| Background:Diabetes mellitus(DM)is a metabolic disease characterized by chronic hyperglycemia caused by the combined effects of genetic and environmental factors,which are essential for the secretion and/or action of insulin.At present,the incidence and prevalence of diabetes worldwide have risen rapidly,and China has the world's largest diabetes patients.Diabetes is mainly divided into type 1 and type 2,regardless of which type of diabetes,functional islet ? cell damage and apoptosis are important mechanisms to induce diabetes.The inflammatory cytokines TNF-? and IL-1? play an important role in diabetes as a cytokine that induces islet cell apoptosis.Thioredoxin-interacting protein(TXNIP)is the only endogenous TRX binding and inhibitory protein that attenuates TRX's anti-free and anti-apoptotic functions.The expression of TXNIP was significantly upregulated in all tissues in diabetes.The study found that high glucose can induce TXNIP m RNA and protein levels significantly increased in INS-1 cells and accompanied by apoptosis.Previous studies confirmed that TXNIP could induce INS-1 cell apoptosis through endoplasmic reticulum stress pathway involving caspase-8,9 and PERK.Thus,TXNIP is emerging as a critical link between hyperglycemia and beta cell death in diabetes.So,whether the inflammatory cytokines TNF-? and IL-1? can induce the expression of TXNIP in INS-1 cells is not clear.The aim of this study was to investigate the effect of TNF-? and IL-1? on TXNIP and its possible mechanism,so as to provide a new target for the prevention and treatment of diabetes mellitus.Objective:INS-1 cells were cultured at normal glucose and lipid concentration and TNF-?(5ng/ml)and IL-1?(10ng/ml)were used to stimulate cells for 24 hours to observe whether TNF-?and IL-1? can induce TXNIP upregulated in INS-1 cells and to analyze the mechanism.Methods:1.INS-1 cells were divided into four groups: normal control group,TNF-? group(5ng/ml,24h),IL-1? group(10ng/ml,24h),TNF-? + IL-1? group(5ng/ml + 10ng/ml,24h).2 INS-1 cells were treated with inflammatory cytokines TNF-? and IL-1?,and cell viability was detected by CCK-8.3.Flow cytometry and cleaved caspase-3 were used to detect the apoptosis of INS-1 cells induced by TNF-? and IL-1?.4.Real-time PCR to test the m RNA expression of TXNIP,NF?B and Ch REBP m RNA in INS-1 cells treated with TNF-? and IL-1?.5.Western blot was performed to detect the protein expression of TXNIP,NF?B,Ch REBP and FOXO1 in INS-1 cells stimulated by TNF-? and IL-1?.6.The m RNA and protein levels of TXNIP,NF?B and Ch REBP in INS-1 cells after pretreatment with NF?B inhibitor PDTC and p38 MAPK inhibitor SB203580 were detected by Real-time PCR and Western blot.Results:1.TNF-? and IL-1? reduce the survival of INS-1 cells TNF-? and IL-1? could induce INS-1 cells damage and decrease the survival rate compared with normal control group after intervention of different concentrations of inflammatory factors in INS-1 cells.TNF-?(5ng/ml,24h)and IL-1?(10ng/ml,24h)decreased the cell viability obviously,so set the concentration of TNF-? and IL-1?respectively were 5ng/ml and 10ng/ml,duration of 24 h.2.TNF-? and IL-1? induce INS-1 cells apoptosis Flow cytometry showed that the apoptotic rates of TNF-? group,IL-1? group and TNF-?+IL-1? group were significantly higher than those of control group(P <0.05),The expression of cleaved caspase-3,which represents apoptosis in these three groups of cells were also increased.It demonstrated that TNF-? and IL-1? could induce apoptosis in INS-1 cells,and play a synergistic effect when combined with the two,and induce a more pronounced apoptotic.3.The expression of TXNIP m RNA in INS-1 cells was upregulated by TNF-? and IL-1?Compared with the control group,at 5ng/ml TNF-? and 10ng/ml IL-1? reached a peak(P<0.05),and the expression of TXNIP m RNA was significantly increased at 24h(P<0.05).Therefore,the follow-up experiments were normal control group,TNF-? group(5ng/ml,24h),IL-1? group(10ng/ml,24h)and TNF-?+IL-1? group(5ng/ml+10ng/ml,24h).Real-time PCR results showed that TXNIP m RNA was significantly higher in the cytokines intervention groups than the control group(P<0.05).4.The expression of TXNIP protein was increased induced by TNF-? and IL-1?Compared with the control group,TXNIP protein levels in TNF-? group,IL-1? group and TNF-?+IL-1? group increased significantly(P<0.05),suggesting that TXNIP mediates inflammation-induced apoptosis in INS-1 cells.5.Detection of p-NF?B,Ch REBP,FOXO1 and other transcription factors5.1 inflammatory factors up-regulate Ch REBP m RNA and protein levels Compared with the control group,the expression levels of Ch REBP m RNA and protein were elevated in cells treated with TNF-? and IL-1?(P<0.05).5.2 inflammatory factors down-regulate FOXO1 protein expression The expression of FOXO1 protein in cells cultured with cytokines was significantly lower than that in normal group(P<0.05).5.3 inflammatory factors activate NF?B in INS-1cells Compared with the control group,TNF-? and IL-1? upregulated the expression of NF?B both at transcription and protein levels(P<0.05).5.4 NF?B inhibitor PDTC downregulates the expression of TXNIP and Ch REBP induced by inflammatory cytokines PDTC significantly inhibited the expression of TXNIP and Ch REBP m RNA and protein(P<0.05),suggesting that inflammatory factors may induce TXNIP expression by regulating NF?B and Ch REBP.NF?B as an upstream transcription factor involved in the regulation of Ch REBP expression.5.5 p38 MAPK inhibitor SB203580 attenuates inflammatory cytokines induced activation of p-NF?B and expression of TXNIP The expression of NF?B phosphorylation and TXNIP in INS-1 cells treated by SB203580 was significantly decreased(P<0.05),suggesting that p38 MAPK as an important protein in the inflammatory response by activating NF?B to induce TXNIP change.Conclusions:1.Inflammatory cytokines TNF-? and IL-1? can induce INS-1 cell apoptosis.2.The expression of TXNIP induced by TNF-? and IL-1? was correlated with the apoptosis of INS-1 cells.3.TNF-? and IL-1? may regulate the expression of TXNIP through the p38MAPK-NF?B-Ch REBP pathway. |
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