| Objective:Atherosclerosis(AS)is an arteriosclerotic vascular disease that begins with endothelium inflammation and injury,characterized by hyperlipidemia and atheroma plaque formation,and has become one of the main causes of death.With the development of researching on the pathogenesis of atherosclerosis,besides the traditional factors such as smoking and obesity,the relationship between autoimmune diseases such as antiphospholipid syndrome(APS)and AS has got more and more attention.Studies have shown that the high incidence of AS in patients with APS may be related to the excessive ?2 glycoprotein I(?2GPI)and anti-?2 glycoprotein I antibody(anti-?2GPI)in their body.In addition,a number of studies have shown that ?2GPI can bind oxidative low-density lipoprotein(ox LDL)to form a stable ox LDL/?2GPI complex and mediate AS-related dysfunction of endothelial cells.The role of ox LDL/?2GPI/anti-?2GPI complex in the above process remains to be further explored.In our previous study,ox LDL/?2GPI/anti-?2GPI complex can promote the foam cell formation of mouse macrophages in vitro.In addition,the complex can promote the expression of IL-6 and IL-1? in human umbilical vein endothelial cells(HUVEC)through the TLR4-dependent way.Based on the previous experiments and related literatures,this study explored the role of ox LDL/?2GPI/anti-?2GPI complex on the expression of HUVEC inflammatory cytokine(TNF-?),adhesion molecules(VCAM-1/ICAM-1)and thrombus-associated molecule(v WF),as well as the role of TLR4 and p38 MAPK played in this process.Methods:(1)Culturing HUVEC and taking 3-8 generations of better cells for the experiment.Total RNA and protein were collected after stimulation by DMEM,ox LDL,ox LDL/?2GPI complex,ox LDL/anti-?2GPI complex,ox LDL/?2GPI/anti-?2GPI complex and LPS respectively.The m RNA levels of TNF-?,ICAM-1,VCAM-1 and VWF were detected by Real-time quantitative PCR(RT-q PCR),and the protein levels of ICAM-1,VCAM-1,v WF and TNF-? were detected by Western blot and Enzyme-linked immunosorbent assay(ELISA).(2)The HUVEC pre-treatmented with TLR4 antagonist TAK-242(5 ?mol/L)and p38 MAPK antagonist SB203580(10 ?mol/L)were stimulated by DMEM,ox LDL/?2GPI/anti-?2GPI complex and LPS respectively.The expression of atherosclerosis-related molecules in HUVEC were detected by RT-q PCR,Western blot and ELISA.(3)THP-1 cell adhesion test and the antagonist pre-treatment methods were employed to evaluate the effect of ox LDL/?2GPI/anti-?2GPI complex on the ability of HUVEC attracting monocytes and the role of TLR4 and p38 MAPK in this process.Results:(1)Compared with media group,the m RNA and protein levels of TNF-?,ICAM-1,VCAM-1 and v WF in HUVEC stimulated by ox LDL/?2GPI/anti-?2GPI complex were significantly higher,and the difference was statistically significant(P < 0.05).(2)The high expression of TNF-?,ICAM-1,VCAM-1 and v WF m RNA and protein stimulated by ox LDL/?2GPI/anti-?2GPI complex and LPS were significantly inhibited by TAK-242 or SB203580 pretreated(P < 0.05).(3)Compared with the media group,the number of mononuclear cells adhering to HUVEC increased significantly in the ox LDL/?2GPI/anti-?2GPI complex group and LPS group,the difference was statistically significant(P < 0.05).TAK-242 and SB203580 pretreatment effectively inhibited this phenomenon(P < 0.05).Conclusions:(1)ox LDL/?2GPI/anti-?2GPI complex could up-regulate the expressions of adhesion molecule VCAM-1 and ICAM-1,proinflammatory cytokines TNF-?,and thrombus-associated molecule v WF in HUVEC and promote the adhesion of THP-1 cells to HUVEC.(2)ox LDL/?2GPI/anti-?2GPI complex could increase the expression of HUVEC atherosclerosis-related molecules through a similar approach to LPS and strengthen their ability to adhere to monocytes.The TLR4/p38 MAPK signaling pathway played an important mediating role in this process. |
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