Effect And Mechanism Of Autophagy In The Inflammation Of Microglia Induced By HIV-1 Gp120V3 Loop

Background With the widespread use of combination antiretroviral therapy(cART)in the treatment of AIDS,The incidence of fatal complications such as opportunistic infection was significantly reduced.However,the incidence of HIV-1-associated neurocognitive disorders(HAND)was still increased due to the prolonged survival of AIDS patients,HAND is characterized by attention,behavioral cognition and memory disorders,which seriously affect the quality of life of patients,and the time from diagnosis to death is only 4-6 months in China.Studies have shown that the main pathogenesis of HAND is the apoptosis of neurons mediated by the release of inflammatory factors such as microglia cells infected with HIV-1,but the specific mechanism is unknown.Therefore,exploring the pathogenesis of HAND has become an urgent problem to be solved.HIV-1 can activate microglia and cause them to release inflammatory cytokines,causing a chronic inflammatory response and damage neurons.HIV-1 envelope glycoprotein gp120(HIV-1 gp120)is a key protein for the virulence of HIV-1 virus,our previous studies have found that gp120 can cause microglia inflammation,and preliminary experiments have found that autophagy plays a role in HIV-1 gp120V3 loop induced microglia inflammation,but the specific mechanism is still unclear.Aim Investigate the effect of autophagy in early and late stage of microglia inflammation induced by HIV-1 gp120V3 loop,as well as the effect and mechanism of differential expression of autophagy substrate protein p62 in early and late stage of microglia inflammation induced by HIV-1 gp120V3 loop.To provide a new perspective and strategy for treatment and prevention of HAND.Method In vitro1.To screen the experimental concentration of HIV-1 gp120V3 loop-induced CHME-5inflammation and determine the trend of inflammatory factors.CCK8 assay was used to determine the effect of HIV-1 gp120V3 loop and random peptide on the survival rate of CHME-5.The 1μg/mL HIV-1 gp120V3 loop and random peptides of the same concentration were selected to act on CHME-5 for 0,6,12,24,36 and 48 hours respectively,the levels of TNF-α,MCP-1 and IL-1β were detected by ELISA.2.To observe the role of autophagy in CHME-5 microglia cell inflammation induced by HIV-1 gp120V3: 1μg/mL HIV-1 gp120V3 loop and random peptide at the same concentration acted on CHME-5 for 0,6,12,24,36 and 48 hours respectively,and the changes of autophagy flux at each time point were detected by RFP-GFP-LC3 double-labeled adenovirus assay.Western blot was used to detect Beclin-1,LC3Ⅱ/Ⅰ and p62 levels of autophagy related proteins at each time point.According to the optimal action concentration of HIV-1 gp120V3 loop as well as the time point of inflammatory changes,the effects of HIV-1 gp120V3 loop at 6 h and 24 h were selected as the early and late study time points.3.The role of autophagy in HIV-1gp120V3 loop-induced microglia inflammation: Autophagy flux was detected by RFP-GFP-LC3 double-labeled adenovirus assay after using autophagy blocker 3-MA and autophagy agonist RAPA,and autophagy related proteins beclin-1,LC3Ⅱ/Ⅰ,p62 levels were detected by Western blot,the levels of TNF-α,MCP-1 and IL-1βwere detected by ELISA.4.To observe the role of p62 differential expression in HIV-1 gp120V3 loop-induced CHME-5 inflammation: according to the part 2,to the observation of p62 levels of HIV-1 gp120V3 loop induced CHME-5 at various time points,it was found that p62 was down-regulated at6 h and up-regulated at 24 h.Therefore,p62 was knockdown at 6 h and 24 h to observe the changes of inflammatory factors.5.To observe the role of Nrf2,the downstream signal molecular of p62,HIV-1 gp120V3 loopinduced CHME-5 inflammation:1)1μg/mL of HIV-1 gp120V3 loop-induced CHME-5 for 0,6,12,24,36 and 48 hours,respectively.Western blot was used to detect Nrf2 protein level changes at various time points.2)According to the previous