| Background:As the lives of the residents improve,the incidence of diabetes in China has increased year by year,and has been the most important health killer in the society.Diabetes mellitus?DM?is a disease caused by genetic and environmental factors,the damage and reduction of islet?cells is the main cause of its deterioration.It has been found in recent years that renin-angiotensin system?RAS?exists in local pancreas,and the most importantly,the level of angiotensin ??Ang ??,the principal member in RAS,is increased after RAS being activated in DM.The elevated Ang ? causes islet dysfunction and islet?cell apoptosis,however,the mechanism is not yet clear.More and more studies confirm that chronic inflammatory response is an important factor in the development of diabetes.NLRP3 inflammasome is a polyprotein complex,which is activated by various pathogens,and then continue to break Pro Caspase-1 to be the active fragment,Caspase-1 p10,which causes the IL-1?to be mature and induce inflammatory response.There's a lot of speculation about the activation mechanism of the NLRP3,whereas oxidative stress triggers the increase of ROS,which in turn promotes TXNIP over-expression or combine with NLRP3 is the most important activation pathway.Therefore,this study was designed to discuss whether TXNIP-NLRP3 inflammasome pathway can play a role in the process of islet lesion by Ang ?.Objective:1.Using Ang ? to stimulate islet of normal rats,to observe its effect on islet?cells secretion.2.Ang ? icubates normal islet and INS-1 cells,to observe its effects on the apoptosis of islet cells and the expression of NLRP3 inflammasome.3.To Explore the specific mechanism of Ang ? affecting the expression of NLRP3 inflammasome.Methods:1.In vivo experiment:Building the model of diabetes,then isolate the islet and test the Ang ? level of it;2.In vitro experiment:?1?Isolating islet in normal rat.ELISA was used to detect the insulin secretion after the islet treated with Ang ?;?2?Western Blot was used to detect the apoptosis and the expression of NLRP3inflammasome induced by Ang ?;?3?CCK-8 was used to test the cell viability in different concentrations(0?1×10-8?1×10-7?1×10-6?1×10-5mol/L)and different time?0?6?12?24?48 h?of Ang ?;?4?Flow cytometry and Western Blot tested the Cleaved Caspase-3/Caspase-3 to investigate the effect of Ang ? on INS-1 cell apoptosis;?5?Western Blot was performed to detect the expression of NLRP3inflammasome,TXNIP,ChREBP and AT1R under the effect of Ang ?;?6?RT-PCR test for the effect of Ang ? on the m RNA level of the TXNIP and ChREBP of INS-1 cells;?7?IF/ICC were used to observe the effect of Ang ? on the expression of TXNIP,ChREBP and AT1R of INS-1 cells.Results:1.Ang ? is elevated in the diabetes ratAfter isolating diabetic rat islet,ELISA showed that Ang ? level of T1DM group was significantly increased?P<0.01,n=7?;Ang ? content of T2DM group was also increased?P<0.01,n=7?.?Figure 1?2.Ang ? affects the function and protein expression of the islet?cell2.1 Ang ? reduces insulin secretionThe detection of pancreatic islet function showed that the insulin secretion index of glucose stimulation in Ang ? group decrease?P<0.05,n=6?,indicating that Ang ? damaged the islet function.?Figure 2A-C?2.2 Ang ? induces islet cell apoptosisWestern Blot showed that the ratio of Cleaved Caspase-3/Caspase-3 in Ang ? group was increased compared with the Control group?P<0.01,n=6?,indicating that Ang ? induced apoptosis of islet cells.?Figure 2D?2.3 Ang ? activates the NLRP3 inflammasome of isletCompared with the Control group,protein expressions of NLRP3,ASC and Caspase-1 p10 in Ang ? group were all increasing?P<0.01,n=6;P<0.01,n=6;P<0.05,n=6?,the secretion of IL-1?in the culture medium was increasing?P<0.01,n=6?,indicating that Ang ? could induce the activation of NLRP3 inflammasome.?Figure 2E?F?3.Ang ? reduces the survival of INS-1?cellsCCK-8 showed that INS-1 cells viability was falling within the increase concentration of Ang ?,the survival rate was significantly decrease in 1×10-6mol/L?P<0.01,n=6?;INS-1 cells were incubated with different time,we found that cell survival