The Role Of Nrf2 Pathway In Methamphetamine And HIV-Tat Protein Synergistically Induced Autophagy And Activated NLRP3 Inflammasome In Microglia

Background:Drug abused and HIV infection have become the public health issues of global concernd that cannot be ignored.Methamphetamine(METH)is the most abused drug in our country.Long-term abused can cause strongly dependence and high neurotoxicity.HIV infection is a common comorbidity of drug abuse and is often associated with increased neurological complications.Compared with HIV(+)individuals who not abused METH,co-exposure of HIV and abused METH have additive/synergistic effects on neurotoxicity.HIV-1 Tat protein is a HIV transactivator of transcription,HIV-1 Tat plays a key role in HIV-1 replication by upregulating the transcription of the 5’long terminal repeat-containing promoter.Co-exposure of CNS cells to HIV-Tat and abused METH could have synergistic effects on neurotoxicity,but the mechanisms leading to neurotoxicity in the CNS remain unclear.So we hypothesized that Nrf2 pathway,oxidative stress,autophagy and neuroinflammation may be involved in the synergistically effect of METH and HIV-Tat proteins in microglia.Objective:The purpose of present study is to explore the role of Nrf2 pathway in METH and HIV-Tat protein synergistically induced autophagy and activated NLRP3 inflammasome in microglia.To clarify the mechanism of METH and HIV-Tat protein synergistically induced autophagy and activated NLRP3 inflammasome in brain cells could provide the theoretical basis and new ideas for an alternative therapeutic approach to HIV(+)and drug abusers.Methods:The study was carry out in human brain tissue samples,in vivo and in vitro experiments.Part 1.Human brain tissue samples:our study choose human striatum from the Forensic Appraisal Center of Kunming Medical University as clinical sample to solve the following scientific questions.The expressions of Nrf2,LC3-Ⅱ and NLRP3 were detected by immunofluorescence and immunohistochemistry,and using TUNEL staining to measure apoptosis in striatum of human brain tissuePart 2.In vivo experiments:our study choose WT-C57BL/6J and Nrf2-KO C57BL/6J mouse as animal model to solve the following scientific questions.After SFN(10 mg/kg.d)activating Nrf2 and knocking out Nrf2 gene in C57BL/6J mouse,intraperitoneal injection of METH(2 mg/kg,12 h interval)and HIV-Tat(10 ug/kg)protein was injected into the right striatum.2.1 Using open field test to analyze behavioral changes in C57BL/6J mouse.2.2 Detected the expression levels of Nrf2-related protein(T-Nrf2,N-Nrf2,HO-1,NQ01),autophagy-related protein(LC3-II,Beclin-1,ATG5,ATG7),NF-kB-related protein(NF-kB and p-NF-kB)and NLRP3 inflammasome-related protein(NLRP3,ASC,Caspase-1,IL-18,IL-1β)by Western blot.2.3 Detected LC3-Ⅱ and NLRP3 expression by immunofluorescence in microglia of C57BL/6J mouse striatum.2.4 Using TUNEL staining to measure apoptosis in striatum of C57BL/6J mouse.Part 3.In vitro experiments:our study choose BV2 cells and primary C57BL/6J mouse microglia as cell models to solve the following scientific questions.After SFN(1.25 μM)activating Nrf2 and silencing Nrf2 gene,microglia was treated with METH(0.1mM)and HIV-Tat protein(50 nM).3.1 Examined the cell viability by CCK8 kit.3.2 The level of MDA,SOD,CAT and GSH-Px were measured using commercial kits in BV2 cells.3.3 Detected the expression levels of Nrf2-related protein(T-Nrf2,N-Nrf2,HO-1,NQ01),autophagy-related protein(LC3-Ⅱ,Beclin-1,ATG5,ATG7),NF,kB-related protein(NF-kB and p-NF-kB)and NLRP3 inflammasome-related protein(NLRP3,ASC,Caspase-1,IL-18,IL-1β)by Western blot.3.4 Detected LC3-Ⅱ and NLRP3 expression by immunofluorescence in BV2 cells and primary microglia.3.5 Using TUNEL staining to measure apoptosis in BV2 cells.Results:1.Human brain tissue samplesThe expression levels of Nrf2,LC3-Ⅱ and NLRP3,and the number of cell apoptosis were significantly increased in METH group and METH+AIDS group,and the microglia were activated in striatum of human brain tissue.2.In vivosMETH and HIV-Tat protein synergistically activated microglia and induced the expression levels of autophagy-related proteins(LC3-Ⅱ,Beclinl,ATG5,ATG7),NF-κB-related proteins(NF-κB,p-NF-κB)and NLRP3 inflammasome-related proteins(NLRP3,ASC,Caspase-1,IL-18,IL-1β)and the number of cell apoptosis were significantly increased in striatum of C57BL/6J mouse.After activating Nrf2 gene,the expression levels of above-mentioned proteins were decreased to varying degrees,and the number of apoptosis was reduced.After knocking out Nrf2 gene,the expression levels of above-mentioned proteins and the number of apoptosis were significantly increased.3.In vitrosMETH and HIV-Tat protein synergistically reduced microglia viability,and increased the expression levels of ROS and MDA and decreased the expression levels of SOD,CAT and GSH-Px.METH and HIV-Tat protein synergistically induced the expression levels of autophagy-related proteins(LC3-Ⅱ,Beclinl,ATG5,ATG7),NF-κB-related proteins(NF-κB,p-NF-κB)and NLRP3 inflammasome-related proteins(NLRP3,ASC,Caspase-1,IL-18,IL-1β)and the number of BV2 cells apoptosis were significantly increased in microglia.After activating Nrf2 gene,the expression levels of above-mentioned proteins were decreased to varying degrees,and the oxidative stress level and the number of BV2 cells apoptosis were reduced.After knocking out Nrf2 gene,the expression levels of above-mentioned proteins and the oxidative stress level and the number of BV2 cells apoptosis were significantly increased.Conclusions:1.METH poisoning or addiction could activate microglia,induce autophagy and activate Nrf2 and NLRP3 inflammasome,co-exposure of METH poisoning or addiction and HIV(+)have additive/synergistic effects on autophagy,activated Nrf2 and NLRP3 in striatum of hunman brain.2.METH poisoning or addiction and co-exposure of METH poisoning or addiction and HIV(+)could induce apoptosis in striatum of hunman brain,respectively.3.Co-exposure of METH and HIV-Tat protein have additive/synergistic effects on the oxidative stress and autophagy,and synergistically activated NLRP3 inflammasome by NF-κB in microglia.4.Nrf2 pathway regulates the synergistic effect of METH and HIV-Tat protein induced autophagy by regulating oxidative stress in microglia.5.Nrf2 pathway regulates the synergistic effect of METH and HIV-Tat protein activated NLRP3 inflammasome by regulating oxidative stress and NF-κB in microglia.6.Nrf2 pathway plays an crucial regulatory role in METH and HIV-Tat protein synergistically inducted microglia apoptosis.