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

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.

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与HIV相关认知功能障碍密切相关。我们研究发现Tat诱导紧密连接（Tjs）破坏和促进Aβ沉积，但作用机制未清楚。本项目阻断Rho/ROCK信号通路和Caveolin-1后研究HIV-1 Tat对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）的表达变化。本项目发现：①Tat抑制hCMEC/D3的Occludin 和LRP表达，同时上调RAGE表达。ROCK通路抑制剂法舒地尔显著抑制Tat诱发的Occludin和LRP1的破坏，同时明显减少RAGE蛋白/mRNA 表达。②Tat可促进紧密连接蛋白ZO-1蛋白和mRNA下调，同时诱导NEP蛋白和mRNA下调，促进细胞内活性氧的生成，可能导致Aβ在脑内沉积增加。阻断Ras通路可抑制Tat诱导的ZO-1和NEP破坏。③抑制Caveolin-1可减弱HIV-Tat诱导的occludin、LRP-1的破坏。④PPARα激动剂非诺贝特和PPARγ激动剂罗格列酮抑制经颈内动脉注入的Tat诱导的脑微血管通透性增加，而PPARγ拮抗剂GW9662 加重通透性破坏。非诺贝特还通过ERK1/2 和 Akt信号通路抑制 Tat诱导的TJS破坏。在MMP-9基因敲除鼠中Tat诱导的脑微血管毒性、胶质细胞增生和神经元丢失明显较对照组减少。⑤罗格列酮可抑制hCMEC/D3及C57BL/6J小鼠脑微血管中Tat诱导的ICAM-1和VCAM-1蛋白/mRNA表达增加，而这种抑制作用可被GW9662和Akt抑制剂KP3721逆转。⑥脂多糖(LPS)可诱导hCMEC/D3中Occludin 和 ZO-1 蛋白破坏及p38MAPK和JNK 磷酸化水平增高，而抑制p38MAPK 和JNK 磷酸化以及MMP-9 的活性可以明显上调 LPS 诱导的 Occludin的表达。另外阻断p38MAPK 和JNK通路可显著降低MMP-2/MMP-9 的高表达。⑦雌二醇可抑制Tat介导 Claudin-1，ZO-1的破坏而对BBB起保护作用。本项目的完成首次阐明MAPKs、Rho/ROCK及MMPs调节Tat诱导BBB破坏的作用机制，进一步研究Rho/ROCK抑制剂、PPAR激动剂、E2及MMPs抑制剂的保护作用，将为HIV-1感染提供新的治疗策略。