Effect Of HIV-1 Tat From HAD And Non-HAD Patients On Oxidative Stress And Autophagy Of U87 And SH-SY5Y Cells

OBJECTIVEHIV-1 associated dementia(HAD)is one of the neurological complications caused by the invasion of HIV-1 into the central nervous system.It usually appears in the late stage of AIDS.It mainly manifests as cognitive and behavioral dysfunction and short-term memory loss.Highly active antiretroviral therapy(HAART)can inhibit virus replication and increase the survival time of HIV-1 infected persons,but the drug cannot penetrate the blood brain barrier(BBB)to protect the nervous system.Therefore,the treatment ultimately causes the prevalence of HAD to continue to increase year by year.In recent years,studies have shown that nerve cell damage in the brain of HAD patients is closely related to oxidative stress and autophagy.Malondialdehyde(MDA),glutathione peroxidase(GPX),and total antioxidant capacity(T-AOC)are commonly used indicators for monitoring cell oxidative stress.p62 and LC3B are key proteins involved in autophagy and are often used to monitor autophagy in cells.HIV-1 Tat protein is an important regulatory protein of HIV-1.In HIV-1 infected cells,Tat protein can interact with transcription factors to transactivate viral genome transcription and promote viral replication.Tat can be secreted from infected macrophages and microglia and enter nerve cells through receptor-mediated endocytosis,and participate in cell inflammation,oxidative stress and autophagy,and aggravate the occurrence of neurological complications.And the amino acid site variation of Tat protein plays an important role in neuropathogenicity.To investigate the amino acid site variation of HIV-1 Tat protein in central and peripheral sites and its role in the pathogenesis of HAD.To study the amino acid site variation of HIV-1 Tat protein in the central nervous system and peripheral parts of a HAD patient and a non-HAD patient.And to study the effect of HIV-1 Tat protein from different parts on the cell activity,oxidative stress and autophagy of human neuroastroglioma cell lines(U87)and human neuroblastoma cell lines(SH-SY5Y).So as to provide a scientific basis for the neuropathic mechanism of HIV-1.METHODS:1.Amino acid sites analysis of HIV-1 Tat proteinSequences of HIV-1 tat gene from peripheral spleen(SPL)and central nervous tissue basal ganglia(BG)with an HIV-associated dementia(HAD)patient(H)and a non-HAD patient(N)were sent to the firm.Amino acid sequence analysis of HIV-1 Tat protein.Amino acid sequence was compared using BLAST,an online tool of NCBI website,and the variation of key amino acid sites was analyzed by MEGA6.2.Expression and identification of HIV-1 Tat in cellsCulture SH-SY5Y and U87 cells routinely,and the recombinant plasmid pEGFP-N1-tat from different parts was transfected into cells with TurboFect Transfection Reagnt and set up a vector and cell control group(only transfection reagent).The expression of green fluorescent protein(GFP)was observed at 12h,24h,36h and 48h after transfection.Cells were harvested at the peak of fluorescence.The expression of HIV-1 Tat protein in SH-SY5Y and U87 cells was detected by Western Blot.3.The effect of HIV-1 Tat protein on cell activityCells were seeded in 96-well plates and transfected with recombinant plasmid pEGFP-N1-tat.The cell activity was determined by CCK-8 method.4.Effects of HIV-1 Tat protein on cellular oxidative stressFirst,verify whether Tat protein induces oxidative stress in cells:N-Acetyl-L-cysteine(NAC)(final concentration of 2mM)was added to SH-SY5Y and U87 cells to intervene for 1h and then transfected with Tat protein.Cell viability was detected after 48h.The SH-SY5Y,U87 cells were seeded in 6-well cell culture plate.After transfection of the recombinant plasmid pEGFP-N1-tat 48h,the cells were collected.The content of MDA and T-AOC in the cells was detected according to the instructions of the oxidative stress kit,and the level of GPX was detected by Western blot.5.The effect of HIV-1 Tat protein on autophagyThe SH-SY5Y,U87 cells were seeded in 6-well cell culture plate.After transfection of the recombinant plasmid pEGFP-N1-tat 48h,the cells were collected.Western Blot was used to detect the expression of LC3-II and p62.The gray value of the target protein was quantitatively analyzed by Image J software.6.Statistical analysisData was processed statistically with SPSS25 software.Each experiment provided with three parallel holes.One-way analysis of variance was used for comparison between groups.RESULTS1.Amino acid sites analysis of HIV-1 Tat proteinAfter BLAST comparison,compared with the standard sequence of Tat proteins from four parts of HAD and non-HAD patients,HAD and non-HAD patients have mutations in the center and periphery,and different parts of the same patient have different mutations.Compared with standard sequence,H59P,Q63T,T64A,H65D,A67G,L69I and K7LN were mutated in all four sequences;N-SPL,H-BG,and H-SPL three sequence mutation sites are I39T,T40K,A42G.Some sequences have unique mutation sites,such as I39M and N61D sites of N-BG;R53G site of N-SPL;K19R,Q54R site of H-BG;T23N site of H-SPL.2.Expression and identification of HIV-1 Tat in SH-SY5Y and U87 cellsAfter the recombinant plasmid pEGFP-N1-tat