The Rolf Of Rho/ROCK Signaling Pathway In HIV-1 Tat Induced Occludin Dysfunction And Amyloid-beta Deposition

PARTⅠ The preliminary study of h CMEC/D3 cell culture and the impact of Hydroxyfasudil on the cell viabilityObjective: To explore the impact of Hydroxyfasudil(HF)on the human cerebral microvascular endothelial cells(h CMEC/D3)cell viability for subsequent researches.Method: h CMEC/D3 viability was determined using the3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)after treatment with different concentrations of Rho/ROCK signaling pathway inhibitor HF.The logarithmic growth phase of h CMEC/D3 treated with different concentrations(0,10,30,50,80,100 umo L/L)of HF.Blank control group was set up and optical density was measured.Results: cells were treated with HF for 24 h.HF at the concentration of30 umo L/L(P>0.05)has no significant effect on h CMEC/D3 cell viability assessed by MTT assay.Conclusion: MTT results show that the concentration of HF at the level30 umo L/L,has no significant effect on h CMEC/D3 cell viability.PARTⅡ The Role of Rho/ROCK signaling pathway in HIV-1 Tat-induced Occludin dysfunction and Amyloid-beta depositionObjective: The main purpose of this paper is to investigate the role of Rho/ROCK signaling pathway in HIV-1 Tat induced Occludin dysfunction and Amyloid-beta deposition.Method: The expressions of Occludin,receptor for advanced glycation end products(RAGE)and receptor for lipoprotein related protein1(LRP-1)in h CMEC/D3 by real-time reverse transcription polymerase chain reaction(RT-PCR),Western Blot(WB)and Immunofluorescence(IF).Results: Treatment with HIV-1 Tat protein for 24 h,the protein levels of Occludin and LRP-1 and mRNA levels in h CMEC/D3 decreased.HIV-1 Tat induced upregulation of protein and mRNA expression of RAGE.However,HF induced upregulation of protein and mRNA expression of Occludin and LRP-1,and attenuated HIV-1 Tat-induced upregulation of RAGE.Conclusions: HIV-1 Tat-induced downregulation of Occludin and LRP-1 and upregulation of RAGE.This may lead to an increase Aβ deposition in the central nervous system.Moreover,this effects were attenuated by the addition of HF.This study shows that Rho/ROCK signaling pathway plays a key role on the protection against HIV-1 Tat-induced dysfunction of Occludin and Amyloid beta deposition.