The Effect Of Endoplasmic Reticulum Stress On ISN-1 Cell Apoptosis Induced By TXNIP Overexpression

Backgroud:Diabetes Mellitus has become a big killer to health in worldwide.It has type 1 and type 2 diabetes,though they are distinct,but autoimmunity and insulin resistance are common feature in the pathogenesis of both types, at last mass of β-cell apoptosis and death,induced islet dysfuncion.we have found in our previous study that,TXNIP expression elevated in diabetic rat model and high glucose and high fat cultured rat myocardial cells,meanwhile,the damage and apoptosis of myocardial increased.Then we use RNA interference to restrain TXNIP expression,significant myocardial cell apoptosis decline were observed.So we are interested in studying the affect of TXNIP to pancreatic islet cell.TXNIP was discovered to be up-regulated in response to glucose in human islet gene study,in the past decades,TXNIP has been thought to be a key role in β-cell physiological function and diabetes therapies.Research has shown that ER stress has influence on β-cell apoptosis in type 2 diabetes.Accumulation of mislead and unfolded proteins in ER leads to activation of the unfolded protein response.If the problem existing,it results in ER stress and apoptosis via 3 pathways involving PERK( protein kinase RNA(PKR)-like ER kinase)、 IRE1α(inositol-requiring protein-1α)、ATF6α(activating transcription factor 6α).But the down stream pathway of TXNIP induced cell apoptosis still unclear, so this study is to observe if TXNIP overexpression alone can cause INS-1 cell apoptosis which cultured in normal glucose and normal lipid concentration conditions.Cell apoptosis is the programed cell death,among three pathway of cell apoptosis,we observe whether TXNIP overexpression can induce ER stress-mediated INS-1 cell apotosis.Objective:To observe if TXNIP overexpression alone can cause INS-1 cell apoptosis, and whether TXNIP overexpression can induce ER stress-mediated INS-1 cell apotosis.To prove the three signaling pathway of ER-stress which has significant effect on cell apoptosis, we use adenovirus carrying TXNIP gene to transfect INS-1 islet cells which cultured in normal glucose and normal lipid concentration conditions.Methods:1、 INS-1 islet cells in logarithmic growth phase were divided into three groups: nomal cultured group,empty adenovirus vector(Ad-e GFP)group,and TXNIP overexpression(Ad-TXNIP-e GFP)group.We transfected cells according to MOI=50,4 hours later,we add 3ml 1640 with fetal bovine.24h、48h after transfection, we changed the medium.2、 Observe the transfection efficiency:We used fluorescence microscope to observe the transfection efficiency, after 48 h, the transfection efficiency of Ad-e GFP group and Ad-TXNIP-e GFP group are equally and reach the mostest,then we collected cells for further assay.3、 Real-time PCR method to test transfected INS-1 cells TXNIP、ATF5、XBP-1 m RNA expression.4、 Western Blot method to test transfected INS-1 cells TXNIP、CHOP、p-PERK、PERK、ATF6、IRE1α、p- IRE1α、ATF5 proteins expression.5、 ELISA method to test the expression of Caspase-3 and Caspase-12.6、 INS-1 cells apoptosis assay by flow cytometry.Results:(1)After 48 h the transfection efficiency reached the peak, and the expression of green fluorescent protein equally.(2)Compared to Ad-e GFP group, Ad-TXNIP-e GFP group the expression of TXNIP m RNA(P<0.01)and protein(P<0.01) increased significantly, so adenovirus-mediated TXNIP overexpression is successful;(3)There also have significant increase of Caspase-3 expression(P<0.01)and INS-1 cells apoptosis(P<0.01);(4)CHOP protein expression(P<0.01) and Caspase-12 expression(P<0.01)increased;(5)ATF6α protein level has no significant change(P>0.05);(6)The phosphorylation of PERK increased(P<0.01),the expression of ATF5 m RNA(P<0.01)and protein also increased;the phosphorylation of IRE1α(P>0.05)has no significant change,the expression of ATF5 m RNA(P>0.05) has no significant change either.Conclusion:The result demonstrate that TXNIP overexpression alone can cause INS-1 cell apoptosis, which were cultured in normal glucose and normal lipid concertration conditions.Further more, TXNIP can cause ER stress-induced INS-1 cell apoptosis, and on the three signaling pathway of ER stress-induced cell apoptosis, PERK signaling pathway may take part in the process.