experiment result,Nrf2 was up-regulated at all time points.Combined with the results of experiment 1,2 and 3,In early and late stage of HIV-1 gp120V3loop-induced CHME-5 inflammation,RNAi technology was used to knock down Nrf2 to observe the changes of inflammatory factors.And the expression levels of p62 and Keap-1were observed in the late stage to verify whether p62 and Keap-1 are Nrf2 target proteins.3)To observe the activation way of Nrf2: the Nrf2 negative regulator Keap-1 were detected by Western blot at each time point.Identification of interaction between p62 and Keap-1 by coimmunoprecipitation in the early and late stage.p62 was knocked down respectively in early and late stage of HIV-1 gp120V3 loop-induced CHME-5 inflammation,Keap-1,Nrf2,HO-1and p62 protein levels were detected by western blot.6.The mechanism of Nrf2-HO-1-iNOS in HIV-1 gp120V3 loop mediated CHME-5 microglia cell inflammation:1)To observe the protein levels of HO-1(the target protein of Nrf2)and iNOS in the early and late stage of HIV-1 gp120V3 loop-induced CHME-5 inflammation.2)HO-1 was knocked down in early and late stage using RNAi technology,and the levels of inflammatory factors in each group were detected by ELISA;Western blot,and immunofluorescence method was used to observe the iNOS and HO-1.3)In the early and late stages of HIV-1 gp120V3 loop-induced CHME-5 inflammation,the protein levels of Nrf2,Keap-1,HO-1 and iNOS were detected by using autophagy blocker3 MA and autophagy agonist RAPA,respectively.In vivo1.Wild type mice(WT)strain C57BL6 J were used as experimental subjects,with 6 mice in each group.A: Control group,B: ACSF sham group,C: Random peptide control group,D:HIV-1 gp120V3 loop 1ng group,E: HIV-1 gp120V3 loop 10 ng group,F: HIV-1 gp120V3 loop 100ng group.Morris water maze experiment was conducted after 3 days of continuous injection to test the spatial memory ability of mice to determine the optimum dose of HIV-1gp120V3 loop;After the animal water maze experiment,whole brain or perfusion fixation samples were taken immediately after the experiment.The levels of inflammatory factors TNF-α,MCP-1 and IL-1β in brain homogenates of mice were detected by ELISA;The microglia marker protein Iba-1 was labeled by immunofluorescence.2.Wild type mouse(WT)strain C57BL6 J was used as the experimental object,with 6 mice in each group,half male and half female and following groups were set : A: Control group,B:NS,C: Brusatol 2 h group,D: Brusatol 6 h group,E: Brusatol 12 h group,F: Brusatol 24 h group,G: Brusatol 48 h group.There were 3 mice in each group,fresh brain tissue homogenates of mice in each group were taken,and Nrf2 protein levels were detected by Western blot to determine the time when Nrf2 expression in brain tissue was blocked by Brusatol.3.Wild-type mouse(WT)strain C57BL6 J was used as the experimental object,after HIV-1gp120V3 loop was intracerebroventricular injected,Brusatol was used to block Nrf2.With 10 mice in each group and the following groups were set as follows,A: Random peptide group,B: Brusatol + Random peptide,C: HIV-1 gp120V3 loop,D: HIV-1gp120V3 loop + Brusatol.The levels of inflammatory factors TNF-α,MCP-1 and IL-1β in brain tissue were detected by ELISA.Expression levels of autophagy related proteins beclin-1,LC3Ⅱ/Ⅰ,autophagy and Nrf2 signaling pathway node p62,and Nrf2 signaling pathway proteins such as Nrf2,Keap-1,HO-1,as well as iNOS were tested by Western blot.The expressions of p62 and Iba-1 were detected by immunofluorescence.Results In vitro1.To screen the experimental concentration of HIV-1 gp120V3 loop-induced CHME-5inflammation and determine the trend of inflammatory factors.CCK8 assay showed no significant difference between the 4 mg/mL random peptide had no significant effect on the survival rate of CHME-5 microglial cells(P>0.05 vs control group).when the concentration of HIV-1 gp120V3 loop reaches 1 mg/mL or higher,the survival rate decreased(P<0.05 vs Random peptide group),therefore,the concentration of