The Role Of TLR4 And Ros In OxLDL/β2GPI/aβ2GPI Complex Induced HUVEC Apoptosis

Objective:β2-glycoprotein I(β2GPI)is an autoantigen in patients of antiphospholipid syndrome(APS),and the anti-β2GPI antibody(aβ2GPI)can participate in the pathogenesis of APS in a TLR4-dependent manner.Patients with APS have a tendency to accelerate the development of atherosclerosis(AS).Oxidized low density lipoprotein(oxLDL)is an independent risk factor for AS,which can form a complex with β2GPI to promote cell oxidation and lipid accumulation.oxLDL/β2GPI has been proved to be a pro-atherosclerosis factor,and it also forms a complex of oxLDL/β2GPI/aβ2GPI with the aβ2GPI to participate in atherosclerosisrelated pathological changes.Our previous studies have proved that oxLDL/β2GPI/aβ2GPI can promote the activation of a series of atherosclerosis-related factors.The mechanism of endothelial cells induced by oxLDL/β2GPI/aβ2GPI is still unclear,while dysfunction of endothelial cells is known to be the origin of AS Factors.Based on the related researches and previous studies,our study aims to investigate whether oxLDL/β2GPI/aβ2GPI can induce human umbilical vein endothelial cells apoptosis,and the role of TLR4 and oxidative stress during this process.Methods:(1)Western blot was used to explore expression of apoptosis-related proteins including Bax,Bcl-2,cleaved caspase-3 in HUVEC stimulated by media,β2GPI,aβ2GPI,β2GPI/aβ2GPI complex,oxLDL,oxLDL/β2GPI complex,oxLDL/β2GPI/R-IgG and oxLDL(50 μg/mL)/β2GPI(100 μg/mL)/aβ2GPI(100 μg/mL)complex.Annexin V-FITC/PI double-label flow cytometry was used to detect apoptosis rate.The cells were pretreated with TLR4 inhibitor and IRAK1/4 inhibitor to observe whether the apoptosis-related proteins and apoptosis rate were changed.(2)Western blot and RT-qPCR were used to detect the expression of TLR4,TLR2,MyD88 and IRAK4 in HUVEC stimulated by media,β2GPI,aβ2GPI,β2GPI/aβ2GPI complex,oxLDL,oxLDL/β2GPI complex,oxLDL/β2GPI/R-IgG and oxLDL/β2GPI/aβ2GPI complex.(3)DCFH-DA reactive oxygen probe,SOD and MDA kit were used to detect ROS,SOD,MDA expression in HUVEC stimulated by media,β2GPI/aβ2GPI complex,oxLDL,oxLDL/β2GPI/aβ2GPI complex.The cells were pretreated with antioxidants to observe whether ROS could be reduced.(4)CCK-8 was used to detect the cell viability.Western blot was used to detect expression of apoptosis-related proteins,and Annexin V-FITC/PI double-label flow cytometry was used to detect apoptosis rate in HUVEC stimulated by oxLDL/β2GPI/aβ2GPI complex after antioxidants pretreatment,aiming to observe the changes of the apoptosis-related proteins and apoptosis rate.Results:(1)After stimulation with oxLDL/β2GPI/aβ2GPI complex,Bax and cleaved caspase-3 protein level have increased in HUVEC,while Bcl-2 protein level has decreased,the apoptosis rate increased detected by flow cytometry,which was significantly different from the blank(p<0.05).The expression of TLR4,TLR2,MyD88 and IRAK4 were increased,which was significantly different from the blank(p<0.05).(2)The apoptosis-related proteins and apoptosis rate in HUVEC induced by oxLDL/β2GPI/aβ2GPI complex can partially reverse after pretreatment with TLR4 inhibitor or IRAK1/4 inhibitor.(3)After stimulation with oxLDL/β2GPI/aβ2GPI complex,cell viability and SOD decreased while ROS and MDA increased in HUVEC.(4)Pretreatment with antioxidants can attenuate the apoptosis induced by oxLDL/β2GPI/aβ2GPI complex and partially restore cell viability in HUVEC.Conclusions:(1)oxLDL/β2GPI/aβ2GPI can induce apoptosis in HUVEC,and apoptosis can be alleviated by TLR4 or IRAK1/4 inhibitor,suggesting that TLR4 plays a role in ox LDL/β2GPI/aβ2GPI induced apoptosis in HUVEC.(2)oxLDL/β2GPI/aβ2GPI can increase the ROS in HUVEC,after antioxidant pretreatment,ROS reduced and the apoptosis partially reversed,suggesting that reactive oxygen species may also plays a role in oxLDL/β2GPI/aβ2GPI induced apoptosis in HUVEC.