Effect Of Fasudil On Lipopolysaccharide Induced Injury Of Human Umbilical Vein Endothelial Cells

Objective The purpose of the present study is to evaluate the effect of fasudil on lipopolysaccharide-induced injury in human umbilical vein endothelial cells and clarify its underlying mechanisms.Methods1. HUVECs were obtained from human umbilical vein, digested with 0.1%collagenase type I and cultivated in endothelial cell medium with 5%FBS,ECGS and P/S. Subculture was attained with trypsin solution when cells were 90%confluent. The cells were detected under the inverted microscope.The cells were identified by VIII monoclonal antibody.2. HUVECs were randomly divided into three groups: Control group(endothelial cell medium was added), LPS group(0.1ug/ml LPS was added)、Fasudil group(fasudil 3.275ug/ml and 0.1ug/ml LPS were added).3. Quantitative real-time PCR and Western blot were performed to measure the m RNA and protein levels of Rho A, ROCK2 and p-ERM respectively.4. The rhodamine-palloidin staining was used to visualize the morphological changes of F-actin cytoskeleton。Results1. The primary cells could adhere to the plate completely within 24 hours an d grow to a monolayer in 3 days. Confluent cells exhibited the characterist ic "Cobblestone" morphology in culture in 3 days.The results of immunocy-tochemical staining of factor VTIII related antigen showed that about the cells were positive,which proved that the cultured cells were human umbi lical vein endothelial cells。2. Compared with the control group, LPS group significantly up-regulated the expression of Rho A,ROCK2 and p-ERM protein and m RNA(P<0.01).Compared with the LPS group, Fasudil group significantly down-regulated ROCK2 and p-ERM protein and m RNA expression(P<0.01), but there was no significant difference in Rho A protein and m RNA expression through fasudil treated.LPS was significantly altered the distribution of F-actin and these changes was attenuated by fasudil。Conclusions1. The primary HUVECs were successfully cultured in the present experiment.2. The Rho/ROCK/ERM plays an important role in LPS-induced the damage of cytoskeletal in HUVECs.3. Fasudil inhibited the LPS-induced cytoskeletal rearrangement was probably mediated by the inhibition of Rho A/ROCK/ERM signaling pathways.