Angiotensin ? Type 1 Receptor Autoantibodies Passes Through NLRP3 Inflammasome-IL-1? Pathway Induce Dysfunction Of ? Cells

Background: Diabetes Mellitus(DM)has become an increasingly common disease,with high morbidity and mortality rates,and the onset of the disease is becoming more and more youthful.With the development of diabetes,it will lead to damage to the functions of the heart,blood vessels and other multiple organs.The key to treating diabetes is to curb damage to islet beta cells.Hence,the ability to reduce islet cell apoptosis due to certain pathological causes,such as inflammation,is critical to preventing the development of diabetes.Angiotensin ? type 1 receptor autoantibodies(AT1-AA)were first detected in the serum of patients with pre-eclampsia.Our previous studies have confirmed that AT1-AA can be combined with AT1 R on islet ? cells to promote apoptosis of islet beta cells,but the specific mechanism is unclear.It is reported that chronic inflammation is linked to pancreatic islet beta cell damage in type 2 diabetes.It is further said that inflammatory factor interleukin 1?(IL-1?)is associated with injury to type 2 diabetic islets.There are many mechanisms to promote the activation of IL-1?,among which the activation effect of NLRP3 inflammasome is the most deeply studied.NLRP3 inflammasome is a complex consisting of a variety of proteins.The activation of NLRP3 inflammatory will promote the IL-1? cutting mature,the formation of active IL-1? and play a role.Studies have reported that the activation of NLRP3 inflammasome mainly has three kinds of pathways,including activated oxygen(ROS),potassium ion outflow and lysosomal destruction.TXNIP is an endogenous TRX combined with inhibitory protein,which can block TRX's anti-free radicals and antiapoptosis function and induce apoptosis.Studies have shown that AT1-AA has the effect of promoting inflammation.The main content of this research is whether AT1-AA can increase TXNIP expression,activate NLRP3 inflammasome,lead to increased IL-1? secretion,and then induce the cells dysfunction.Objective: 1.AT1-AA active immune rats were established to observe the rat islet morphological and function,and the change of TXNIP?NLRP3?IL-1?;2.Confirm TXNIP-NLRP3-IL-1? signaling pathways in the role of the islet ? cells injury induced by AT1-AA.Methods: 1.In rats with biologically synthesized AT1R-EC? antigen peptide segment Active immune Experiment Group,mixed liquid immune control group made from immune adjuvant and saline was used in rats.2.Immunohistochemical staining was observed to observe the expression levels of TXNIP,NLRP3 inflammasome,IL-1? and other proteins.3.CCK-8 kit detects cell survival.4.The apoptosis level was detected by flow cytometry.5.ELISA detected the amount of IL-1? secretion.6.Western blot detected the expression level of TXNIP?NLRP3?ASC?Caspase1?Cleaved Caspase3/Caspase3.7.Real time PCR tested the expression level of TXNIP m RNA.Results: 1.Successful establishment of AT1-AA active immune rat model The serum antibody titer of rats was detected by SA-ELISA method using synthetic Antigen peptide segment active immune rats.The results showed that,with the prolongation of immune time,the antibody titer of immunized rats increased with the control group,increased significantly in the 4th travels(P<0.01),and by the 8th week,the antibody Titer peaked(P<0.01),and the model was successfully constructed(Fig.1).2.AT1-AA active immune Rat islet tissue structure and function may be impaired The results showed that four weeks before the immune group rats weight and the control group no difference between the weight of rats,and the weight of immunized rats group was consistently lower at the beginning of the 10 th week,and weight decreased obviously appeared at the 16 th week(P<0.05)(Fig.2A).The day before the immunization,two groups of rats were tested for fasting blood glucose.The results showed that there was no statistically significant blood glucose in the two groups of rats in the early stage of immunity,and the fasting blood glucose in immune rats group was significantly higher than that in the same period from the beginning of week 8th to the 16 th week(P<0.05)(Fig.2B).The results of glucose tolerance test showed that the blood glucose value of immunized rats did not drop to the original level after 2h in 12 weeks and 16 weeks(P<0.05)(Fig.2C).The results of insulin tolerance test showed that insulin was injected into the abdominal cavity of the control rats,the blood glucose decreased rapidly at 30 min,and the blood glucose returned to normal level at 2h,and 12 weeks and 16 weeks of immune rats were injected with insulin,and there was no significant change of blood glucose in rats(P<0.05)(Fig.2D).With the prolongation of immune time,the results of HE staining in rat islet tissue showed that the pancreatic islet tissue structure was seriously disordered(Fig.2E).The above results suggest that AT1-AA long-standing can lead to damaged islet function and structure.3.Increased apoptosis of pancreatic islet cells in AT1-AA active immune rats.TUNEL staining results showed that,compared with the control group,the nucleus of islet tissue in immune rats group became larger and the brown-yellow granules increased with the prolongation of immune time(Fig.3A).Western blot results showed that