HIV-1 gp120V3 loop selected in this study was 1 mg/L,and a random peptide of 1 mg/L was used as the control.At 6h,12 h,24h,36 h and 48 h,the expression levels of inflammatory factors in each group were detected by ELISA at different time points.No significant increase in TNF-α,MCP-1 and IL-1β was observed in the inflammatory factors in the HIV-1 gp120V3 loop treatment group at 6 hours(P>0.05 vs Random peptide group).At time point 24 h,the levels of TNF-α,MCP-1 and IL-1β in HIV-1 gp120V3 loop treatment group were significantly increased(P<0.001 vs Random peptide group).2.To observe the role of autophagy in CHME-5 microglia cell inflammation induced by HIV-1gp120V3 loop.Combined with the detection of adenovirus RFP-GFP-LC3 assay and Western bolt results showed that compared with the random peptide group,at 6h and 12 h,the HIV-1gp120V3 loop treatment group showed enhanced autophagy flux(P<0.05,P<0.001 vs Random peptide group;P<0.05,P<0.01 red puncta vs yellow puncta),while at 24 h,36 h and48 h,the autophagosome increased and suppression at autophagy late stage(P<0.001 vs Random peptide group;P<0.001,P<0.01 red puncta vs yellow puncta).Combined with the changes in the levels of inflammatory cytokines and autophagy at various time points,the working concentration of HIV-1gp120V3 loop was selected in this study as 1μg/mL,and the early time point of HIV-1gp120V3 loop induced CHME-5 microglial cell inflammation was 6h,and the late time point was 24 h.3.The role of autophagy in HIV-1gp120v3 loop-induced microglia inflammation:1)In the early stage of HIV-1gp120V3 loop-induced CHME-5 microglia cell inflammation.Western blot assay indicate: compare with control group,Beclin-1 and LC3Ⅱ/Ⅰ were upregulated in the model group(P<0.05 vs Random peptide group),and p62 protein level was down-regulated(P<0.01 vs Random peptide group).Compared with the model group,Beclin-1 and LC3Ⅱ/Ⅰ were down-regulated in HIV-1gp120V3 loop + 3-MA group(P<0.05,P<0.01 vs Random peptide group),and p62 protein level was up-regulated(P<0.01 vs HIV-1gp120V3 loop group).Autophagy flux assay found: Compared with the control group,there were significantly more red puncta in the model group(P<0.05 vs Random peptide group),and more red puncta than yellow puncta in the model group(P<0.05 red puncta vs yellow puncta).Compared with the model group,The red puncta of HIV-1gp120V3 loop + 3-MA was significantly reduced(P<0.05 vs HIV-1gp120V3 loop group),and the red puncta was more than the yellow puncta(P<0.05 red puncta vs yellow puncta).Compared with the control group,there was no significant change in inflammatory factors in the model group.Compared with the model group,inflammatory factors in the HIV-1gp120V3 loop + 3-MAgroup were significantly up-regulated(P<0.05,P<0.01 vs HIV-1gp120V3 loop group).In summary,compared with the control group,autophagy flux was enhanced in the model group,p62 was decreased,and no significant changes in inflammatory factors were observed in the model group.Compared with the model group,in the HIV-1gp120V3 loop + 3-MA group,autophagy flux was decreased,p62 was up-regulated,and cytokine release was significantly up-regulated.2)At the late stage of CHME-5 microglia inflammation induced by HIV-1gp120V3 loop,Western bolt found that Beclin-1,LC3Ⅱ/Ⅰ and p62 protein levels were up-regulated in the model group(P<0.05 vs.Random peptide group)and up-regulated in the model group(P<0.05 vs.HIV-1gp120V3 loop +RAPA group),compared with the model group,Beclin-1,LC3Ⅱ/Ⅰ were up-regulated in the HIV-1gp120V3 loop +RAPA group(P<0.05 vs.HIV-1gp120V3 loop group)and down-regulated in p62 level(P<0.05 vs.HIV-1gp120V3 loop group);Autophagy flux assay found: Compared with the control group,there were significantly more yellow puncta in the model group(P<0.001 vs Random peptide group),and more yellow puncta than red puncta(P<0.05 red puncta vs yellow puncta),compared with the model group,there were significantly more red puncta of HIV-1gp120V3 loop +RAPA(P<0.01 vs HIV-1gp120V3 