Role Of RhoA/ROCK Signaling Pathway In The Endothelial Cell Injury Induced By Lipopolysaccharide And The Intervention Of Stilbene Glucoside (TSG)

Objective In order to analyze the role of Rho A/ROCK signaling pathway in regulating endothelial dysfunction triggered by lipopolysaccharide(LPS),we constructed endothelial dysfunction model in vitro by using human umbilical vein endothelial cells(HUVECs).Methods HUVECs were purchased and cultured with DMEM.The approporate time and concentration for LPS treatment were cheked by MTT assay,flow cytometry and LDH(lactate dehydrogenase)detection kit.Cells were divided into the following group: control group,TSG-treated group(100 ?M,24 h),model group(100 ?g/ml LPS for 24 h),TSG(10?25?50?100 ?M,24 h)+LPS-treated group(cells were pretreated with TSG for 24 h and then were treated with 100 ?g/ml LPS for another 24 h).The cell viability was measured by MTT assay,and cell apoptosis was detected by flow cytometry via PI/Annexin V-FITC double staining.The release of LDH was determined by LDH(lactate dehydrogenase)detection kit,and the morphology and distribution of F-actin were detected by immunofluorescence assay.The expression of Rho A,ROCK and the activation of ROCK were determined by Western Blot analysis.The expression of Rho A,ROCK1 and ROCK2 in the vascular endothelial cells were knockdowned by si RNA technology,and then identified by Western Blot analysis.Cells were also divided into the following group:control group,model group(100 ?g/ml LPS for 24 h),si RNA or Y27632-pretreated group,si RNA or Y27632 pretreatment + LPS group.Subsequently,the cell viability and apoptosis was measured by MTT assay and flow cytometry via PI/Annexin V-FITC double staining,respectively.The release of LDH and morphology of F-actin were determined by LDH detection kit and immunofluorescence,respecyively.Results The results of MTT,flow cytometry and LDH assary showed that TSG reduced Lps-induced endothelial cell apoptosis and LDH release,and improved cell viability by a dose-dependant manner.The optimal time and concentration of TSG was100?g/ml TSG treatment for 24 h,which was utilized in the subsequent experiments.Western Blot showed that TSG significantly reduced the expression of Rho A and Rock and the activation of ROCK induced by LPS.The suppression of Rho A/Rock signal obviously decreaed Lps-induced endothelial apoptosis and LDH release,and dramatically improved cell viability.Immunofluorescence assary illustrated that LPS significantly induced actin lytoskeleton rearrangement which was inhibited by 50?M or 100?M TSG treatment and Rho A/Rock signaling suppression.Conclusions Rho A/ROCK signaling pathway participates in the LPS-induced the injury of endothelial cell,which relates with the destruction of F-actin skeleton.In the meanwhile,TSG could inhibit F-actin remodeling mediated by LPS through blocking Rho A/ROCK signaling pathway,and thus greatly reduce LPS-induced endothelial toxicity.