The Role And Mechanism Of TXNIP In Aging Related Dysfunction Of Islet ? Cell

PART 1: HIGH-FAT DIET AGGRAVATES THE AGED-INDUCED DISFUCTION OF PANCREASE ISLETSAND ACCELERATES THE EXPRESSION OF TXNIPWITH AGEObjective: To investigate the effects of senescence on the age-induced dysfunction of pancreas islets and the expression of TXNIPMethods: Three aged-periods(youth,mid-aged,elderly)human normal pancreas tissue were collected,and the expression of TXNIP protein was measured.4-week-old C57BL/6J mice(n=48)were randomly divided into two groups.One was fed on a normal chow diet(NC),the other was fed on a high fat diet(HFD).each group was fed for four periods(3 months,6 months,9 months,12 months).The general condition were recorded during feeding period.GTT,ITT and basic physiological metabolic indexes were measured.Meanwhile,the protein expression of TXNIP,TRX,p16 of pancreas were measured.Results: The expression of TXNIP protein was gradually up-reguated with age in the human normal pancreas tissue.The FBG was significantly increased with age(p <0.05).The insulin secretory function was remarkably reduced with age(p <0.05),while the insulin resistance was increased with age(p <0.05).Compared with the age-matched NC group,the FBG was significantly higher in 6,9,12 months HFD mice;the insulin secretory function was also better,and the insulin resistance was milder among the each HFD group.The serum TC?TG?LDL were gradually increased with age in HFD groups(p <0.01).The protein expression of TXNIP,p16 of pancreas were up-regulated with age(p <0.05),but the protein expression of TRX were down-regulated with age(p <0.05).Compared with the age-matched NC group,The protein expression of TXNIP was up-regulated in the 9 and 12 months HFD groups(p<0.05).Among each month HFD groups,the protein level of p16 was up-regulated,but the protein expression of TRX were down-regulated,compared with the age-matched NC group.Conclusion: high fat diet aggravates the aged-induced dis-function of pancreas islets and accelerates the expression of TXNIP protein with age.PART 2: TXNIP AGGRAVATES THEAGED-INDUCED DYSFUCTION OF PANCREASEISLETSObjective: To investigate the effects of TXNIP on the age-induced dysfunction of pancreas islets,and to explore the molecular mechanism involved.Methods: 4-week-old C57BL/6J mice(n=24)and TXNIP-/-mice(n=24)were fed for four periods(3 months,6 months,9 months,12 months)on a normal chow diet.The general condition were recorded during feeding period.GTT,ITT and basic physiological metabolic indexes were measured.Meanwhile,the protein expression of TXNIP,TRX,p16 of pancreas were measured.Results: Compared with the age-matched WT mice,the FBG was significantly lower(p<0.05),the insulin secretory function was also better(p<0.05),and the insulin resistance was milder among each TXNIP-/-groups(p<0.05).Meanwhile,the weight and the serum TG?LDL were also lower in TXNIP-/-groups to some extent(p<0.05),Compared with the age-matched WT mice.The protein expression of p16 was down-regulated in the 6,9 and 12 months TXNIP-/-mice(p<0.05),but the protein expression of TRX were up-regulated,Compared with the age-matched WT mice.Conclusion: TXNIP aggravates the aged-induced disfuction of pancreas islets,accelerates the metabolic disorders,and progress the expression of p16 protein with age.PART 3 TXNIP PROMOTE THE SENESCENCE OF MIN6?-CELL VIA P38 MAPK PATHWAY.Objective:To investigate the effects of TXNIP and EZH2 on the senescence of MIN6 ?-cell and the mechanism involved.Methods :EZH2 over-expression,TXNIP over-expression and TXNIP silence lentivirus was used to transfect MIN6 ?-cell within logarithmic growth phase,the transfection efficiency was tested with both the fluorescence microscope and Western blot.The MIN6 ?-cell were divided into three groups: empty lentivirus vector(LV-GFP)group,TXNIP over-expression(LV-TXNIP)group and TXNIP silence(LV-TXNIP si RNA)group,respectively.The cell cycle was measured by flow cytometry.CCK-8 assay was used to reflect the proliferation of cells;the apoptosis of MIN6 cells was measured by FITC-Annexin V /PI double staining;the ROS level was measured with DHC;The protein expressions of TXNIP,TRX,p 16,Cleaved-Caspase and the p38 MAPK signal pathway were determined by using Western-blot.Results : After 72 h of transfection,that lentivirus-madiated EZH2 over-expression,TXNIP over-expression and TXNIP silence was successfully achieved.Compared with LV-GFP group,the cell cycle arrested at G1,the cell viability was significantly decreased,while the cell apoptosis and ROS level was increased in LV-TXNIP group(p<0.01).Additionally,there was markedly increased p 16,Cleaved-Caspased3 and the phosphorylation of p38 MAPK in LV-TXNIP group compared with that in LV-GFP group(p<0.01).However,the cell viability was significantly increased,the cell apoptosis and ROS level was significantly decreased in LV-TXNIPsi RNA group and LV-EZH2 group(p<0.01).thep16 and Cleaved-Caspased3 expression down-regulated in LV-TXNIPsi RNA group(p<0.01).While the expression of TXNIP andp16 was down-regulated in LV-GFP-EZH2 group(p<0.01),the expression of TRX was up-regulated in LV-GFP-EZH2 group(p<0.01).Both the p16 and Cleaved-Caspased3 expression induced by TXNIP were obviously reversed by p38 MAPK inhibitor(p <0.01).Conclusion:TXNIP might promote the senescence of MIN6?-cell via p38 MAPK pathway,EZH2 over-expression might reduce the senescence of MIN6?-cell via down-regulating TXNIP..