| ObjectiveTo observe the effects of Fasudil Hydrochloride on the injury of HUVECs induced by AngiotensinⅡ( AngⅡ), which influence on the change of cytoskeleton and the expression of Rho associated kinase(ROCK) and Phospho-myosin light chain(P-MLC), and explore the possible molecular mechanisms of Fasudil Hydrochloride in cytoskeleton of the injuried HUVECs.MethodsNeonatal umbilical cord were digested into the primary human vascular endothelial cell(HUVECs)by collagenaseII, HUVECs was cultured. According to the morphological characteristics and immunohistochemical detection of intracellular von willebrand factor (vWF), we determined the cultured cells were endothelial cells. Endothelial cells used in experiments when they spread to 3 5 generation. Experiments were divided into four groups: normal control group; AngⅡgroup(final concentration of AngⅡwas 10-7mol/L); specific blocker group(final concentration of Y-27632 was 30umol/L , final concentration of AngⅡwas 10-7mol/L); Fasudil Hydrochloride group(final concentration of fasudil hydrochloride was 50umol/L , final concentration of AngⅡwas 10-7mol/L). The cell activities were detected by MTT method. Morphous and distribution changes of F-actin were observed by Immunofluorescence, Expression of ROCK and P-MLC were measured by Western blot. Results1,Identification of endothelial cells:The cultured endothelial cells are flat, polygonal and short spindle, which showed typical cobblestone morphology characteristic in Inverted microscope; With immunohistochemical method to detect theⅧ-related antigen antibody, it presents a lot of brown and strong granular accumulates in the cell plasma.2,To observe the effects of Fasudil Hydrochloride on the injury of HUVECs induced by AngⅡ, which influence on the vitality and functioning of HUVECs:①The cell activities were detected by MTT method: Compared with control group, AngⅡhad significant inhibitory effect to endothelial cells vigor(P<0.01), the specific blocker group and the Fasudil Hydrochloride group significantly weaken the production of endothelial cells vigor (P<0.01).②The functioning on cell were detected by nitrate reduction test method: Compared with control group, AngⅡhad significantly decreased the production of NO in endothelial cells (P<0.01), and compared with AngII group, the specific blocker group and the drug group significantly increased the production of NO (P<0.01).3,To observe the effects of Fasudil Hydrochloride on the injury of HUVECs induced by AngⅡ, which influence on the cytoskeleton of HUVECs: Morphous and distribution changes of F-actin by imunofluorescence, compared with control group, The rupture of stree fibers contructed by F-actin, deformation and digitation of HUVEC were exhibited.4,To observe the effects of Fasudil Hydrochloride on the injury of HUVECs induced by AngⅡ, which influence on the expression of ROCK: Western blot demonstrated:①Compared with AngⅡgroup, the specific blocker group and the Fasudil Hydrochloride group restrained the expression of ROCK remarkably (0.538±0.053%, 0.529±0.016% VS 1.374±0.015%, P<0.01), but they were also higher than the control group (P< 0.01),comparison between the two groups,the Fasudil Hydrochloride group compare with the specific blocker group P>0.05.②Compared with AngⅡgroup, the specific blocker group and the Fasudil Hydrochloride group restrained the expression of P-MLC remarkably (0.599±0.040%,0.608±0.022% VS 1.339±0.042%,P<0.01), but they were also higher than the control group (P<0.01),comparison between the two groups, the Fasudil Hydrochloride group compare with the specific blocker group P>0.05.Conclusions①The vitality,functioning and cytoskeleton of HUVECs are significant damaged by AngⅡ.Through obviously enhancing the vitality of HUVECs, increasing the production of NO, improving cytoskeleton of the HUVECs, Fasudil Hydrochloride can protect the injury of of HUVECs;②Fasudil Hydrochloride can protect the injury of of HUVECs which were induced by AngⅡ, through obviously controlling the expression of ROCK and P-MLC in the Rho/Rho kinase signaling pathways, thus weakened the damage in cytoskeletal and plaied a better protective effect on vitality,functioning of HUVECs.
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