The Role Of Ras Signaling Pathway In HIV-1 Tat Induced Dysfunction Of ZO-1 And Neprilysin

Objective: To evaluate the role of Ras singnaling pathway in HIV-1Tat-induced dysfunction of tight junction protein Zonula Occludens-1(ZO-1)and neprilysin(NEP,endogenous A?-degrading enzyme)in the blood-brain barrier(BBB).Methods: Human cerebral microvascular endothelium cells(HBEC-5i)were exposed to different concentrations of HIV-1 Tat and Ras inhibitor farnesylthiosalicylic acid(FTS)for 24 h,the viability of cells was tested by MTT assay.HBEC-5i cells were treated with HIV-1 Tat or FTS respectively,or co-treated with HIV-1 Tat and FTS for 24 h prior to treating with FTS for 3h.The levels of protein or mRNA of ZO-1 and NEP in HBEC-5i were evaluated with Western blot or Real-time reverse transcription polymerase chain reaction(qRT-PCR)assay respectively.Also,HIV-1 Tat induced alteration ofimmunoreactivity of ZO-1 and NEP were detected by Immunofluorescence(IF),and intracellular levels of reactive oxygen species(ROS)were assayed by2?,7?-dichlorodihy-drofluorescein diacetate(DCFH-DA)fluorescence probe,respectivrly.Meanwhile,intracellular accumulations of exogenous A? were detected by a fluorescently labelled A?(A?(1-40)Hilyte).Results: The concentrations of HIV-1 Tat and Farnesylthiosalicylic acid under 1?g/mL and 20?mol/L respectively had no significant effect on the HBEC-5i cell viability(P > 0.05)as determined by the MTT assay.With treatment of HIV-1 Tat,the protein(P<0.01,P<0.05)and mRNA(P<0.01,P<0.01)levels of ZO-1 and NEP,the immunoreactivity of ZO-1 and NEP were decreased in HBEC-5i cells,and also intracellular ROS levels(P<0.001)and accumulation of exogenous A? were increased.However,pretreated with FTS for 3h effectively protected against HIV-1 Tat-induced downregulation of ZO-1and NEP expression in both protein(P<0.01,P<0.05)and mRNA(P<0.01,P< 0.01)levels,strengthened the immunoreactivity of ZO-1 and NEP,and decreased intracellular ROS(P<0.001)levels and accumulation of exogenous A?.Conclusion: These results show that HIV-1 Tat-induced downregulation of ZO-1 and neprilysin in both protein and mRNA levels,facilitating the production of intracellular ROS,may result in accumulation of A? in the brain.Inhibition of Ras signaling pathway can restrain HIV-1 Tat-induced dysfunctionof ZO-1 and neprilysin,decrease the production of ROS in cerebral microvascular endothelial cells,and inhibit accumulation of A? in the brain.Ras signaling pathway plays a key role on the protection against HIV-1 Tat-induced dysfunction of ZO-1,neprilysin and A? accumulations.