Role Of Rho/ROCK Signaling Pathway In HIV-1 Tat-induced Blood-brain Barrier Dysfunction And Aβ Clearance Deficiency In Mice

Objective: HIV-1 transactivator of transcription(HIV-1 Tat)induced bloodbrain barrier(BBB)dysfunction and amyloid-beta(Aβ)deposition in brain,which contributed to the development and progression of HIV-1 associated neurocognitive disorder(HAND).In this study,it would be explored that the roles of Rho / ROCK signaling pathway on HIV-1 Tat-induced the disruption of zonula occludes-1(ZO-1),occludin,claudin 5,neprilysin(NEP),low density lipoprotein receptor related protein 1(LRP-1)and receptor for advanced glycation end products(RAGE).Method:(1)C57BL/6 mice(n=36,8 weeks old,male)were weighed and randomly divided into 4 groups(n=9 each): control group,HIV-1 Tat 25μg/kg group,HIV-1 Tat 50μg/kg group,HIV-1 Tat 100μg/kg group.The control group was injected with 100μl sterile saline via tail vein,while the treatment group was injected with 25μg/kg,50μg/kg and 100μg/kg HIV-1 Tat respectively.22 hours after treatment,six mice in each group were injected with Evans blue(EB)solution(2%,5ml/kg)intraperitoneally and allowed it to circulate for 2 hours.After anesthetized,mice were transcardially perfused with frozen sterile saline.The amount of EB leakage was measured by tissue homogenate method and fluorescence microscopy of frozen sections.24 hours after treatment,three mice in each group were decapitated and their brains were harvested,and then the brain microvessels of mice were isolated and the expression of ZO-1 protein was detected by Western blotting(WB).(2)C57BL/6 mice(n=48,8 weeks old,male)were randomly divided into four groups(n=12 each): control group,HIV-1 Tat group,hydroxyfasudil(HF)group,HIV-1 Tat + HF group.In the control group,after injected intraperitoneally with 100μl sterile saline 1 hour,the mice were injected with 100μl sterile saline via tail vein.In the HIV-1 Tat group,after injected intraperitoneally with 100μl sterile saline 1 hour,the mice were injected with 100μl of HIV-1 Tat 100μg/kg and sterile saline through tail vein.In the HF group,after injected intraperitoneally with 100μl of 10mg/kg HF and sterile saline1 hour,the mice were injected with 100μl sterile saline via tail vein.In the HIV-1Tat + HF group,after injected intraperitoneally with 100μl of 10mg/kg HF and sterile saline 1 hour,the mice were injected with 100μl of HIV-1 Tat 100μg/kg and sterile saline through tail vein.The above treatment was daily for seven consecutive days.All mice were sacrificed 24 h after the last treatment and their brains were harvested.The amount of mouse brain EB leakage was evaluated by the blue staining color of mouse brain surface,tissue homogenate method and fluorescence microscopy of frozen sections.The protein and m RNA levels of ZO1,occludin,claudin 5,NEP,LRP-1 and RAGE in the mouse brain microvessels were detected by WB and quantitative real-time polymerase chain reaction(q RT-PCR),respectively.Results:(1)With the dosage of HIV-1 Tat growing,the mouse brain in blue,the amount of EB leakage,the mouse brain fluorescence intensity were gradually enhanced;however,the protein level of mouse brain microvessels ZO-1 was gradually reduced.HIV-1 Tat at 100μg/kg significantly increased EB leakage and EB fluorescence intensity compared to the control group(***P < 0.001)and reduced the expression of mouse brain microvessels ZO-1 compared to the control group(**P < 0.01).HIV-1 Tat increased the BBB permeability in a dosagedependent manner.(2)Compared with the control group,the mouse brain was stained in blue;the leakage amount of EB,EB fluorescence intensity were significantly increased in HIV-1 Tat group(***P < 0.001,**P < 0.01),meanwhile the protein and m RNA levels of ZO-1,occludin,claudin 5,NEP,LRP-1 were significantly downregulate and the RAGE protein and m RNA levels were remarkable upregulated in mouse brain microvessels(* P < 0.05,** P < 0.01).Compared with HIV-1 Tat group,the blue staining of the mouse brain surface became lighter;the EB leakage amount,EB fluorescence intensity were decreased significantly in the HIV-1 Tat + HF group(### P < 0.001,##P < 0.01),the protein and m RNA levels of ZO-1,occludin,claudin 5,NEP,LRP-1 in mouse brain microvessels were upregulate and the RAGE protein and m RNA levels in mouse brain microvessels were downregulated in the HIV-1 Tat + HF group(#P < 0.05,##P < 0.01).Conclusion: the permeability of BBB is decreased by HIV-1 Tat via reducing the expression of ZO-1,occludin and claudin 5 at the level of transcription and translation.The clearance function of Aβ in mouse brain microvessels is inhibited by HIV-1 Tat through downregulation of NEP,LEP1 meanwhile upregulation of RAGE.HIV-1 Tat-induced dysfunction of tight junction and Aβ clearance in mouse brain microvessels is attenuated by inhibition of Rho/ROCK signaling pathway.