loop group),and more red puncta than yellow puncta(P<0.05 red puncta vs yellow puncta).Compared with the control group,the inflammatory factors in the model group were significantly up-regulated(P<0.01,P<0.001 vs.Random peptide group),compared with the model group the inflammatory factors in the HIV-1gp120V3 loop +RAPA group were significantly up-regulated(P<0.05,P<0.01 vs HIV-1gp120V3 loop group).To sum up: compared with the control group,the model group had more autophagosomes and the autophagy flow was blocked at late stage.p62 was upregulated and inflammatory factors were significantly up-regulated.Compared with the model group,in the HIV-1gp120V3 loop +RAPA group,autophagy flux was enhanced,p62 was down-regulated,and cytokine release was significantly down-regulated.4.To observe the role of p62 differential expression in HIV-1 gp120V3 loop-induced CHME-5inflammation: At early stage,knockdown of p62 had no effect on HIV-1 gp120V3 loopinduced CHME-5 microglia inflammation(P>0.05 vs Random peptide group),while at late stage,knockdown of p62 can significantly attenuate HIV-1 gp120V3 loop-induced CHME-5microglia inflammation(P<0.05,P<0.01 vs HIV-1gp120V3 loop group).5.To observe the role of Nrf2,a downstream factor of p62,in the early and late stage of HIV-1gp120V3 loop-induced CHME-5 microglia inflammation:1)Western blot assay showed that Nrf2 level was significantly up-regulated compared with the random peptide control group at each time point(P<0.001 vs Random peptide group).2)ELISA assay showed that knockdown of Nrf2 at early stage could significantly promote the release of TNF-α,MCP-1 and IL-1β(P<0.05,P<0.01,P<0.001 vs HIV-1gp120V3 loop group),while at late stage,knockdown of Nrf2 can significantly attenuate HIV-1 gp120V3loop-induced CHME-5 microglia inflammation(P<0.05 vs HIV-1gp120V3 loop group).Western blot assay showed that compared with the control group,Keap-1 and p62 levels increased in the model group(P<0.05,P<0.001 vs.Random peptide group),Compared with the model group,Keap-1 and p62 levels decreased significantly in the HIV-1 gp120V3 loop +si Nrf2 group(P<0.05,P<0.01 vs.Random peptide group).It is suggested that Keap-1 and p62 are also Nrf2 target proteins.Knocking down Nrf2 can down-regulate the expressions of Keap-1 and p62 and inhibit the positive feedback loop of p62-Nrf2.3)To observe the activation way of Nrf2: Combined with relevant detection,it was found that early 6h: Keap-1 level was down-regulated(P<0.05 vs Random peptide group),p62 had no interaction with Keap-1,and knockdown of p62 had no significant effect on Nrf2 signal activation;in the late stage,Keap-1 level was up-regulated(P<0.001 vs Random peptide group),p62 co-precipitated with Keap-1,and the knockdown of p62 significantly weakened the activation of Nrf2 signal(P<0.05,P<0.01 vs HIV-1gp120V3 loop group).6.The mechanism of Nrf2-HO-1-iNOS in the early and late stagy of CHME-5 microglia cell inflammation mediated by HIV-1 gp120V3 loop.1)Western bolt analysis of HO-1(the target protein of Nrf2),and iNOS protein level in the early and late stage.The results showed that,compared with the control group,the levels of HO-1in 6 h and 24 h model group were significantly up-regulated(P<0.05 vs Random peptide group),while the levels of iNOS in HIV-1 gp120V3 loop 6h group were not significantly changed(P>0.05 vs Random peptide group),and the levels of iNOS in HIV-1 gp120V3 loop24 h group were significantly up-regulated(P<0.01 vs Random peptide group).2)At the early stage,compared with the control group,there was no significant change in inflammatory cytokines in the model group(P>0.05 vs Random peptide group),and Western bolt showed significant up-regulation of HO-1(P<0.05 vs Random peptide group),no significant change in iNOS,and the immunofluorescence results were consistent with Western bolt.Compared with the model group,after HO-1 knockdown,inflammatory factors were significantly up-regulated(P<0.05,P<0.01 vs HIV-1gp120V3 loop group),Western bolt