Effect Of Pitavastatin On Biological Behavior And Expression Of ENOS And MiR-155 In Human Umbilical Vein Endothelial Cell

BackgroundAtherosclerosis has become one of the major diseases that seriously threaten the health of the residents in our country.In the study of atherosclerosis,vascular endothelial cell injury is the initiating factor of atherosclerosis.It is great significance to construct an in vitro culture system that can faithfully and steadily to represent the physiological of vascular endothelial cells in the body.Pitavastatin is a new statin drug which has the reputation of "super statins".The study of the effect of pitavastatin on the function of endothelial cells can provide scientific interpretations for the non lipid lowering effect of pravatin in the prevention and treatment of atherosclerosis.ObjectiveScreen the optimum medium in primary culture and subculture of human umbilical vein endothelial cells(HUVECs).Pitavastatin(Pit)has been proved to efficiently inhibit the onset and progression of atheroscleosis.However the mechanism by which Pit exert non-lipid related effects,such as anti-inflammatory actions,is not quite clear.Our study aimed at investigating the effect of Pit on the expression of eNOS and miR-155 in LPS-stimulated HUVECs to reveal the anti-inflammatory mechanism of pitavastatin.MethodCells were obtained by 0.02% type II collagenase perfusion of umbilical vein to digest15 min and cultured in different medium.After 24 h according to observe cell adherence status,calculate the number of cells,and then identify marker protein vWF,we can select the optimal culture medium.After subculture,the effect of different culture solution on the activity of HUVECs was compared by the MTT method.CCK-8 assay,scratch assay andtuberculosis experiment were used to determine the effect of pitavastatin on the biological behavior of HUVECs.Cells were treated with LPS(0.05,0.1,1 ?g/L)or LPS(0.1 ?g/L)+Pit(0.01,0.1,1?mol/L),untreated cells were used as control.For LPS + Pit induction,cells were firstly incubated with Pit for 1 h before co-incubation with LPS for 24 h.eNOS mRNA and miR-155 were detected by RT-PCR and western blotting was used to detect protein expression of eNOS.ResultECM medium containning of ECGS and 20% FBS in primary culture can harvest the largest number of HUVECs in contrast to other groups;HUVECs were subcultured by M199 medium containing 30 ng/mL VEGF165 and 10% FBS,but the difference of HUVECs viability was not obvious compared with the ECM group.High concentration of Pit(10 ?mol/L)could significantly inhibit the proliferation of HUVECs cells;Different concentrations of Pit(0.01,0.1,1 ?mol/L)had no significant effect on the migration ability of HUVECs;Different concentrations of Pit(0.01,0.1,and 1 ?mol/L had significant inhibitory effects on the ability of HUVECs to form tubes;Treatment of HUVECs with LPS enhanced the expression of miR-155 and reduced the expression of eNOS in mRNA and protein level in a dose-dependent manner as revealed by RT-PCR and western blotting respectively.Pitavastatin ameliorated LPS-induced endothelial dysfunction through up-regulation of eNOS expression and down-regulation of miR-155 expression.ConclusionOptimized medium was used to primary culture and subculture of HUVECs to ensure the quality and quantity of HUVECs and reduce the cost of passage culture by 60.8%.High dose of Pit can inhibit the proliferation and tube formation ability of HUVECs;But Pit had no effect on the migration ability of HUVECs;Pitavastatin increases eNOS expression through inhibition of LPS-induced miR-155 expression.