Effects Of Aging And Stress On Islet Cell Dysfunction And Protective Effect Of LBP

Research background: Type 2 diabetes has brought huge economic burden to individuals and society.Aging and stress play an important role in the occurrence of type 2 diabetes.So exploring its pathogenesis is of great significance for the prevention and treatment for type 2 diabetes.Objective: To investigate the role and mechanism of aging and stress in the development of islet cell dysfunction,as well as the protective effect of Lycium barbarum polysaccharide(LBP).Method:1.The effects of aging and stress on pyruvate dehydrogenase complex(PDC)(1)Etoposide,sodium dithionite palmitic acid were used to intervene ?-TC6 cells for 24 h,respectively,in oder to establish aging,hypoxia and hyperlipidemia models.In the meanwhile MTT method was used to detect the survival rate of cells after modeling,so as to determine the optimal concentration of aging,hypoxia and hyperlipidemia for modeling.(2)Then the E1 activity detection kit was used to determine the activity of E1 after modeling to determine the effects of aging and stress on pyruvate dehydrogenase complex(PDC).2.The effect of PDC status on islet cell function and its mechanism(1)Silencing or overexpression of E1 gene in ?-TC6 cells were induced by siRNA or lentivirus infection,respectively.(2)The distribution and expression of E1 after intervention were observed by immunofluorescence and Western Blot in order to detect the effects of silencing or expression;(3)The level of acetyl-CoAproduction in the nucleus was detected after intervention by ELISA.(4)RT-PCR and Western Blot were used to detect the expression levels of insulin gene Islet1,Ins,Pdx1 and those protein after intervention.3.Protective mechanism of Lycium barbarum polysaccharide(LBP)on islet cell dysfunction caused by PDC injury(1)50?g/mL LBP was combined with Etoposide,bisulfite,glucose and palmitic acid to culture for 24 h,respectively.(2)Western Blot was used to detect the expression of ISLET1,INS,PDX1 and INSR in ?-TC6 cells after LBP interventionResult:1.The effects of aging and stress on pyruvate dehydrogenase complex(PDC).(1)MTT experiments found that when the concentration of Etoposide was 40?g/mL,sodium dithionite was 4mmol/L anoxia for 2h,and palmitic acid was0.25mmol/L,which were the most suitable for modeling;(2)The activity of E1 in each group was detected with the E1 kit,and the results showed that the activity of E1 in the aging group,the hypoxia group,the high glucose group and the hyperlipidemia group was significantly lower than that in the control group(P<0.05).2.The effect of PDC status on islet cell function and its mechanism(1)Gene silencing results showed that the production of acetyl-CoA in the nucleus decreased significantly after E1 silenced by E1a-722 site,Insulin secretion related genes Islet1,Ins and Pdx1 promoter region of DNA expression significantly lower than the blank group and Scrambled siRNA group and the relative proteins expression level of ISLET1,INS,PDX1 and INSR also significantly lower than the blank group and Scrambled siRNA group(P<0.05);(2)After E1 overexpression,the relative proteins expression levels of ISLET1,INS,PDX1 and INSR in cells were significantly higher than those in the blank group and the empty vectorcontrol group(P<0.05).3.Protective mechanism of Lycium barbarum polysaccharide(LBP)on islet cell dysfunction caused by PDC injury(1)Examination of E1 activity and protein expression showed that after using LBP intervention,E1 activity and relative protein expression levels were significantly increased compared with the aging,hypoxia and hyperlipidemia,but still lower than those in the control group(P<0.05);The activity and protein expression levels of E1 after LBP intervention under low sugar,high sugar and fluctuation of sugar concentration were detected.It was found that,from the perspective of the activity of E1,whether under low sugar,high sugar or fluctuation of sugar,there was no statistical difference in the activity of E1 per unit protein concentration after LBP intervention compared with that before the intervention(p>0.05).However,from the point of view of the protein expression level of E1,the addition of LBP in a simple low-sugar and high-sugar environment inhibits the protein expression level of E1;Under normal conditions or when sugar concentrations fluctuate,adding LBP can increase the expression level of E1 protein;(2)The detection of insulin secretion-related proteins showed that the relative proteins expression levels of ISLET1,INS,PDX1 and INSR after LBP intervention were significantly higher than those in the aging group,the hypoxia group and the hyperlipidemia group(P<0.05);When high glucose and sugar fluctuated,the expression of PDX1 increased,but the expression of ISLET1 and INSR protein decreased through the intervention of LBP.It shows that LBP intervention has certain protective effect on the insulin secretion-related proteins caused by PDC damage caused by aging and stress.Conclusions: Aging and stress will damage PDC,and then inhibit the production of acetyl-CoA in the nucleus,hinder the acetylation of histones,inhibitthe Ins gene transcription initiation,and inhibit its related regulatory genes,which will eventually cause INS secretion disorders.LBP may improve the secretion of insulin-related proteins by promoting PDC damage,and promote insulin secretion,thereby achieving the goal of improving T2 DM insulin secretion disorders.