Rac1/MAPK/ERK 通路介导脂多糖LPS 诱导的人内皮细胞 单层通透性增高机制研究

目的:探讨LPS诱导的人内皮细胞单层通透性改变的分子机制。方法:应用逆转录病毒为载体,感染并筛选稳定表达持续活化型Rac1和主导抑制型Rac1的人HUVEC细胞,应用LPS刺激并观察细胞骨架蛋白F-actin和HUVEC单层通透性的改变。同时应用Western blot方法检测LPS刺激前后细胞中MAPK/ERK信号通路的改变及加入PD98059阻断ERK表达后,细胞内F-actin的改变情况。结果:与正常HUVEC相比较,LPS刺激后,感染活化型Rac1和主导抑制型Rac1的HUVEC中F-actin重构并形成大量应力纤维,细胞单层通透性显著增加。而抑制型Rac1感染后的HUVEC中F-actin无重构现象,同时细胞单层通透性无明显增加。LPS刺激前后,各组细胞中ERK1/2总蛋白均无明显改变。LPS刺激后,感染活化型Rac1的HUVEC中,p-ERK增加。经PD98059阻断后,细胞内p-ERK表达下降同时伴随F-actin解聚发生。结论:LPS诱导的内皮细胞通透性增加是经过Rac1-MAPK/ERK通路介导的。

LPS Regulates the Enhancement of HUVEC PermeabilityMediated by Rac1- MAPK/ERK Signal Pathway

Objective:To investigate the mechanism of LPS regulating the enhancement of endothelium permeability in vitro.Methods: The HUVECs in vitro were infected by the retrovirals mediated domain-negative N17Rac1 and activate Q61Rac1.The increase of endothelial permeability was induced by addition of LPS and the change of F-actin cytoskeleton was investigated by phallodin-Rhodamin staining.The expressions of pERK and total ERK were detected by western blot with and without addition of LPS.Subsequently,F-actin cytoskeleton of HUVEC was investigated by phallodin-Rhodamin staining when cells was incubated by PD98059 to block the activation of pERK.Results: Compared with that in the normal HUVECs,the formation of actin stress fibers was investigated associated with the enhancement of endothelial permeability in cells infected with Q61Rac1 with and without treatment of LPS.In contrast,there were no changes in cells infected with N17Rac1.Although there was change in the total ERK1/2 with or without LPS treatment in HUVECs,the expression of pERK significantly increased in cells treated by LPS by Western blotting.Meanwhile,the expression of pERK enhance in Q61Rac1-infected HUVECs with or without LPS stimulation.Reversely,the cells infected by N17Rac1 inhbited the activation of pERK induced by LPS.Moreover,when cells were treated with PD98059,a pERK inhibitor,LPS induced F-actin stress fibre disformated and the endothelial permeability was inhbited in Q61Rac1-infected cells.Conclusion: Rac1-MAPK/ERK is involved in the regulation of LPS-indcued endothelial permeability and F-actin stress fiber formation.