麝香乌龙丸对人脐静脉内皮细胞的细胞连接及RhoA/ROCK信号通路的影响

Effects of Shexiang Wulong Wan(麝香乌龙丸)on Cell Junction and RhoA/ROCK Signaling Pathway of Human Umbilical Vein Endothelial Cells

目的:观察麝香乌龙丸对高浓度1-磷酸鞘胺醇(S1P)诱导下人脐静脉内皮细胞(HUVEC)的细胞连接及RhoA/RhoA激酶(ROCK)信号通路的影响,探讨麝香乌龙丸治疗类风湿关节炎的作用机制。方法:将HUVEC分为4组:对照组,高浓度S1P组,高浓度S1P+麝香乌龙丸组,高浓度S1P+S1PR2拮抗剂JTE013组。采用免疫荧光检测HUVEC中细胞连接相关蛋白ROCK、ZO-1的变化,Western blot检测RhoA、ROCK、磷酸化RhoA激酶(p-ROCK)、ZO-1蛋白的表达水平。结果:高浓度S1P干预后,HUVEC中RhoA、p-ROCK、ROCK蛋白表达均上调,ZO-1表达下调,与对照组比较,差异有统计学意义(P<0.05)。麝香乌龙丸能够下调RhoA、ROCK、p-ROCK蛋白的表达,上调ZO-1表达,与高浓度S1P组比较,差异有统计学意义(P<0.05)。结论:麝香乌龙丸能够通过RhoA/ROCK通路调控HUVEC细胞连接相关蛋白ROCK、p-ROCK及ZO-1的表达,稳定HUVEC的血管屏障,这可能是麝香乌龙丸治疗类风湿关节炎的机制之一。

Objective:To observe the effect of Shexiang Wulong Wan(麝香乌龙丸,SXWLW)on the cell junction and RhoA/ROCK signal pathway of human umbilical vein endothelial cells(HUVEC)induced by high concentration of sphingosine 1-phosphosphingol(S1P),and to explore the mechanism of SXWLW in the treatment of rheumatoid arthritis.Methods:HUVEC was divided into four groups:the control group,the high-concentration S1P group(the HS1P group),the high concentration S1P+SXWLW group(the HS1P+SXWLWgroup),and the high concentration S1P+S1PR2 antagonist JTE013 group(the HS1P+JET013 group).Immunofluorescence was used to detect the changes of cell junction related proteins ROCK and ZO-1 in HUVEC.The expression levels of RhoA,ROCK,phosphorylated RhoA kinase(p-ROCK)and ZO-1 were detected by Western blot.Results:Under the intervention of high concentration of S1P,the expressions of RhoA,p-ROCK and ROCK in HUVEC were up-regulated and ZO-1 was down regulated,the difference was statistically significant compared with the control group(P<0.05).SXWLW could down regulate the expression of RhoA,ROCK,p-ROCK protein,and upregulate the expression of ZO-1,and the difference was statistically significant com-pared with the HS1P group(P<0.05).Conclusion:SXWLW can regulate the expressions of ROCK,p-ROCK and ZO-1 and stabilize the vascular barrier of HUVEC through RhoA/ROCK pathway,which may be one of the mechanisms of SXWLW in treating rheumatoid arthritis.