Role Of Caveolin-1Involves In The Rearrangement Of F-actin In Endothelial Cells Induced By Lipopolysaccharide

Part one: Selection of the most effective small interfering RNA whichinhibits the expression of Caveolin-1in endothelial cells and detection ofthe cytotoxicity of transfection complexObjective：Aim to pick out the siRNA which could most effectively inhibit theexpression of caveolin-1in endothelial cells and to detect the cytotoxicity of thetransfection complex.Methods：Four siRNAs were chemically synthesized: siRNA422, siRNA548, siRNA710were used to inhibit caveolin-1expression in endothelial cells, FAM-labeled mismatchsiRNA was as a negative control. They were all transfected into endothelial cells,respectively. Caveolin-1mRNA was detected after transfection by Real-Time PCR and itsprotein expression was measured by Western blot. The cytotoxicity was analyzed with themethod of the Sulforhodamine B （SRB）.Results:①The transfection rates are all above85%under the transfection conditionof siRNA (20,30or40nmol L^-1) combined with the same dose of siRNAFect （P<0.01）;②The survival rates of transfected cells are all above80%under the transfection condition ofsiRNAFect1.3μL combined with siRNA (10or20nmol L^-1)（P<0.05）;③The expressionof caveolin-1mRNA in EA.hy926cells was significantly decreased after siRNA548treatment （P<0.01）;④Compared with siRNA422, siRNA710,vehicle or mismatchsiRNA-treated group, the expression of caveolin-1protein was the least after siRNA548treatment48hours later （P<0.01）.Conclusions:①The siRNA548can inhibit caveolin-1expression most effectively in endothelial cells.②siRNA (20nmol·L^-1) combined with siRNAFect （1.3μL） has lowercytotoxicity and higher transfection efficiency, which is suitable for transfection.Part Two: Caveolin-1involves in the rearrangement of F-actinthrough NF-κB pathway in endothelial cells induced bylipopolysaccharideObjective: To explore the role and possible mechanism of Caveolin-1in therearrangement of F–actin in endothelial cell induced by lipopolysaccharides （LPS）.Methods: Human umbilical vein endothelial cell strain（EA.hy926cell strain）wasselected and cultured in special condition. When the cells had grown stable, and after theirsidewall fusion, they were divided into4groups according to different treatment, withevery group in6holes: control group （group C）, LPS10μg/ml group （group L）,cav-1-siRNA548pre-treatment group （group S） and cells were treated with cav-1-siRNA548and L-NAME10μM （group N）. Both group S and group N were further treated with LPS（10μg/ml）. According to the time of treatment with LPS, every group was further dividedinto1h,3h,6h and12h subgroups. The expression of peNOS-s1177and NF-κB wasmeasured in every group by Real-Time PCR, meanwhile the change of F-actin in everygroup was observed by immunofluorescence staining.Results:（1）The expression of peNOS-s1177in group L and group S increasedsignificantly compared with in group C in every time point （P<0.01）. Similarly, theexpression of peNOS-s1177in group S increased remarkably compared with group L inevery time point（P<0.01）. The highest level of peNOS-s1177was found in6h after LPStreatmen（tP<0.01）, while the level began to decrease in12h after LPS（P<0.05）in groupL. However, the highest level of peNOS-s1177was found in3h after LPS （P<0.05）, andbegan to decrease step by step in6h and12h after LPS in group S （P<0.01）.（2） Theexpression of NF-κB increased significantly in group L, S and N in every time point compared with group C （P<0.01）. And there were more significant increase in theexpression of NF-κB in group L and N in every time point than group S （P<0.01）, whilethe expression level has no significant difference between group L and N（P>0.05）. Theexpression of NF-κB decreased significantly in3,6and12h after LPS compared with1hsubgroup（P<0.01）.（3） immunofluorescence staining showed that EA.hy926cell strainmanifested F-actin depolymerization and reconstitution in6h after LPS treatment, and thephenomenon became apparently following the longer time. Conversely, in the used of thecav-1-siRNA548pretreatment cell strain, the occurrence of F-actin depolymerization andreconstruction was delayed and to a lesser degree. And L-NAME may reverse theresponse.Conclusions:（1） Caveolin-1may have a role in the modulation of LPS-inducedendothelial F–actin depolymerization.（2） Caveolin-1may modulate F-actindepolymerization and reconstitution through eNOS-NF-κB pathway.