Xiaokeping treatment with the concentration of 100 flod dilution or 1000 flod dilution or 10000 flod dilution were detected by immunofluorescence test.The autophagy level of MIN-6 cells under Xiaokeping in 100 flod or 1000 flod dilution was significantly increased.Western blot teat shows that after treated with Xiaokeping,the LC3 II /LC3 I ratio was increased,P62 level was decreased,and the phosphorylation level of S6 K was decreased,but the phosphorylation level of AMPK remains unchanged in MIN-6 cells,indicating that Xiaokeping induced autophagy in MIN-6 cells via mTOR pathway.3.MIN-6 cells under high glucose conditions were treated with XKP,the apoptosis rate of which was detected by flow cytometry.The results showed that the apoptosis rate of high glucose + XKP group was significantly decreased than that of high glucose group.The autophagy level of high glucose group was no difference from that of control group,and the autophagy level of high glucose+XKP group was significantly higher than that of the two group.When 3-MA,a autophagy inhibitor,was added,the results has been changed.The autophagy level of high glucose + XKP +3-MA group was significantly lower than that of high glucose + XKP group,while the apoptosis level of former was significantly increased than that of latter.All together,we draw the conclusion: under high glucose concentration condition,XKP protect pancreatic ? cell from apoptosis through inducing autophagy via mTOR pathway.