Ghrelin Inhibits High Glucose And High Lipid-induced Islet ? Cell Apoptosis By Promoting Autophagy

Ghrelin has been found to protect islet ? cells,which can reduce apoptosis and increase cell viability in the long-term state of high glucose and fat.On the other hand,ghrelin has also been studied in the field of autophagy.It can induce protective autophagy,protect Cardiomyocytes from oxidative stress and hypoxia,and inhibit the proliferation of cancer cells by inducing autophagy.In general,It is clear that ghrelin has a protective effect on islet cells,but it is not clear that this protection is related to autophagy,and whether ghrelin has a pro-autophagy effect on islet beta cells is not clear,if it has the effect of promoting autophagy,and then reduce the occurrence of apoptosis by this action?Therefore,this study intends to establish an in vitro apoptosis model induced by high glucose and high lipid(HGHL)environment to study(1)whether ghrelin can enhance autophagy of cells;(2)whether ghelin is related to its role in promoting autophagy in the protection of islet cells;(3)ghrelin regulates the interaction between apoptosis and autophagy in a HGHL environment.It provides a new experimental theoretical basis for the clinical protection of islet function and diabetes medication.Part 1.Construction of a model of apoptosis induced by high glucose and high lipid in islet NIT-1 cellsObjective:To establish a model of apoptosis induced by HGHL in vitro,and provide a basis for subsequent experiments.Methods:An in vitro apoptosis model was constructed with glucose and palmitic acid(PA),grouped into control group(5.6 mmol/L glucose+0.5%bovine serum albumin(BSA))and HGHL group(33.3 mmol/L glucose+0.5 mmol/L PA).Tested whether the apoptosis model was successfully constructed:(1)The islet cell proliferation viability was detected by Cell Counting Kit-8(CCK-8).(2)Annexin v-fitc/PI double staining and flow cytometry were used to detect the apoptosis rate.(3)Hoechst 33258 staining labeled nuclei to observe the morphology of apoptosis.(4)qPCR was used to determine the expression levels of Bcl-2 and Bax mRNA in each group.Results:The cell apoptosis model induced by HGHL was successfully established.(1)CCK-8 showed that the cell viability of HGHL group was lower than that of control group(P<0.05).(2)The total apoptotic rate of Annexin V-FITC/PI double staining and Hoechst 33258 showed that compared with the control group,the apoptosis of HGHL group increased(P<0.05).(3)Bcl-2 expression was down-regulated after HGHL induction(P<0.05),and Bax expression was up-regulated(P<0.05).Conclusion:The apoptosis model of islet beta cells induced by HGHL was successfully constructed by using 33.3 mmol/L glucose and 0.5 mmol/L PA,which provided a basis for subsequent experiments.Part 2.Effect of ghrelin on apoptosis and autophagy of islet ? Cells under glycolipid toxicityObjective:To investigate the effect of Ghrelin on islet cell apoptosis and autophagy.Methods:Firstly,a model of apoptosis induced by HGHL was constructed and divided into different treatment groups by ghrelin intervention:control group(glucose 5.6 mmol/L+0.5%BSA)and ghrelin group(glucose 5.6 mmol/L+0.5%BSA+100 nmol/L ghrelin),HGHL group(glucose 33.3 mmol/L+0.5 mmol/L PA),HGHL+ghrelin group(glucose 33.3 mmol/L+0.5 mmol/L PA+100 nmol/L ghrelin).(1)Islet cell proliferation activity was measured by CCK-8.(2)Detection of apoptosis:Annexin V-FITC/PI double staining flow detection for apoptosis rate,Hoechst 33258 staining nucleus,qPCR detection of Bcl-2,Bax mRNA expression.(3)Autophagy detection:Dansylcadaverine(MDC)autophagy vacuole fluorescence staining,microtubule-associated protein 1 light chain 3(LC3)B immunofluorescence,electron microscopy detection of autophagosomes,Western Blot detection of autophagy-related proteins Beclin-1,P62,LC3?/LC3? conversion rate,qPCR was used to detect the expression of Atg6 and LC3B mRNA.Results:(1)There was no significant difference in cell viability between control group and ghrelin group.Compared with the HGHL group,the cell viability of the HGHL+ghrelin group increased(P<0.05).(2)The total apoptotic rate detected by Annexin V-FITC/PI double staining and Hoechst 33258 showed that compared with the control group,the apoptosis of ghrelin group decreased(P<0.05),but the expression of Bax and Bcl-2 was not significantly different.On the basis of HGHL,apoptosis decreased after ghrein application(P<0.05),up-regulated Bcl-2 expression(P<0.05),and down-regulated Bax expression(P<0.05).(3)MDC staining and autophagy protein LC3B immunofluorescence and electron microscopy showed that compared with the control group,autophagy increased in the ghrelin group and the HGHL group.Beclin-1 protein,LC3?/LC3? conversion,P62 protein,Atg6 and LC3B mRNA expression were increased in HGHL group(P<0.05).Beclin-1 protein expression was increased in ghrelin group(P<0.05).Compared with the HGHL group,autophagy in the HGHL+ghrelin group was further increased.Ghrelin up-regulated Beclin-1 protein,LC3II/LC3I conversion and Atg6,LC3B mRNA expression(P<0.05),and there was no significant difference in P62 protein expression.Conclusion:(1)HGHL induce cell apoptosis and Simultaneously enhance autophagy,which may be related to the increase of autophagy initiation and the decreased degradation.(2)Ghrelin can inhibit the apoptosis induced by HGHL,and protect cells from glycolipid toxic damage.(3)Ghrelin has no effect on autophagy of islet cells under normal conditions,but can enhance autophagy under glycolipid toxicity.Part 3.The role of autophagy in ghrelin's anti-apoptosis of islet ? cellsObjective:To study the effects of ghrelin in regulating the interaction between apoptosis and autophagy under glycolitic toxicity.Methods:Grelin and chloroquine(CQ)were used to intervene in the apoptotic model of glycolipid and divided into different treatment groups:HGHL group(33.3 mmol/L+0.5 mmol/L PA),HGHL+ghrelin group(33.3 mmol/L+0.5 mmol/L PA+100 nmol/L ghrelin),HGHL+CQ group(33.3 mmol/L+0.5 mmol/L PA+25 ?mol/L CQ),HGHL+CQ+ghrelin group(33.3 mmol/L+0.5 mmol/L+100 nmol/L ghrelin+25 ?mol/L CQ).Cell viability,apoptosis and autophagy were detected respectively.The method was the same as the part 2.Results:Chloroquine inhibited cell activity and reversed the protective effect of ghrelin on cells.The autophagy was blocked by chloroquine,an autophagy inhibitor,showing the accumulation of autophagy vesicles,lysosome and autophagy protein LC3B,and the expression of P62 was increased.Since the autophagy degradation pathway was blocked,after combined application of ghrelin and chloroquine resulted in increased apoptosis and decreased activity compared with the HGHL+ghrelin group,and the anti-apoptotic effect of ghrelin was inhibited.Conclusion:Apoptosis is increased when the autophagy of ghrelin is blocked,it is reasonable to believe that ghrelin promotes autophagy of islet ?cells to regulate its resistance to glycolipid-induced apoptosis.