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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。
Objective: To investigate the molecular mechanism of LPS-induced monolayer permeability changes in human endothelial cells. Methods: The retrovirus was used as a vector to infect and screen human HUVEC cells stably expressing sustained-activated Rac1 and dominant-negative Rac1. LPS stimulation was used to observe the change of monolayer permeability of cytoskeleton proteins F-actin and HUVEC. At the same time, Western blot was used to detect the changes of MAPK / ERK signal pathway before and after LPS stimulation and the change of F-actin in cells after adding PD98059 to block the expression of ERK. RESULTS: Compared with normal HUVEC, F-actin in HUVECs infected with activated Rac1 and dominant inhibitory Rac1 restructured and formed a large number of stress fibers after LPS stimulation, and cell monolayer permeability increased significantly. However, there was no remodeling of F-actin in HUVECs after inhibition of Rac1, but no significant increase of cell monolayer permeability. Before and after LPS stimulation, there was no significant change in total ERK1 / 2 protein in all groups. After LPS stimulation, p-ERK increased in HUVECs infected with activated Rac1. After PD98059 was blocked, the expression of p-ERK in cells decreased accompanied with depolymerization of F-actin. CONCLUSION: Increased endothelial cell permeability induced by LPS is mediated through the Rac1-MAPK / ERK pathway.