Study On The Mechanism Of NLRP3 Inflammasome In HIV-1 Gp120-induced Central Nervous System Injury

BackgroundHuman immunodeficiency virus 1(HIV-1)infection not only impairs immune function,but also causes neuronal injury,leading to HIV-associated neurocognitive disorders(HAND).Although highly active antiretroviral therapy(HAART)effectively decreases the HIV burden in the central nervous system(CNS),mild and moderate forms of HAND remain prevalent and affect the quality of life.This phenomenon indicates that HAART fails to provide effective protection against HIV-associated neuronal damage and suggests a need for effective neuroprotective agents.Numerous studies demonstrated that neuroinflammation is involved in gp120-induced neuronal damage.Interleukin-1?(IL-1?)is a critical proinflammatory mediator that initiate the inflammation and destructive responses in CNS upon exposure to gp120.pro-IL-1? becomes proteolytically matured via cleavage by activated caspase-1.The activity of caspase-1 is controlled by a cytosolic protein complex,namely NLRP3 inflammasomes.The relationship between gp120 and NLRP3 inflammasomes and the exact mechanism by which induction of IL-1?production and subsequent neuronal damage remain unclear.OurStudy aims at the mechanism of NLRP3 inflammasome in HIV-1 gp120-induced central nervous system injury.ObjectiveThe purpose of this study was to investigate the neurological damage caused by the gp120-NLRP3-caspase-1-IL-1? pathway.The NLRP3 inhibitor MCC950 and caspase-1 inhibitor Z-YVAD-fmk can reduce the release of neurotoxic factors and chemokines,such as 1L-1beta.nitric oxide(NO),tumor necrosis factor-alpha(TNF-alpha)and cyclooxygenase-2(COX-2),also can reduce nerve injury and improve the role and mechanism of neurocognitive impairment.It provides a new research direction for providing theoretical basis and selection of clinical therapeutic targets for the pathogenesis of HAND.Methods1.Identification and proliferation of gp120 transgenic(Tg)mouse model3-week-old mice bred from gp120 Tg mice,DNA was extracted from the tail tissue of the mouse,and after PCR,nucleic acid agarose gel electrophoresis was used to identify gp120-positive mice.Marking the birth date and proliferate to 6-14 months for subsequent experiments.2.Treatment of microglia BV2 and neurons with gp120Treatment of BV2 cells or neuronal cells with p120 concentration gradient and time gradient,and Cells were treated with inhibitors of NLRP3,caspase-1,K+,ROS,cathepsin B,potassium voltage-gated channels Kvl.3,CXCR4,IL-1 receptors and/or corresponding siRNA gene silencing techniques.Cell supernatant and cell protein were collected,and the expression levels of IL-1?,NLRP3,caspase-1,ASC,neurotoxic factors and neuronal cell damage induced by gp120 were detected by Enzyme-Linked Immuno Sorbent Assay(ELISA),Western blot(WB)and immunofluorescence.3.Intraperitoneal injection of MCC950 and Z-YVAD-fmk inhibitors into gp120 Tg micegp120 Tg mice(11-12 months)and WT were divided into 4 groups(9 mice per group):WT+vehicle,WT+MCC950,gp120 Tg+vehicle and gp120 Tg+MCC950.Mice were intraperitoneally injected with 200 ?L sterile PBS(vehicle)or MCC950(10 mg/kg body weight in PBS)every third day for 80 days.Follow-up Behavioral Analysis.gp120 Tg mice(9-10 months)and WT were divided into four groups:WT+vehicle,WT+Z-YVAD-fmk,vehicle+gp120 Tg and Z-YVAD-fmk+gp120 Tg.Mice were intraperitoneally injected with 200 !uL sterile PBS(vehicle,containing 1%vol/vol DMSO)or z-YVAD-fmk(10 mg/kg body weight in 1%DMSO,diluted in PBS)every second day for 30 days.Afterward,euthanasia,the brain was removed and the cells were homogenized.The supernatant was harvested by centrifugation and IL-1? release was measured by ELISA and Western blotting.The supernatant was centrifuged and harvested,IL-lbeta release was measured by ELISA and Western blotting.4.Behavioral analysis of miceMorrris water maze and open field experiments were conducted in four groups of mice injected MCC950 intraperitoneally.Animal movements were tracked using the TSE-VideoMot2 video tracking system.And automatically record the total distance moved,Swimming speed,time spent in each quadrant,number of crossings of platform positions,the distance moved in the center area,the time spent in the center area,and the number of central area entries.After euthanasia,the supernatant of blood and brain cell lysate was collected,the expression of IL-lbeta was analyzed by ELISA,and the expression of inflammatory and chemokines in cerebral cortex and hippocampus was analyzed by immunohistochemistry.Results1.Thegp120 Tg mice expressed gp120 stably,and the number of mice proliferated and the month-old met the experimental needs.2.The expression of IL-1?,caspase-1 and NLRP3 in BV2 cells treated with gp120 was increased.Z-YVAD-fmk(a specific inhibitor of caspase-1)could inhibit the release of IL-1 beta induced by gp120 in a dose-dependent manner in vitro and in vivo.It was demonstrated that gp120-induced IL-1? release is dependent on caspase-1 activation.3.The oligomer formation of ASC was detected in BV2 cells treated with gp120,suggesting that gp120 can induce ASC inflammatory body assembly.Higher IL-1? release was still detected in NLRP3 knockdown BV2 cells than in controls,suggesting that non-caspase-1 treatment may be involved in processing pro-IL-1?.The knockdown of ASC or caspase-1 also significantly reduced the activation of caspase-1 and the production of IL-1 beta and reduced by MCC950.gp120 can promote the degradation of I?Ba and nuclear translocation of NF-?B p65,and enhance the expression of NLRP3 and pro-IL-1 beta.The results showed that the production of IL-1? induced by gp120 was regulated by NLRP3-ASC-caspase-1 and caused cell death.4.When high concentration of K+ inhibited K+ efflux in cells,IL-1? production by gp120 decreased in a dose-dependent manner.Pretreatment of BV2 cells with ROS inhibitors also inhibited the production of IL-lbeta induced by gp120.The results indicate that both K+ efflux and ROS production are required for inflammatory body activation by gp120.Activation of NLRP3 in BV2 cells was attenuated with Kv1.3 inhibitor or Kvl.3-siRNA,indicating that gp120-induced NLRP3 activation requires the CXCR4-Kvl.3 pathway.gpl20-induced IL-1?release and increased expression of NO,TNF-? and COX-2 neurotoxic factors,but significantly inhibited its expression with NLRP3 and IL-1 inhibitors.5.Neuronal cells were cultured with supernatant of BV2 cells treated with gp120,causing significant dendritic damage.but neurotoxicity was blocked with MCC950,indicating that gp120-induced neuronal damage is mediated by the NLRP3-IL-1? axis and MCC950 Protected.6.Treatment with MCC950 can blocked NLRP3 inflammatory body activation and IL-1? production,improved neuroinflammation,promoted M2 microglia polarization,attenuated neuronal damage and improvement and cognitive function of gp120 Tg mice.Conclusions1.gp120 causes neurological damage through the gp120-NLRP3-caspase-1-IL-1?pathway.2.Inhibition of NLRP3 reduces activation of the gp120-caspase-1-IL-1? pathway and attenuates neurological damage.3.MCC950 improves neurotoxicity,cognitive function and neuroprotection in gp120 Tg mice.