shows no significant effect on iNOS protein level(P>0.05 vs Random peptide group),immunofluorescence results were consistent with Western bolt.At the late stage,compared with the control group,the inflammatory factors in the model group were significantly upregulated(P<0.001 vs Random peptide group),and Western bolt showed significant upregulation of HO-1 and iNOS(P<0.05,P<0.01 vs Random peptide group),i immunofluorescence revealed that the levels of HO-1 and iNOS were up-regulated and colocalized.Compared with the model group,after HO-1 knockdown,the inflammatory factors were significantly down-regulated(P<0.05 vs HIV-1gp120V3 loop group),Western bolt showed the iNOS level was significantly decreased(P<0.05 vs HIV-1gp120V3 loop group),immunofluorescence revealed that the levels of HO-1 and iNOS were down-regulated.It is suggested that late HO-1 may promote the up-regulation of iNOS and promote inflammation by interacting with iNOS.3)Western bolt revealed that at the early stage,compared with the control group,Nrf2 and HO-1were significantly up-regulated and Keap-1 was down-regulated in the model group(P<0.01 vs Random peptide group),with no significant changes in iNOS,compared with the model group,Nrf2 and HO-1 were significantly down-regulated and Keap-1 and iNOS were upregulated in HIV-1 gp120V3 loop +3MA group(P<0.05 vs HIV-1gp120V3 loop group).It suggested that early inhibition of autophagy weakened the canonical activation of Nrf2 and up-regulated the level of iNOS.At the late stage,compared with the control group,the levels of Nrf2,Keap-1,HO-1 and iNOS in the model group were significantly up-regulated(P<0.05 vs.Random peptide group).Compared with the model group,the levels of Nrf2,Keap-1,HO-1 and iNOS in the HIV-1 gp120V3 loop +RAPA group were significantly down-regulated(P<0.05 vs.HIV-1gp120V3 loop group),suggesting the activation of autophagy.The noncanonical activation of Nrf2 was weakened and the iNOS protein level was down-regulated.In vivo1.Compared with the blank control group,there was no significant change in the latency of the sham surgery group.Compared with the sham group,the latency of the Random peptide group was not significantly changed.Compared with Random peptide group,the latency of HIV-1gp120V3 loop 100 ng group was significantly prolonged on the 5th and 6th days(P<0.05,P<0.01 vs Random peptide group).ELISA results showed that all inflammatory factors in the HIV-1gp120V3 loop 100 ng group were significantly up-regulated(P<0.01,P<0.001 vs Random peptide group).Immunofluorescence assay showed that the expression of Iba-1 in HIV-1gp120V3 loop 100 ng group was up-regulated.The above results show that HIV-1 gp120V3 loop 100ng/mouse/day,three consecutive days of intracerebroventricular injection can cause learning and memory dysfunction in mice,and microglia cells activation and releasing inflammatory cytokines such as TNF-α,MCP-1 and IL-1β.2.The level of Nrf2 protein in brain tissue of mice detected by Western blot showed that intraperitoneal injection of Brusatol(2mg/kg)for 2 h-48 h significantly reduced the expression of Nrf2 protein in brain tissue of mice(P<0.001 vs Random peptide group).3.After the use of Nrf2 blocker Brusatol,the relevant experimental results were as follows:1)Behavioral experiment(1)Water maze experiment results show that: compared with the control group,the latency on the5 th and 6th days of the model group was significantly prolonged(P<0.01 vs Random peptide group),the time in target quadrant and the number of platform crosses were reduced(P<0.05,P<0.01 vs Random peptide group).In the HIV-1 gp120V3 loop +Brusatol group,the latency at 5th and 6th days was significantly shortened(P<0.05,P<0.01 vs HIV-1gp120V3 loop group),and the time in target quadrant and the number of platform crosses were increased(P<0.05,P<0.01 vs HIV-1gp120V3 loop group).