Ras信号通路在HIV-1 Tat诱导ZO-1及脑啡肽酶破坏的作用

The role of Ras signaling pathway in HIV-1 Tat induced dysfunction of ZO-1 and Neprilysin

目的探讨Ras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(BBB)中紧密连接蛋白Zonula Occludens(ZO)-1及脑啡肽酶(Neprilysin,NEP)破坏的作用。方法以不同浓度Ras信号传导通路抑制剂法尼基硫代水杨酸(farnesylthiosalicylic acid,FTS)刺激培养好的人脑微血管内皮细胞(human cerebral microvascular endothelium cells,HBEC-5i),并设立对照组,观察其对细胞活力的影响。分别予以HIV-1 Tat、FTS刺激细胞,以蛋白免疫印迹法及实时反转录聚合酶链式反应(realtime reverse transcription polymerase chain reaction,RT-PCR)检测HIV-1 Tat诱导的紧密连接蛋白ZO-1及NEP(脑内降解β淀粉样蛋白Amyloid-beta,Aβ的限速酶)的蛋白和mRNA表达的变化。结果 FTS在20μmol/L(0.35±0.06)以下时对HBEC-5i活力无明显影响(P>0.05),HIV-1 Tat能抑制HBEC-5i的ZO-1、NEP蛋白(0.53±0.07,P<0.01;0.40±0.04,P<0.05)与mRNA(0.42±0.10,P<0.05;0.31±0.09,P<0.05)的表达。用FTS阻断Ras信号通路后可显著增加ZO-1、NEP蛋白(1.20±0.22,P<0.01;0.58±0.03,P<0.05)及mRNA(1.10±0.19,P<0.05;0.97±0.38,P<0.05)的表达。结论 HIV-1 Tat可促进紧密连接蛋白ZO-1蛋白和mRNA下调而导致血脑屏障破坏,同时诱导脑内脑啡肽酶NEP蛋白和mRNA下调,可能导致Aβ在脑内沉积增加。阻断Ras信号传导通路可抑制HIV-1 Tat诱导的ZO-1和NEP破坏,可能减少Aβ在脑内的沉积。

Objective To evaluate the role of Ras singnaling pathway in HIV-1 Tat-induced dysfunction of ZO-1 and neprilysin （ NEP, endogenous Ap-degrading enzyme） in human cerebral microvascular endothelium cells （ HBEC-5i）. Methods HBEC-5i viability was tested by MTT assay,with the exposure of different concentrations of Ras inhibitor farnesyl- thiosalicylic acid （FTS）. The levels of protein and mRNA of ZO-1 and NEP in HBEC-5i were evaluated with Western blot and Real-time reverse transcription polymerase chain reaction （qRT-PCR） assay respectively. Results Farnesylthiosalicylic acid at 20 jjimol/L （0.35 ± 0.06, P〉 0.05） or less had no significant effect on the HBEC-5i cell viability as determined by the MTT assay. Treatment with HIV-1 Tat decreased protein and mRNA levels of ZO-1 （0.53 ±0.07,P 〈0.01 in protein levels; 0.42 ±0. 10〈0. 05 in mRNA levels） and NEP （0.40 ± 0. 04〈0. 05 in protein levels ;0. 31 ± 0.07,P〈0.05 in mRNA levels）. While inhibition of Ras by FTS effectively protected against HIV-1 Tat-induced downregulation of ZO-1 （1. 20 ± 0. 22,P 〈0.01 in protein levels;1. 10 ±0.19,P 〈0. 05,in mRNA levels） and NEP（0. 58 ±0. 03,P 〈0. 05 in protein levels; 0. 97 ± 0. 38,P 〈 0. 05 in mRNA levels） expression. Conclusion These results show that HIV-1 Tat induces dysfunction of ZO-1 and neprilysin in both protein and mRNA levels and these effects can be attenuated by FTS. Ras signaling pathway plays a role in HIV-1 Tat-induced ZO-1 dysfunction and neprilysin in cerebral microvascular endothelial cells.