The Role Of TLR4/NF-?B Pathway In OxLDL/?2GPI/anti-?2GPI Complex Induced HUVEC Inflammatory Cytokines Expression

Objective: The dysfunction of endothelial cells is one of the major factors for atherosclerosis(AS),which is closely related to the high incidence and mortality of AS.Oxidized low density lipoprotein(oxLDL)can accelerate inflammatory response and the development of AS by activating endothelial cells in the arterial intima,upregulating the expression of chemokines and inflammatory factors.The ?2-glycoprotein I(?2GPI)is a key antigen of antiphospholipid syndrome(APS),which can combine with oxLDL to form oxLDL/?2GPI complex and participate in the pathogenesis of AS.Our previous studies have shown that toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-?B)could play an important role in oxLDL/?2GPI/anti-?2GPI complex induced mouse peritoneal macrophage transforming to the foam cells.However,the effect of oxLDL/?2GPI/anti-?2GPI complex on endothelial cells which take part in the early process of AS remains unclear.Based on our previous work and related reports,we further explored the role of oxLDL/?2GPI/anti-?2GPI complex in the expression of some inflammatory factors which are closely related to AS in human umbilical vein endothelial cells(HUVEC),and involvement of TLR4 / NF-?B.Methods:(1)The migration ability of HUVEC treated with oxLDL,oxLDL/?2GPI complex,oxLDL/anti-?2GPI complex,oxLDL/?2GPI/anti-?2GPI complex and lipopolysaccharides(LPS)was observed by Scratch test,and the cells migration distance was analized by Image J software.(2)The m RNA and protein levels of MCP-1,IL-1?,IL-6 and TNF-? after oxLDL,oxLDL/?2GPI complex,oxLDL/anti-?2GPI complex,oxLDL/?2GPI/anti-?2GPI complex and LPS treatments were measured by Real-time quantitative PCR(RT-q PCR)and Enzyme-linked immunosorbent assay(ELISA).(3)The m RNA and protein levels of TLR4 in HUVEC treated with the above stimulants were measured by RT-q PCR and Western blot.(4)HUVEC was pretreated with TAK-242 or PDTC and the expression of MCP-1,IL-1?,IL-6 and TNF-? induced by above stimulants was measured by RT-q PCR and ELISA,in order to investigate the roles of TLR4/NF-?B.Results:(1)The migration distance of HUVEC significantly increased after treatment with oxLDL(40 ?g/m L)/?2GPI(100 ?g/m L)/anti-?2GPI(100 ?g/m L)complex,compared with media(p<0.05).(2)The m RNA and protein levels of MCP-1,IL-1?,IL-6 and TNF-? were significantly increased after treatment with oxLDL/?2GPI/anti-?2GPI complex(p<0.05 vs media).(3)The m RNA and protein levels of TLR4 significantly increased after treatment with oxLDL/?2GPI/anti-?2GPI complex(p<0.05 vs media).(4)The expression of MCP-1,IL-1?,IL-6 and TNF-? stimulated with oxLDL/?2GPI/anti-?2GPI complex was inhibited by TAK-242 or PDTC(p<0.05),and the inhibitory effect of TAK-242 is more stronger than that of PDTC.Conclusions:(1)Compared with media,oxLDL,oxLDL/?2GPI,oxLDL/anti-?2GPI,oxLDL/?2GPI/anti-?2GPI and LPS could promote HUVEC migration and MCP-1,IL-1?,IL-6,TNF-? expression.However,the migration distance of HUVEC in oxLDL/?2GPI/anti-?2GPI complex group was the most obvious and the expression of inflammatory factors was also the highest,suggesting that oxLDL/?2GPI/anti-?2GPI complex played a significant role in HUVEC inflammatory response.(2)oxLDL/?2GPI/anti-?2GPI complex induced TLR4 high expression in HUVEC inflammatory response.TAK-242 or PDTC could decrease the expression of inflammatory factors in stimulated group,moreover,the inhibitory effect of TLR4 inhibitor was stronger than that of NF-?B inhibitor,which suggested that TLR4 / NF-?B mediates the expression of inflammatory cytokines in HUVEC and TLR4plays a major role in this process.Decreasing TLR4 level may prevent the AS accompanied by APS.