Rho/ROCK信号传导通路在HIV-1 Tat蛋白诱导血脑屏障破坏与β淀粉样蛋白沉积中的作用

我们研究发现HIV-1 Tat诱导紧密连接（Tjs）破坏和促进Aβ沉积，但作用机制尚未清楚。本项目将阻断Rho/ROCK信号通路及Caveolin-1（Cav-1）后，蛋白印迹和免疫荧光等技术检测人脑血管内皮细胞Tat治疗后Tjs蛋白、Neprilysin（NEP）、脂蛋白受体相关蛋白（LRP）和晚期糖基化终产物受体（RAGE）的表达；血脑屏障（BBB）体外模型观察Rho/ROCK通路和Cav-1对Tat调节Aβ转运的影响；动物验证Rho/ROCK通路和Cav-1对Tat诱导的脑微血管Tjs蛋白、LRP、RAGE的表达、NEP活性、Aβ沉积以及神经认知功能的影响。检测HIV感染者BBB受损与脑脊液/血液NEP、LRP、RAGE、Aβ的水平及与神经认知功能的关系。本项目将首次阐明Rho/ROCK通路及Cav-1调节Tat诱导BBB破坏及Aβ沉积作用机制，将利于HIV相关性神经认知功能损害治疗

HIV-1 Tat is an important regulating protein for HIV-1 encoding. Our previous data showed that HIV-1 Tat induced tight junction（Tjs） disruption and enhanced Aβdeposit，however, the involving mechanism is unknown. By blocking the Rho/ROCK signaling pathway and inhibiting Cav-1 (Cav-1) with specific siRNA or shRNA or inhibitor，this proposal will explore the interrelation of tight junction protein (ZO-1/Occludin/Claudin-5) Cav-1, Neprilysin（NEP）、lipoprotein receptor-related protein (LRP), receptor for advanced glycation end products (RAGE) as well as proteins of Rho kinase signaling in endothelial cells （hCMEC/D3）with expose of HIV-1 Tat via Western Blotting, Immunoprecipitation and Immunofluorescent staining. The effect of HIV-1 Tat, Rho/ROCK signaling pathway and Cav-1 on Aβtransport through endothelium will be investigated with blood brain barrier (BBB) model in vitro. The role of Rho/ROCK signaling pathway and Cav-1 on HIV-1 Tat induced Tjs dysfunction, the expression of LRP/RAGE, NEP activity, Aβdeposit and neuroeognitive function will be studied in Cav-1 knockout mice and its control C57BL/6 mice. Moreover, the level of neuron specific enolase, albumin, NEP, LRP, RAGE, Aβin cerebrospinal fluid and serum as well as neuroeognitive examination will also be tested in patients with HIV-1 infection. This proposal will firstly provide data on the mechanism of Rho/ROCK signaling pathway and Cav-1 on HIV-1 Tat induced Tjs dysfunction, which benefits on the treatment of HIV-1 associated neuroeognitive disorder.