Study On The Mechanism Of Vascular Endothelial Cell Injury By Ox-LDL Complex Through NF-κB-mediated Inflammatory Signaling Pathway

Objective:The oxidative stress of human umbilical vein endothelial cells(HUVECs)induced by oxidized low-density lipoprotein(Ox-LDL)complex was used to investigate the mechanism of Ox-LDL complex on vascular endothelial cells,and to find a new therapeutic target for atherosclerosis.Methods:1.Lipoprotein biomarkers levels were collected from The Second Hospital of Jilin University,among which diagnosed as coronary atherosclerosis(n=300)and age-matched healthy people(n=300).Including TG,TC,LDL-C,apo B,HDL-C,apo A,FFA and Lp(a).2.The effects of Ox-LDL and Ox-LDL complex on the proliferation activity of HUVECs were measured by MTT assay.The expressions of TRAF3IP2,PPARγ,Cleaved Caspase-3,Bcl-2,Phospho-IKKγ and Phospho-NF-κB p65 were measured by Western Blot.3.The effects of different stimulants on the migration ability of HUVECs were detected by cell scratch test.4.The changes of reactive oxygen species(ROS)in HUVECs were observed by chemical fluorescence probe under fluorescence microscope.5.Phospho-NF-κB p65 nuclear translocation was observed by immunofluorescence staining.The m RNA and protein levels of ET-1,IL-1β and TNF-α were detected by real-time PCR and ELISA.Results:1.Compared with controls,the peripheral venous TG,TC,LDL-C,apo B,FFA and Lp(a)levels in patients with atherosclerosis all increased(P<0.01),and HDL-C and apo A levels decreased(P<0.001).2.Ox-LDL,Ox-LDL/β2GPI and Ox-LDL/β2GPI/anti-β2GPI complex incubated with HUVECs 12 h induced significant oxidative stress injury,ROS level increased,and cells migration ability decreased(P<0.05),among which Ox-LDL/β2GPI/anti-β2GPI had the strongest effect(P<0.001).Atorvastatin could relieve these injuries(P<0.05).3.After treatment with Ox-LDL/β2GPI and Ox-LDL/β2GPI/anti-β2GPI complex,the m RNA and protein levels of ET-1,TNF-α and IL-1β were increased(P<0.05),and atorvastatin could inhibit the inflammatory response(P<0.05).4.Ox-LDL/β2GPI/anti-β2GPI complex showed the highest expression levels of TRAF3IP2,Phospho-IKKγ and Phospho-NF-κB p65(P<0.001).Atorvastatin inhibited the inflammatory response induced by Ox-LDL/β2GPI/anti-β2GPI(P<0.01).5.Zinc could reduce the oxidative stress,reduce ROS level,improve cells migration ability(P<0.01),weaken inflammatory response induced by Ox-LDL on HUVECs,and reduce m RNA and protein levels of ET-1,TNF-α and IL-1β(P<0.05).6.Zinc pre-treatment group inhibited Ox-LDL inflammatory signaling,decreased the expression levels of Cleaved Caspase-3 and Phospho-NF-κB p65,up-regulated the expression levels of PPARγ and Bcl-2(P<0.01).Cell fluorescence confocal assay showed that Zn could attenuate the nucleation of Phospho-NF-κB p65 induced by Ox-LDL.Conclusions:1.Ox-LDL/β2GPI/anti-β2GPI complex could induce maximum vascular endothelial injury,which possibly mediated by TRAF3IP2/IKKγ/NF-κB signaling pathway.2.Atorvastatin has obvious inhibitory effects on the decrease of HUVECs cell activity,cells migration,inflammation and oxidative stress injury induced by Ox-LDL/β2GPI/anti-β2GPI complex.3.Zinc has significant inhibitory effects on Ox-LDL-induced HUVECs inflammatory response and oxidative stress,which may be inhibited by PPARγ/NF-κB signaling pathway.