Oleanolic Acid By LOX-1 / NADPH Oxidase / ROS, Nrf2 / HO-1 Pathway Inhibition OxLDL-induced HUVEC Cell Damage

Objective To repeat oxLDL-induced injury model of human umbilical vein endothelial cells (HUVEC), construct a lentivirus-LOX-1-shRNA expression vector and investigate the mechanisms of Oleanolic Acid (OA) on protecting HUVEC cells from oxLDL-induced injury through LOX-1/NADPH oxidase/ROS signaling pathway and Nrf2/HO-1signaling pathway. Method OxLDL-induced injury model of HUVEC cells was established by200μg/mL oxLDL and HUVEC cells co-incubated for24h and cells were divided into four groups:control group, oxLDL model group, OA treatment group(5,10,20μmol/L), and positive control group (200μmol/L vitamin E). Cell viability was investigated by MTT; the generation of ROS was detected by2’,7’-Dichlorofluorescin diacetate (DCFH-DA); LOX-1and NADPH oxidase subunits (p22phox, gp91phox, Racl) mRNA levels were detected by real-time PCR; LOX-1and NADPH oxidase subunits (p47phox, p67phox, gp91phox) protein levels after coincubated with oxLDL at diffferent time point (0,3,6,12,24h) were detected by western blot. Results OxLDL-induced injury model of HUVEC cells was successfully constructed. Compared with the control group, after oxLDL treatment cell viability was notably decreased; the levels of LOX-1mRNA and protein were significantly increased; NADPH oxidase subunits (gp91phox, p22phox, Racl) mRNA and (p47phox, p67phox, gp91phox) protein levels were significantly increased; the generation of ROS was markedly increased as well as Nrf2nuclear protein expression and HO-1protein expression. Western blot analysis in oxLDL treated HUVEC cells showed that LOX-1and HO-lprotein expression level as well as ROS release significantly increased following oxLDL treatment and reached peak at3h and24h post treatment; NADPH oxidase subunits (p47phox, p67phox, gp91phox) protein levels significantly increased following oxLDL treatment and reached a plateau between3h and12h, then reached peak at24h (p<0.05).5-20μmol/L OA,200μmol/L vitamin E and lentivirus mediated LOX-1-shRNA groups significantly inhibited oxLDL induced cell viability decreased, upregulation of LOX-1mRNA and protein expression, upregulation of NADPH oxidase subunits (gp91phox, p22phox, Racl) mRNA and (p47phox, p67phox, gp91phox) protein expression as well as the generation of ROS.5-20μmol/L OA and200μmol/L vitamin E groups markedly further increased oxLDL induced upregulation of Nrf2nuclear protein expression and HO-1protein expression (p<0.05); however lentivirus mediated LOX-1-shRNA group inhibited oxLDL induced upregulation of Nrf2nuclear protein expression and HO-1protein expression (p<0.01). Conclusion200μg/mL oxLDL induced HUVEC cells injury through activation of the LOX-1/NADPH oxidase/ROS signaling pathway; OA inhibited oxLDL induced HUVEC cells injury through inhibition of the LOX-1/NADPH oxidase/ROS signaling pathway and activation of the Nrf2/HO-1signaling pathway; and the expression of LOX-1is essential for Nrf2/HO-1activation in oxLDL induced injury model.