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

The role of Rho / ROCK signaling pathway in HIV-1 Tat induced blood-brain barrier dysfunction and Amyloid-beta deposition

目的探讨Rho/ROCK信号传导通路在人类免疫缺陷病毒-1型反式转录激活因子(HIV-1 transactivator of transcription,HIV-1 Tat)诱导血脑屏障破坏及对β淀粉样蛋白(Amyloid-beta,Aβ)沉积中的作用。方法将培养的人脑微血管内皮细胞(human cerebral microvascular endothelial cells,h CMEC/D3)予以不同浓度的Rho/ROCK信号传导通路抑制剂盐酸法舒地尔(Hydroxyfasudil,HF)刺激细胞,并设立对照组,观察其对细胞活力的影响,分别予以HIV-1 Tat、HF刺激细胞,以蛋白免疫印迹法和实时反转录聚合酶链式反应(real-time reverse transcription polymerase chain reaction,RT-PCR)检测h CMEC/D3中紧密连接蛋白Occludin、晚期糖基化终产物受体(receptor for advanced glycation end products,RAGE,转移血液Aβ到脑组织)的蛋白和mRNA表达的变化。结果 HF在30μmol/L(0.17±0.02)以下时对h CMEC/D3活力无明显影响(P>0.05)。HIV-1 Tat能抑制h CMEC/D3的Occludin蛋白(0.71±0.03、P<0.001)与mRNA(0.41±0.05、P<0.05)表达,同时上调RAGE蛋白(0.83±0.01、P<0.001)和mRNA(1.93±0.66、P<0.05)表达。HF预处理细胞2 h后与HIV-1 Tat共培养可以显著增加Occludin蛋白与mRNA的表达(0.93±0.03、P<0.001;1.04±0.45、P<0.05),同时可以明显减少RAGE蛋白与mRNA的表达(0.54±0.01、P<0.001,1.08±0.43、P<0.05)。结论 HIV-1 Tat可使紧密连接蛋白Occludin蛋白和mRNA表达下调导致血脑屏障破坏,诱导Aβ转运受体RAGE的蛋白和mRNA过度表达,从而可能导致Aβ在脑内沉积增加;阻断Rho/ROCK信号传导通路可抑制HIV-1 Tat诱导血脑屏障破坏作用,阻止RAGE蛋白和mRNA的过度表达。

Objective To evaluate the role of Rho/ROCK signaling pathway in HIV-1 Tat-induced Occludin dysfunction and Amyloid-beta deposition in human cerebral microvascular endothelial cells （hCMEC/D3）. Methods hC- MEC/D3 viability was tested by MTT assay ,with the exposure of different concentrations of Rho/ROCK inhibitor hydroxyfasudil （HF）. Respectively Western blot and real-time reverse transcription polymerase chain reaction （qRT-PCR） assay Occludin and advanced glycation end product receptor （RAGE,which regulates Aβ transfer from the blood stream into the central nervous system） protein and mRNA expression in hCMEC/D3. Results Hydroxyfasudil at 50 μmol/L （0.17 ± 0.02） or less had no significant effect on the hCMEC/D3 cell viability as determined by the MTT assay （ P 〉 0.05 ）. Treatment with HIV-1 Tat decreased protein expression of Occludin （0.71 ±0.03,P 〈0. 001 ） in hCMEC/D3 as well as mRNA （0.41±0.05,P 〈0.05） levels, and also activated the expression of RAGE （0.83 ± 0.01,P 〈 0. 001 in protein levels, 1.93 ±0.66,P 〈 0.05 in mRNA levels）. While inhibition of Rho by HF effectively protected against HIV-1 Tat-induced downregulation of Occludin protein （0.93 ± 0.03,P 〈 0. 001 ） and mRNA （ 1.04 ± 0.45,P 〈 0. 05 ） expression. At the same time, the effect on RAGE protein （ 0.54 ± 0.01 ,P 〈 0. 001 ） and mRNA （ 1.08 ± 0.43 ,P 〈 0.05 ） were attenuated by pretreatment with hydroxyfasudil. Conclusion HIV-1 Tat induces dysregulation of Occludin and Aβ transport RAGE receptor both in protein and mRNA levels. These effects were abolished by the addition of HF. These results show that Rho/ ROCK signaling pathway plays an important role in HIV-1 Tat-induced Occludin dysfunction and Amyloid-beta deposition in cerebral microvascular endothelial cells.