Study On The Mechanism Of TXNIP Promoting The Aging Of Islet ? Cells

Background:With the development of science and technology and the improvement of people's living standards,diabetes has become a serious global health problem threatening human health.The results of a large epidemiological survey showed that diabetes mellitus is closely related to age,and the prevalence of diabetes was higher in the elderly,especially in the elderly over the age of 60.In recent years,the role of islet ?-cells senescence in the pathogenesis of diabetes has attracted much attention.The elimination of aging cells and the improvement of the microenvironment of aging cells are expected to be one of the therapeutic methods for diabetes mellitus.It has been shown that cell senescence can be divided into replicating senescence and stress senescence.Oxidative stress is one of the important causes of cell senescence.Thioredoxin as a widespread redox enzyme,plays a role in reducing oxidative protein and scavenging free radicals during oxidative stress.Thioredoxin interacting protein(TXNIP)is the only endogenous binding inhibitor of Trx,which inhibits Trx activity and thus induces oxidative stress.Some studies have shown that the expression of TXNIP in islet ?-cells of diabetic patients and diabetic model mice is significantly up-regulated,and inhibiting the expression of TXNIP can reduce the occurrence and development of diabetes mellitus.The decrease of cell proliferation ability is the main manifestation of cell senescence,so we speculate that TXNIP expression may be increased during the development of diabetes mellitus,which may promote the aging of islet ?-cells and weaken the proliferation ability of islet ?-cells.The number of ?-cells was decreased due to the deficiency of ?-cells renewal.In addition to inhibiting Trx,it has been found that TXNIP has the characteristics of inhibiting proteins,which regulate cell activity by binding to some key proteins.Therefore,the mechanism of TXNIP is the mechanism of Trx and Trx.So,what way does the TXNIP affect the aging of pancreatic islet cells?In this study,we first observed the expression of TXNIP in the pancreatic tissue of diabetic rats and the changes in aging.Lentiviral vector was used to construct INS-1 islet?-cells lines over-expressing TXNIP,and to simulate the upregulation of TXNIP expression induced by diabetes mellitus in order to analyze the effect of TXNIP itself on islet ?-cells aging.In order to analyze the effect of TXNIP-dependent and non-dependent Trx pathway on islet ?-cells senescence and possible mechanism,a cysteine-247 th point-mutated TXNIP over-expressed INS-1 cells lines was constructed.The TXNIP-silent INS-1 cells lines was constructed to further determine the role of TXNIP in the senescence of pancreatic islet cells.Part one: increase of TXNIP expression induced by Diabetes Mellitus promotes Pancreatic AgingObjective:The increase of TXNIP(thioredoxin interacting protein)expression in islet ? cells can be observed in diabetes mellitus,and TXNIP can promote apoptosis,promote inflammation and inhibit proliferation.The aim of this study is to investigate whether the increased expression of TXNIP in diabetes affected islet ?-cells senescence.Methods:1.db/m,db/db mice were fed.2.Detection of fasting Blood glucose in db/db,db/m mice.3.Western blot was used to detect the expression of TXNIP protein and senescence-related indexes(p16,p21,Rb)in pancreatic tissue.4.?-galactosidase staining was used to detect the number of senescent cells in pancreatic tissue.Results:db/db mice can observe the expression of TXNIP protein in pancreatic tissue and induce cell senescence.Conclustions:The expression of TXNIP is up-regulated and the aging of pancreatic tissue is increased in diabetic mice.Part 2: lentiviral transfection of INS-1 islet ?-cells to construct TXNIP over-expression and interfere with stable cell linessObjective:A stable and efficient over-expression and silencing model of TXNIP was constructed by lentiviral vector-mediated transfection in rat pancreatic islet ?