was transfected into SH-SY5Y and U87 cells,it was observed that the GFP fluorescence was the strongest at 48h.Western blot showed there were specific bands at about 14 kD.However,the plasmid and cell control group did not express.The results showed that the Tat protein was successfully expressed in cells.Gray value analysis showed that there was no significant difference in the expression of Tat protein from different sources in cells(P>0.05).3.The effect of HIV-1 Tat protein on activity in SH-SY5Y and U87 cellsThe results show that 48h after transfection of recombinant plasmid pEGFP-N1-tat.There was no significant difference between the plasmid control group and cell control(P>0.05);The cell viability of the four experimental groups N-BG,N-SPL,H-BG,and H-SPL were significantly lower than that of the plasmid control group and cell control(P<0.05).The results showed that Tat protein could decrease the cell activity.4.Effects of HIV-1 Tat protein on oxidative stress in SH-SY5Y and U87 cellsCell activity with the antioxidant NAC added in advance is normal.Cell damage caused by Tat protein can be reversed by the antioxidant NAC.Oxidative stress plays an important role in cell damage.(1)In SH-SY5Y cells:①GPX protein content:there was no significant differencebetween the plasmid control group and cell control group.Compared with the control group,the expression of GPX protein were decreased in the four experimental groups N-BG,N-SPL,H-BGS and H-SPL(P<0.05)②MDA level:There was no significant difference in MDA level between the plasmid control group and cell control group.Compared with the control group,the level of MDA were increased in the four experimental groups N-BG,N-SPL,H-BG,and H-SPL(P<0.05).The level of MDA in BG group of both HAD and non-HAD patients was higher than that of SPL group(P<0.05).The level of MDA in the SPL group of HAD patients was higher than that of non-HAD patients(P<0.05).③T-AOC level:There was no significant difference in T-AOC level between the plasmid control group and cell control group(P>0.05).Compared with the control group,the level of T-AOC were decreased in the four experimental groups N-BG,N-SPL,H-BG,and H-SPL(P<0.05).The level of T-AOC in the BG group of HAD patients was lower than that of non-HAD patients(P<0.05).(2)In U87 cells:①GPX protein content:there was no significant difference in GPX protein content between the plasmid control group and cell control group.Compared with the control group,the expression of GPX protein were decreased in the four experimental groups N-BG,N-SPL,H-BG,and H-SPL.The expression of GPX protein in BG group of both HAD and non-HAD patients was lower than that of SPL group(P<0.05).②MDA level:There was no significant difference in MDA level between the plasmid control group and cell control group(P>0.05).Compared with the control group,the level of MDA were increased in the four experimental groups N-BG,N-SPL,H-BG,and H-SPL(P<0.05).③T-AOC level:There was no significant difference in T-AOC level between the plasmid control group and cell control group(P>0.05).Compared with the control group,the level of T-AOC were decreased in the four experimental groups N-BG,N-SPL,H-BG,and H-SPL(P<0.05).The level of T-AOC in the H-BG group was lower than that in the N-BG,N-SPL,and H-SPL groups(P<0.05).5.The effect of HIV-1 Tat protein on autophagy in SH-SY5Y and U87 cells(1)In SH-SY5Y cells:①there was no significant difference in LC3-II protein content between the plasmid control group and cell control group(P>0.05).Compared with the control group,the expression of LC3-II protein were increased in the four experimental groups(P<0.05),but there was no significant difference between the groups(P>0.05);And after CQ treatment,compared with the control group,the expression of LC3-II protein were increased in the four experimental groups(P<0.05),but there was no significant difference between the groups(P>0.05).②There was no significant difference in the expression of p62 protein between the four experimental groups and the plasmid control group and cell control group(P>0.05).(2)In U87 cells:①there was no significant difference in LC3-II protein content between the plasmid control group and cell control group(P>0.05).Compared with the control group,the expression of LC3-II protein were increased in the four experimental groups,the expression of LC3-II protein in the H-BG group was lower than that in the N-BG,N-SPL,and H-SPL groups(P<0.05).And after CQ treatment,compared with the control group,the expression of LC3-II protein were increased in the four experimental groups(P<0.05),and the expression of LC3-II protein in the H-BG group was lower than that in the N-BG,N-SPL,and H-SPL groups(P<0.05).②There was no significant difference in the expression of p62 protein between the four experimental groups and the plasmid control group and cell control group(P>0.05).CONCLUSION1.There are differences in the key amino acid sites of Tat protein in peripheral and central nervous system between HAD and non-HAD patients,.2.HIV-1 Tat protein from the peripheral and central nervous system betweenHAD and non-HAD patients can reduce the activity of SH-SY5Y and U87 cells.And Tat protein from the basal ganglia of HAD patients showed stronger cytotoxicity.