大黄素对脂多糖诱导的内皮细胞损伤Rho/ROCK信号通路的影响

目的:探讨大黄素对脂多糖(LPS)诱导损伤的人脐静脉血管内皮细胞(HUVECs)骨架及通透性影响的作用机制。方法:HUECs分为五组:正常对照组、内皮炎症损伤模型组(0.2μg/m L LPS)、大黄素干预组(10μmol/L大黄素+0.2μg/m L LPS)、抑制剂对照组(0.2μg/m L LPS+50μmol/L Y27632)、西药对照组(10-5mol/L缬沙坦+0.2μg/m L LPS)。首先,以内皮细胞生物学功能PCR基因芯片技术筛选可能的基因改变。结合基因芯片结果和文献中各基因功能,拟定观察Rho/ROCK通路相关的和可能涉及调节细胞骨架的基因,如纤连蛋白(FN)、整合素A5(ITGA5)、Ras同源基因家族成员(Rho A)、肌球蛋白轻链磷酸酶(MLCP)、磷脂酰肌醇-4,5-二磷酸肌醇3-激酶(PI3K)、黏着斑激酶(FAK)、血管内皮生长因子(VEGF)、血管内皮生长因子受体2(VEGFR2)的m RNA表达水平,进行实时荧光定量PCR验证,同时以Western blotting对ITGA5、Rho A、4,5-二磷酸磷脂酰肌醇(PIP2)和磷酸化的肌球蛋白轻链(PMLC)进行蛋白水平的研究。结果:1LPS组Rho/ROCK通路相关基因(FN、ITGA5、Rho A、MLCP、PI3K、FAK、VEGF、VEGFR2)表达显著升高,ITGA5、Rho A、PIP2、PMLC的蛋白表达亦升高,与正常对照组比较,差异均有统计学意义(P0.05)。2与LPS组比较,Y27632组、缬沙坦组的FN、ITGA5、Rho A、PI3K、FAK、VEGF、VEGFR2基因表达下降,MLCP表达上升,差异均有统计学意义(P0.05);ITGA5、PIP2的蛋白表达下降;缬沙坦组PMLC表达降低,差异有统计学意义(P0.05)。3与LPS组比较,大黄素组FN、PI3K、FAK表达降低,MLCP表达上升,差异均有统计学意义(P0.05);PIP2蛋白表达下降,差异有统计学意义(P0.05)。结论:LPS可激活Rho/ROCK信号通路,成功构建HUVECs内皮损伤模型;Y27632和缬沙坦可阻断Rho/ROCK通路,对细胞骨架有较强的保护作用;大黄素可轻度抑制Rho/ROCK通路,同时可能通过调节PI3K、MLCP表达而影响其他通路(如AKT和/或FAK-PI3K信号通路)从而发挥对内皮细胞损伤的干预作用。

Protective Effect and Mechanism of Emodin on Rho/ROCK Signal Pathway in Endothelial Injury Induced by Lipopolysaccharide

Objective:To explore the mechanism of emodin influencing the skeleton and permeability of human umbilical vein endothelial cells(HUVECs) induced by lipopolysaccharide(LPS). Methods:HUVECs were divided into 5 groups :normal control group,endothelial injury model group(0.2 μg/ml LPS),emodin group(10 μmol/L emodin+0.2 μg/m L LPS),antagonist group(0.2 μg/m L LPS+50 μmol/L Y27632) and western medicine control group(10-5mol/L valsartan+0.2 μg/m L LPS). First,a gene chip was employed to screen the genes possibly involving in vascular endothelium function. According to the gene chip screening results,and literature review,genes associated with cytoskeleton remolding,or involving Rho/ROCK signal pathway were studied. For example,m RNA levels of fibronectin(FN),integrin A5(ITGA5),Ras homolog gene family member A(Rho A),myosin light chain phosphatase(MLCP),phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K),focal adhesion kinase(FAK),vascular endothelial growth factor(VEGF),vascular endothelial growth factor receptor2(VEGFR2) were determined with realtime fluorescent quantitative-PCR,and then and western blotting was applied to determine the protein level of ITGA5, Rho A, 4, 5-phosphatidylinosital biphosphate( PIP2) and phosphorylation of myosin light chain(PMLC). Conclusion: 1 In LPS group,expression levels of Rho/ROCK signal pathways related gene(FN,ITGA5,Rho A,MLCP,PI3 K,FAK,VEGF and VEGFR2) significantly in-creased,the protein expression of ITGA5,Rho A,PIP2,PMLC also increased. Compared with the control group,the difference was statistically significant(P<0.05). 2In the antagonist group and western medicine control group,compared with the endothelial injury model group,FN,ITGA5,Rho A,PI3 K,FAK,VEGF and VEGFR2 gene expression decreased,and the expression of MLCP increased,the difference was statistically significant(P <0.05);in terms of protein expression,ITGA5 and PIP2 expression decreased;Rho A in the antagonist group and PMLC in western medicine control group decreased,with statistical significance(P<0.05). 3The emodin group compared with the endothelial injury model group,the expression of FN,PI3 K,FAK decreased,MLCP increased,with statistical significance(P<0.05). The protein expression of PIP2 decreased,with statistical significance(P <0.05). Conclusions:LPS activated Rho/ROCK signal pathway and successfully established HUVECs endothelial injury model. Y27632 and valsartan blocked the Rho/ROCK signal pathway,showing the protection effects on cytoskeleton. The emodin could inhibit Rho/ROCK pathway mildly,also may affect other pathways,such as AKT and/or FAK-PI3 K signal pathway by adjusting PI3 K,MLCP,to interfere the damage to the endothelial cells.