(2)Step-Through Passive avoidance test show that: Compared with the control group,the latency of the model group was shortened(P<0.01 vs.Random peptide group)and the number of errors was increased(P<0.001 vs.Random peptide group),compared with the model group,the latency of gp120V3 loop +Brusatol group was prolonged(P<0.05 vs HIV-1gp120V3 loop group),the number of errors decreased(P<0.05,P<0.01 vs HIV-1gp120V3 loop group).These results suggest that Brusatol can reduce the learning and memory impairment induced by HIV-1 gp120V3 loop in mice.2)ELISA detection of inflammatory cytokines and immunofluorescence detection of microglia marker Iba-1 showed that,compared with the control group,the levels of inflammatory cytokines in the model group were significantly up-regulated(P<0.05,P<0.001 vs.Random peptide group),and the expression of Iba-1 was up-regulated.Compared with the model group,the levels of all inflammatory factors in the gp120V3 loop +Brusatol group were down-regulated(P<0.05,P<0.01 vs HIV-1gp120V3 loop group),and the expression of Iba-1was decreased,suggesting that Brusatol could significantly reduce the inflammatory factors and Iba-1 expression induced by HIV-1 gp120V3 loop in mice brain tissue.3)Western blot and immunofluorescence showed that compared with the control group,Beclin-1,LC3Ⅱ/Ⅰ,p62,Nrf2,Keap-1,HO-1,iNOS levels were up-regulated in the model group(P<0.05,P<0.01 vs Random peptide group),Immunofluorescence showed that p62 and microglial marker Iba-1 and were up-regulated and co-localized.Compared with the model group,HIV-1gp120V3 loop +Brusatol group showed significantly decreased expressions of Iba-1/ p62 tested by immunofluorescence,Western blot showed that Beclin-1 and LC3Ⅱ/Ⅰ was significantly up-regulated,autophagy and Nrf2 signaling pathway node p62 was downregulated,Nrf2,Keap-1,HO-1,iNOS was down-regulated(P<0.05,P<0.01 vs HIV-1gp120V3 loop group).These results suggest that Brusatol can improve autophagy dysfunction and inhibit the activation of p62-dependent Nrf2 signaling pathway induced by HIV-1gp120V3 loop in mice brain tissue.Conclusion:1.In the inflammatory response induced by HIV-1gp120V3 loop in CHME-5 microglia,the release of inflammatory factor TNF-α,MCP-1 and IL-1 peaked from 12 h to 24 h,and then decreased.2.In the early stage of inflammatory response induced by HIV-1gp120V3 loop in CHME-5microglia,autophagy was enhanced and the autophagy function was intact,and p62 was down-regulated to inhibit inflammation.In th late stage,autophagy dysfunction,p62 accumulation promotes inflammation.3.In the early stage of inflammatory response induced by HIV-1gp120V3 loop in CHME-5microglia: Nrf2 canonical activation,HO-1 up-regulated,the iNOS level remains unchanged,inhibit inflammation;in late stage,p62-dependent Nrf2 is non-canonical activated,and the target protein HO-1 is up-regulated,which promotes the up-regulation of iNOS and thus promote inflammation by interacting with iNOS.Knockdown of Nrf2 can down-regulate p62,inhibit p62-Nrf2 positive feedback loop,and inhibit inflammation.4.Intracerebroventricular injection injection of HIV-1gp120V3 loop 100ng/mouse/day for three consecutive days can cause cognitive dysfunction in mice,leading to up-regulation of inflammatory cytokines in brain tissue and dysfunction of autophagy.Brusatol can inhibit the activation of p62-dependent Nrf2 signaling pathway,improve microglial autophagy dysfunction,down-regulate the expression of iNOS,reduce the level of inflammatory cytokines in the brain tissue of mice,and alleviate the cognitive impairment caused by HIV-1gp120V3 loop in mice.In summary,HIV-1gp120V3 loop induces inflammatory responses in CHME-5 microglia,at early stage: p62 is down-regulated and inflammatory factors are not significantly changed,the mechanism of which is the classical activation of Nrf2.At late stage,p62 upregulation promotes inflammation and its mechanism is the non-canonical activation of p62-dependent Nrf2,and there is a positive feedback loop of p62-Nrf2,resulting in cognitive dysfunction in mice,which provides experimental basis for the prevention and treatment of HAND.