The Protective Role Of Nrf2-Trx1/TXNIP-NLRP3 Inflammasome Axis In Acute Alcohol Exposure-induced Pancreatic β-Cell Damage

Objective:Alcohol is a psychoactive substance that causes dependence,and health problems associated with excessive alcohol consumption are a major challenge for the public health system.Epidemiological investigations have shown that alcohol consumption is strongly associated with the development of type 2 diabetes.However,the specific biological mechanisms by which alcohol consumption contributes to the development of type 2 diabetes remain unclear.There are two main causes of the development of type 2 diabetes,pancreatic β-cell damage and insulin resistance.Our study focused on the damage and its associated mechanisms to pancreatic β-cell caused by acute alcohol exposure.Oxidative stress and innate immunity-mediated inflammatory responses are key factors in type 2 diabetes development.NLRP3 inflammasome are important components of innate immunity,and their over activation is closely related to the development of type 2 diabetes.Nrf2 is a central regulator of the intracellular antioxidant response and directly regulates the expression of several antioxidant enzymes including Trx1.Our group has demonstrated that Nrf2 gene deficiency mice have more severe pancreatic β-cell damage induced by acute alcohol exposure;however,further studies are needed to investigate the molecular mechanism underlying the protective role of Nrf2 in pancreatic β-cell damage induced by acute alcohol exposure.In this study,we used the normal and Nrf2-KD MIN6 and Nrf2 pancreatic β-cell-specific gene knockout mice to investigate pancreatic β-cell damage induced by acute alcohol exposure and the related mechanisms,as well as the protective role of Nrf2 on pancreatic β-cell damge induced by acute alcohol exposure and the related mechanisms.Finally,we applied the Nrf2 activator DMF to explore the prevention and treatment strategies for pancreaticβ-cell damage caused by acute alcohol exposure.Methods:Part 1(1)Apply different doses and times of alcohol treatment to MIN6 cells and observe the cell morphology by inverted microscope;MTS method to detect cell survival rate;Western blot to detect cleaved-Caspase-3 protein level;and TUNEL staining method to observe the number of apoptotic cells.(2)Gene expression levels of Nlrp3,Txnip,Asc,Caspase-1,IL-1β and IL-18 were detected by RT-q PCR,and protein levels of NLRP3,TXNIP,ASC,cleaved-Caspase-1 and mature IL-1β were detected by Western blotting.(3)Apply the NLRP3 inflammasome inhibitor MCC950 and different doses of alcohol to co-treat MIN6 cells,and detect the cell survival rate by the MTS method,observe the number of apoptotic cells by TUNEL staining,and detect IL-1β and IL-18 levels in cell culture supernatant by ELISA.(4)MIN6 cells were co-treated with antioxidant NAC and different doses of alcohol,and ROS levels in MIN6 cells were measured by flow cytometry and confocal microscopy after exposure to different doses of alcohol.NLRP3,cleaved-Caspase-1,and mature IL-1β levels were measured by Western blotting,the cell survival rate was measured by MTS,the number of apoptotic cells was observed by TUNEL staining,and the levels of IL-1β and IL-18 in the cell culture supernatant were measured by ELISA.TUNEL staining was performed to determine the number of apoptotic cells.(5)6-8 week old C57BL/6 male mice were selected as experimental subjects,and islets were isolated in vitro and treated with different doses of alcohol after 2 days of culture.The level of IL-1β in the supernatant,inverted microscopy to observe the morphological and quantitative changes in islets,and TUNEL staining were used to observe the number of apoptotic cells.(6)RT-q PCR to detect the gene expression levels of Nrf2,Trx1,Gclc and Gclm;Western blotting to detect the protein level of Nrf2;Immunofluorescence to observe the expression and nuclear translocation of Nrf2.Part 2(1)Nrf2-KD MIN6 cells were produced by applying lentivirus as vector sh RNA,the gene level of Nrf2 was detected by RT-q PCR technique;and protein level of Nrf2 was detected by Western blotting.(2)Scr and Nrf2-KD cells were treated with different doses of alcohol and the cell status was observed by inverted microscopy;the cell survival rate was detected by MTS;The cleaved-Caspase-3 protein level was detected by Western blotting;the number of apoptotic cells was determined by TUNEL staining.(3)Scr and Nrf2-KD cells were treated with different doses of alcohol and ROS levels were observed by confocal microscopy(4)Scr and Nrf2-KD cells were treated with different doses of alcohol and protein levels of Nrf2 were detected by Western blot;RT-q PCR technique was used to detect the Nrf2,Trx1,Gclc and Gclm gene expression levels.