rate were gradually reduced,and cell activity was significantly decreased at24h?P<0.01,n=6?.?Figure 3?4.Ang ? induces INS-1?cells apoptosisThe results of flow cytometry analysis showed that the apoptotic rate of Ang ? group was higher than Control group?P<0.05,n=6?.Western Blot was used to detect the ratio of Cleaved Caspase-3/Caspase-3,we found that the ratio in Ang ? group was increased?P<0.05,n=6?.?Figure 4?5.Ang ? induces INS-1 cells apoptosis through the NLRP3 inflammasomeCompared with Control group,the protein expressions of NLRP3 and ASC in Ang ? group were upregulated?P<0.05,n=6?;p10,the active components of Pro Caspase-1,was significantly increased?P<0.05,n=6?;the amount of IL-1?in cell culture was increased too?P<0.01,n=6?.?Figure 5A?B?MCC950 is a NLRP3 inflammasome inhibitor,we found it inhibited the activation of NLRP3 inflammasome,Caspase-1 p10 fragment's expression and IL-1?secretion in cell supernatant significantly decrease meanwhile?P<0.01,n=6?.At the same time,compared with the Ang ? group,the ratio of Cleaved Caspase-3/Caspase-3in MCC950+Ang ? group was falling obviously?P<0.01,n=6?.It was indicated that Ang ? could induce INS-1 cells apoptosis by activating NLRP3 inflammasome.?Figure 5C?D?6.TXNIP mediates apoptosis and the overexpression of NLRP3 inflammasome induced by Ang ?Ang ? stimulated the expression of TXNIP in mRNA and protein level,and has a dose and time-dependent manner,which increased at 1×10-6mol/L?P<0.01,n=6?and24h significantly?P<0.05,n=6?;immunocytochemical staining also showed that the expression of TXNIP was increased in Ang ? group compared with the Control group.?Figure 6A-E?After shRNA inhibited the expression of TXNIP,compared with Scramble shRNA+Ang ? group,the expression of NLRP3,ASC and p10 in TXNIP shRNA+Ang ? was significantly reduced?P<0.01,n=6?,and IL-1?in the cell supernatant was decreased?P<0.01,n=6?.It was suggested that TXNIP was involved in the activation of NLRP3 inflammasome and the apoptosis of INS-1 cells induced by Ang ?.?Figure 6F-I?7.ChREBP involves in INS-1 cell apoptosis induced by Ang ?The mRNA and protein expression levels of ChREBP in Ang ? group were upregulated compared with the Control group?P<0.05,n=7?.Immunofluorescence showed that ChREBP of Ang ? group was more than that of the Control group,and inversioned from cytoplasm into the nucleus,indicating that Ang ? could activate the ChREBP and increase its expression.?Figure 7?8.AT1R mediates Ang ?-induced NLRP3 inflammasome and INS-1 cell apoptosisWestern Blot showed that the expression of AT1R in Ang ? group was significantly higher than that in the Control group?P<0.05,n=6?.The immunofluorescence picture showed that the red fluorescence of the Ang ? group was significantly higher than that in the Control group.?Figure 8A?B?The use of AT1R specific inhibitors Telmisartan,to pretreat INS-1 cell for 1h,then the cell viability was clearly increased?P<0.01,n=6?,and the ratio of Cleaved Caspase-3/Caspase-3 is lower than the Ang ? group?P<0.05,n=6?.The above results indicated that Ang ? reduced the activity of INS-1 cells and apoptosis through AT1R.?Figure 8C?D?In order to further explore the role of AT1R in the activation of NLRP3inflammasome and TXNIP in Ang ?,we explore the expression of NLRP3inflammasome,TXNIP and ChREBP in the use of Telmisartan.The results showed that the expression of NLRP3 and ASC in TM+Ang ? group was decreased compared with Ang ? group?P<0.05,n=6?,Caspase-1 p10 fragment expression?P<0.05,n=6?and the amount of IL-1?in the cell culture was significantly reduced?P<0.01,n=6?.At the same time,the mRNA and protein levels of TXNIP were significantly decreased?P<0.01,n=6?,ChREBP mRNA and protein level were also inhibited by Telmisartan?P<0.05,n=6?.To sum up,Ang ? plays a role of in INS-1 cells through AT1R.?Figure 8E-J?Conclusion:1.Ang ? can inhibit insulin secretion and induce islet?cell apoptosis.2.TXNIP-NLRP3 inflammasome pathway mediates Ang ?-induced INS-1?cell apoptosis.3.AT1R mediates Ang ?-induced activating of NLRP3 inflammasome and INS-1?cell apoptosis. |
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