-cells.Methods:1.A lentiviral vector(Ad-GFP),a TXNIP over-expression lentiviral vector(Ad-TXNIP-GFP)and a TXNIP cysteine 247 site with a green fluorescent protein(GFP)were constructed and the lentiviral vector(Ad-TXNIP-c247s-GFP)was mutated.2.Empty lentivirus vector(Scramble Sh-RNA)containing red fluorescent protein(merry)and TXNIP silencing lentivirus vector(TXNIP Sh-RNA-1,TXNIP Sh-RNA-2,TXNIP Sh-RNA-3)were constructed.3.Normal group was directly added to 1ml RPMI 1640 complete medium,Ad-GFP,Ad-TXNIP-c247s-GFP,Scramble Sh-RNA,TXNIP Sh-RNA-1,TXNIP Sh-RNA-2,TXNIP Sh-RNA-3 each group was added with a mixture of 30?l of the corresponding virus and1 ml of RPMI 1640 complete medium,respectively.After the lentivirus was transfected for4 h,the virus-containing medium was discarded,washed three times with 1ml of PBS.Each group culture 48 h was supplemented with 4ml RPMI 1640 complete medium,Screening of Resistance in RPMI 1640 complete medium containing 3 ?g/ml puromycin,lasting for 7days,the new screening culture medium is replaced every 2-3 days,and the observation is carried out by using a fluorescence microscope.When the fluorescence rate of the cell reaches more than 90%,each group is switched back to normal RMPI 1640 complete culture medium,and the stable-rotation TXNIP silence and the over-expression cell lines are constructed successfully.4.The expression of fluorescent protein in each group was observed under a fluorescence microscope.5.The expression level of TXNIP m RNA was detected by Real-time PCR.6.Immunoprecipitation to verify whether the TXNIP overexpression of c247 s mutation binds to Trx.Results:1.The successful construction of the stable cell lines was observed under fluorescence microscope.2.Compared with the Ad-GFP group,the m RNA and protein expression of TXNIP in Ad-TXNIP-GFP group increased significantly(P <0.01).3.Screening effective TXNIP silencing sequence,Compared with Scramble ShRNA group,the third interference sequence had the best effect,and the expression level of m RNA and protein in TXNIP was significantly lower than control group(P < 0 01).4.The overexpressing TXNIP of the cysteine 247 site mutation cannot be combined with Trx.Conclustions:The over-expression of TXNIP and the construction of silencing cell lines of lentivirus vector were successful.The over-expressing TXNIP of the cysteine 247 site mutation cannot be combined with Trx.Part three: the effect of TXNIP on the senescence of INS-1 islet ?-cellsObjective:Diabetes can increase the expression of thioredoxin-interacting protein in islet ?-cells,and TXNIP can promote apoptosis,promote inflammation and inhibit proliferation.The aim of this study was to investigate whether up-regulation of TXNIP can promote islet?-cells senescence and explore its possible mechanism.Methods:1.Cell senescence was detected by ?-galactosidase staining.2.The expression levels of p16,p21 and Rb proteins were detected by Western blot.3.p38 MAPK phosphorylation and p53 protein expression were detected by Western blot.Results:1.The over-expression of TXNIP and TXNIP-c247 s protein promoted the expression of p21,p16,Rb protein and promoted the senescence of INS-1 cells.TXNIP silencing could significantly inhibit the expression of p21,p16,Rb protein and inhibit the senescence of INS-1 cells.2.Over-expression of TXNIP and TXNIP-c247 s promoted the phosphorylation of p38 MAPK and the expression of p53 protein.TXNIP silencing inhibited the phosphorylation of p38 MAPK and the expression of p53 protein.3.Pretreatment of cells with p38 MAPK inhibitor SB203580,p53 inhibitor Pifithrin-?,It was found that both p38 MAPK and p53 inhibitor could inhibit the protein expression of p21,p16,Rb and alleviate cell senescence.It was further found that p38 MAPK inhibitor could inhibit the expression of p53 protein,but p53 inhibitor did not affect the phosphorylation of p38 MAPK.Conclustions:TXNIP can promote the senescence of pancreatic islet ?-cells,and the mechanism of TXNIP is related to the up-regulation of the expression of p21,p16,and Rb.TXNIP can promote p38 MAPK phosphorylation and follow-up p16 and p53 expression up-regulation through Trx-dependent pathway,The expression of p53 protein can also be increased by the Trx non-dependent way,and the expression of p21 and Rb protein can be increased,thus leading to the senescence of the INS-1 cell.