(5)Scr and Nrf2-KD cells were treated with different doses of alcohol,and the gene expression levels of Nlrp3,Asc,Caspase-1,and IL-1β were detected by RT-q PCR;the protein levels of NLRP3,TXNIP,ASC,cleaved-Caspase-1 and mature IL-1β;Using ELISA for IL-1βand IL-18 in cell culture supernatant.(6)Immunoprecipitation technique was used to detect the interaction of Trx1/TXNIP,TXNIP/NLRP3,and NLRP3/ASC.(7)Ins2-Cre tool mice and Nrf2-Flox tool mice were bred to obtain Nrf2 pancreatic β-cell-specific knockout mice.Morphological and body weight changes were observed,and the islets,tails,liver,spleen,and kidney were used for subsequent experiments.Knockout stability was verified by agarose electrophoresis and the effect of tissue-specific knockout was verified by RT-q PCR.Morphological and quantitative changes in the islets were observed using inverted microscopy after applying different doses of alcohol.(8)Nrf2-KD MIN6 cells were co-treated with DMF and different doses of alcohol,and the cell survival rate was determined using the MTS assay.The number of apoptotic cells was determined by TUNEL staining,the level of ROS in islets was detected by confocal microscopy,and the gene expression levels of Trx1,Txnip,Nlrp3 and Caspase-1 were detected by RT-q PCR.Western blotting was used to detect the protein levels of Nrf2,NLRP3,TXNIP,cleaved-Caspase-1 and mature IL-1β.Results:Part 1(1)Acute alcohol exposure alters MIN6 cell morphology,with a dose-dependent decrease in cell survival and a dose-dependent increase in apoptosis.(2)Acute alcohol exposure activated NLRP3 inflammasomes in MIN6 cells in a dose-dependent manner,peaking at 6-12 hours and decreasing thereafter.(3)Co-treatment of MCC950 with alcohol effectively inhibited NLRP3 inflammasome activation,increased cell survival and reduced the number of apoptotic cells.(4)The level of ROS in MIN6 cells increased after acute alcohol exposure,and NAC treatment effectively reduced intracellular ROS levels,inhibited NLRP3 inflammasome activation,increased cell survival,and reduced the number of apoptotic cells.(5)Acute alcohol exposure increased intracellular ROS levels and induced NLRP3 inflammasome activation in islets.IL-1β levels were increased in islet culture supernatants.Optical microscopic observation of islet morphology revealed a decrease in the number of islets,disruption of membrane structural integrity and a significant increase in the number of apoptotic cells,further validating the experimental findings obtained in MIN6 cells.(6)Acute alcohol exposure activated the Nrf2 signalling pathway in MIN6 cells,increased Nrf2 nuclear translocation and promoted Nrf2 downstream antioxidant gene expression.Part 2(1)Nrf2 gene and protein levels were maintained at low levels in Nrf2-deficient cells compared to Scr controls.(2)The survival rate of Nrf2-deficient cells was further reduced,and apoptosis increased in comparison with that of the Scr control group.(3)Nrf2 gene deletion exacerbated the increase in ROS levels in MIN6 cells compared to that in the Scr control group.(4)Nrf2-KD MIN6 cells showed reduced expression of Nrf2 and its downstream genes and decreased accumulation of Nrf2 protein following acute alcohol exposure.(5)Nrf2 gene deletion exacerbated NLRP3 inflammasome activation in MIN6 cells compared to that in Scr controls.(6)Increased binding of TXNIP,ASC,and NLRP3 in MIN6 cells after acute alcohol exposure demonstrated the involvement of TXNIP in NLRP3 inflammasome activation.Trx1 dissociation from TXNIP was further increased and TXNIP binding to NLRP3 and NLRP3 to ASC was further increased in Nrf2-KD cells compared to Scr controls.(7)DNA agarose electrophoresis showed that the Cre-Loxp system was successfully established and that Nrf2 gene expression was significantly suppressed in islets compared to organs such as the liver,spleen,and kidney.In addition,the body weight and morphology of Nrf2(β)-KO mice were similar to wild-type mice,with no significant changes.Compared with WT mice,the islets of Nrf2(β)-KO mice were more severely damaged after acute alcohol exposure.(8)Co-treatment of DMF with alcohol effectively increased Scr cell survival,reduced apoptosis,decreased intracellular ROS levels,and reduced the gene and protein levels of various components of NLRP3 inflammasomes,but none of the changes were statistically significant in Nrf2-KD cells.(9)Draw summary map.Conclusion:(1)ROS-dependent NLRP3 inflammasome activation is involved in acute alcohol-induced pancreatic β-cell damage.(2)Nrf2 inhibits NLRP3 inflammasome activation and attenuates acute alcohol-induced pancreatic β-cell damage by regulating the Trx1/TXNIP complex.(3)DMF antagonizes acute alcohol-induced pancreatic β-cell damage by activating the Nrf2 signaling pathway.