Role Of Autophagy In TXNIP Overexpression Induced Apoptosis Of INS-1 Islet Cells

Background:Along with the development of aging population,the morbidity of the chronic disease is rising significantly.Diabetes has become one of the most important diseases which threaten to the health of people.In the years of 2010 and 2013,the researches of Yang W and Ning Guang professors had shown that China had overtaken India became the country whose number of diabetes patients was the highest,and the incidence of diabetes was still rising rapidly,and to the young and middle-aged,and even children.No matter type 1 and type 2 diabetes,the reduction of the number of islet beta cells and dysfunction are the important reasons of the occurrence and development.In the study of the pathogenesis of diabetes,Trx interacting protein(TXNIP)is getting more and more attention.It is the only one known endogenous Trx combined proteins,and was showed high expression in the patients’ serum and tissue.Our team and the others are confirmed simple overexpress TXNIP can induce the apoptosis of INS-1 by endoplasmic reticulum stress pathway and death receptor pathway and mitochondrial apoptosis pathway.Autophagy is a phenomenon of life,it often observed in most eukaryotic cells,on the one hand,autophagy can remove the degradation cells,damaged organelles and unwanted biological macromolecules to maintain the intracellular environment and reduce apoptosis of cells.But studies have shown that the excessive activation of autophagy can promote the occurrence of apoptosis,and cause additional damage.Then,whether the rise of TXNIP can affect the level of autophagy and whether the autophagy can induce the apoptosis of islet beta cells by upregulated TXNIP are not known.Therefore,we designed the research subject.Object:To explore if TXNIP overexpression can cause INS-1 cell autophagy in the normal glucose and lipid concentration conditions,and analyze its effect on the apoptosis of INS-1 cell induced by TXNIP overexpression.Methods:1.Using adenovirus transfection technique to make the overexpression of TXNIP in INS-1 islet cells.Choosing INS-1 cells which in logarithmic phase,culturing them in T25 bottles,when cells were grown to 80% of the view,the cell number is about 2x106,then discarding the previous culture medium and using PBS to wash twice,dividing the cells into 3 groups randomly,named the normal group(Control),adenoviruses empty of TXNIP group(Ad-e GFP),TXNIP expressing group(Ad-TXNIP-e GFP).The amount of virus is calculated according to MOI = 50,then adding the virus to 1ml1640,and adding 3ml 1640 with 10% fetal bovine serum and 1%penicillin-streptomycin after 4h,collecting cells after 48 h,the time when the transfection is the most efficiency.2.Determination of indicators2.1 Applying the Real-time PCR and Western blot to detect the m RNA expression and protein expression of TXNIP.2.2 Apoptosis of INS-1 cells were detected by Annexin V-APC/7-AAD double staining flow cytometry.2.3 Applying Western blot to test the determination of LC3-Ⅱ/LC3-Ⅰ,Beclin-1,P62 and cleaved caspase 3 / caspase 3.2.4 Using laser confocal microscope to observe the autophagy body.Results:1.The overexpression of TXNIP is builded successfully.Compared with the Control group,the m RNA and protein levels of Ad-e GFP group TXNIP were not seen obvious difference.Compared with the Ad-e GFP group,the m RNA(Figure 1)and protein levels of Ad-TXNIP-e GFP group TXNIP were significantly higher(Figure 2),showing that the transfection was successful..2.The upregulation of TXNIP causes INS-1 islet cell apoptosis increase.With Annexin V-APC/7-AAD dyed by flow cytometry,it was showing that the apoptosis rate was increasing in Ad-TXNIP-e GFP group: compared with Ad-e GFP group,the apoptosis of Ad-TXNIP-e GFP group was increased(Figure 4),and the Ad-e GFP group had no difference with the Control group.In Ad-TXNIP-e GFP group,the ratio of cleaved caspase 3/caspase 3 was increased: the Control group compared with the Ad-e GFP group had no statistical significance,compared with the Ad-e GFP group,Ad-TXNIP-e GFP group had the higher ratio(Figure 3),further confirm the overexpression of TXNIP could cause INS-1 islet cell apoptosis.3.The overexpression of TXNIP could cause elevated levels of autophagy in INS-1islet cell.Compared with the Control group,the Ad-e GFP group did not show obvious difference,compared with the Ad-e GFP,Ad-TXNIP-e GFP group LC3-Ⅱ/LC3-Ⅰprotein levels were elevated(Figure 5),Beclin-1 protein levels were also increased(Figure 6),showing the higher level of autophagy.Because P62 is the substrate of autophagy,its level will reduce with the strengthening of autophagy,so P62 is a commonly marker to detect autophagy.Compared with the Control group,the Ad-e GFP group had no statistical difference,compared with the Ad-e GFP group,Ad-TXNIP-e GFP group had lower level of P62(Figure 7),showing that autophagy lysosome degradation increased,autophagy reaction was increased.4.Observe the increasing autophagy body in the Ad-TXNIP-e GFP group by laser confocal microscope.Compared with the Control group and Ad-e GFP group,the Ad-TXNIP-e GFP group had highest fluorescence intensity,there was an obvious red fluorescent cells,showing that TXNIP overexpress caused autophagy body generate.(Figure 8)5.3-MA(5 m M)inhibits the apoptosis caused by the overexpression of TXNIP.5.1 3-MA(5 m M)successfully inhibits the autophagy caused by the TXNIP overexpressed.To confirm the autophagy played a role in the INS-1 cells which overexpress TXNIP,the autophagy inhibitor 3-methyladenine(3-MA)was used,divided group into: the Control group,the Ad-e GFP group,Ad-TXNIP-e GFP group and the Control+ 3-MA group,Ad-e GFP +3-MA,Ad-TXNIP-e GFP+3-MA group.The results showed that the Control+3-MA compared with the Control group had no statistical difference;Ad-e GFP+3-MA group compared with the Ad-e GFP group also had no statistical difference;Ad-TXNIP-e GFP+3-MA group compared with the Ad-TXNIP-e GFP group,Ad-TXNIP-e GFP+3-MA group LC3-Ⅱ/LC3-Ⅰratio significantly decreased(Figure 9),Beclin-1 expression decreased obviously((Figure 10),and P62 increased significantly(Figure 11),the above results indicate that 3-MA inhibits the autophagy occurrence in INS-1 cell overexpressed TXNIP.5.2 The apoptosis reduced after autophagy was inhibited.Flow cytometry showed that the Control+3-MA group compared with the Control group had no statistical difference;Ad-e GFP+3-MA group compared with the Ad-e GFP group had no statistical difference;Ad-TXNIP-e GFP+3-MA group compared with Ad-TXNIP-e GFP group,the apoptosis of INS-1 cell(Figure 13)and cleaved caspase 3/caspase 3 ratio(Figure 12)were significantly decreased.Conclusion:1.Overexpression of TXNIP in INS-1 cell induces autophagy;2.TXNIP induced INS-1 cell autophagy can promote the occurrence of apoptosis.