the ratio of cleaved caspase3/caspase3 in immune rats group increased compared with that in control group(P<0.05)(Fig.3B).The results suggest that AT1-AA longstanding can lead to increased islet tissue apoptosis.4.Increased IL-1? secretion in serum of AT1-AA active immune rats Immunohistochemical staining showed that,compared with the control group,the more brown particles in the islet tissue of immunized rats,the darker the color(Fig.4A).The results of IL-1? secretion showed that the secretion of IL-1? in serum of immunized rats increased with the prolongation of immune time(P<0.05)(Fig.4B)5.Increased expression of inflammatory NLRP3 in pancreatic islet tissue of AT1-AA active immune rats The results showed that the increase of brown-yellow granules in pancreatic islet tissue of immunized rats group compared with the control group.(Fig.5A)Western blot showed that the expression of NLRP3 and ASC in the pancreatic islet tissue of immunized rats group increased compared with that in the control group.(P<0.01)(Fig.5B)6.Increased expression of TXNIP in pancreatic islet tissue of AT1-AA active immune rats The results showed that,compared with the control group,the brown yellow granules of the islet tissue of the immunized rats group increased and the coloring deepened.(Fig.6A)The results showed that the expression level of TXNIP protein in the pancreatic islet tissue of immunized rats group increased compared with the control group(P<0.05).(Fig.6B)7.AT1-AA dose-and time-dependently reduces the INS-1 cell viability CCK-8 showed that compared with Negative-Ig G group,the survival rate of INS-1 cells decreased with the increase of AT1-AA concentration.(P<0.05).(Fig.7A)Compared with Negative-Ig G group,the survival rate of cells decreased significantly at 24h(P<0.01)(Fig.7B)8.AT1-AA induces INS-1 cells apoptosis Flow cytometry results show that compared with Negative-Ig G group,the apoptosis rate of AT1-AA experimental group increased obviously(P<0.01)(Fig.8A)The Western blot showed that compared with the Negative-Ig G control group,the ratio of cleaved caspase3/caspase3 in AT1-AA Group increased significantly(P<0.01)(Fig.8B)9.AT1-AA can increase the amount of INS-1 cell IL-1? secretion. The results of ELISA show that compared with Negative-Ig G group,AT1-AA can increase the secretion of cell IL-1? significantly(P<0.01)(Fig.9)10.NLRP3 inflammasome-IL – 1? is involved in the INS-1 cells apoptosis induced by AT1-AA.The results showed that the expression level of NLRP3 inflammatory small body in AT1-AA group was higher than that of Negative-igg control group(P<0.05).(Fig.10A)Incubation of cells using inhibitors of NLRP3 inflammatory small bodies MCC950,Western blot showed that the cleaved caspase3/caspase3 ratio of INS-1 cells in AT1-AA+MCC950 group decreased compared with that of AT1-AA group(P<0.05)(Fig.10B)The secretion of IL-1? decreased(P<0.05)(Fig.10C)11.TXNIP-NLRP3 inflammasome-IL-1? is involved in AT1-AA induced INS-1 cells apoptosis Detection of TXNIP expression by real-time PCR and Western blot,the TXNIP m RNA(P<0.01)and protein expression levels(P<0.01)in AT1-AA group were significantly higher than those in the Negative-Ig G control group.(Fig.11A)TXNIP sh RNA infected INS-1 cells with a slow virus package,the results showed a significant decrease in TXNIP m RNA(P<0.05)and protein expression(P<0.01)in the TXNIP sh RNA group compared with the Scramble sh RNA group(Fig.11B)Compared with Scramble sh RNA Group,the apoptosis rate and cleaved caspase3/caspase3 ratio of Scramble sh RNA+AT1-AA group were significantly increased(P<0.01);Compared with Scramble Sh RNA+AT1-AA group,the apoptosis rate and cleaved caspase3/caspase3 ratio of TXNIP sh RNA+AT1-AA group were significantly reduced(P<0.01)(Fig.11C).Results suggest TXNIP participated in the INS-1cells apoptosis induced by AT1-AA.ELISA showed that compared with Scramble sh RNA group,the IL-1? secretion of Scramble sh RNA+AT1-AA Group increased obviously(P<0.05);Compared with the Scramble Sh RNA+AT1-AA group,the IL-1? secretion of the TXNIP sh RNA+AT1-AA group decreased significantly(P<0.05)(Fig11.D) Western blot showed that compared with Scramble Sh RNA group,the expression of NLRP3 inflammasome protein in Scramble sh RNA+AT1-AA group was significantly increased(P<0.05);Compared with Scramble Sh RNA+AT1-AA group,the expression of protein in NLRP3 inflammasome of TXNIP Sh RNA+AT1-AA group decreased(P<0.05)(Fig11.E).The above results suggest TXNIP is involved in the activation of NLRP3 inflammasome and IL-1? secretion.12.Telmisartan can inhibit TXNIP-NLRP3 inflammasome-IL-1? to reduce AT1-AA induced INS-1 cells apoptosis Western blot showed that compared with AT1-AA group,the ratio of cleaved caspase3/caspase3 in AT1-AA+TM group decreased(P<0.05)(Fig12.A)The secretion of IL-1? decreased(P<0.05)(Fig12.B)The protein expression levels of NLRP3 inflammasome decreased(P<0.05)(Fig12.C).TXNIP protein expression is also reduced(P<0.05)(Fig12.D)Conclusion: 1.AT1-AA exist for a long time can lead to damaged islet structure,functional disorders,and can increase rat islet tissue TXNIP,NLRP3 inflammasome and IL-1? levels at the same time;2.TXNIP-NLRP3-IL-1? pathway is involved in the islet ? cells injury induced by AT1-AA.3.ARB by inhibiting TXNIP-NLRP3-IL-1? pathway to improve islet ? cells induced by AT1 – AA.