The Effects Of Various Titers Of Antiphospholipid Antibody And TNF-α On E-selectin, IL-6, IL-8, MCP-1Secretion By Endothelial Cell

Antiphospholipid syndrome (APS) is a systemic autoimmune disease, the presence of antiphospholipid antibody (aPL) in the serum and generalized prothrombotic state are the two major characteristics, and this serological characteristics are closely related to embolism and pathological pregnancy. The pathogenesis of APS is still unclear, prothromboticstate is one of the important factors, and the important factor required for the formation of prothromboticstate is endothelial cells’damage and activation. About the relation between the concentration of aPL, infection and the incidence and prognosis of APS, some foreign scholars believe that the higher the concentration, the worse the prognosis, infection can make APS worse. What makes this phenomenon? Is the activation of endothelial cell involved in it? No such research has been seen in China.One of the major receptors for aPL is β2-glycoprotein I (β2GPI). aPL/β2GPI complex further binds to annexin A2, then combines with phospholipids on the cell membrane, however, annexin A2does not have the transmembrane structure, cell signal cannot be conducted. Many researches have been focused on toll-like receptor (TLR) signaling pathway and found that TLR2mediated the signal transduction, disagreement was existed about the role of TLR4, no such research in China. After activation, the synthesis and secretion of adhesion molecule such as E-selectin (SELE) significantly increased, a number of inflammatory cytokines such as interleukin-1β(IL-1β), interleukin-6(IL-6),interleukin-8(IL-8), tumor necrosis factor-a (TNF-a), monocyte chemoattractant protein-1(MCP-1) also increased significantly, resulting in a proinflammatory and procoagulant state. Among them, E-selectin、 IL-6、IL-8、MCP-1are the most sensitive and frequently used factors in monitoring the endothelial cells’activation extent. The consistent change of SELE mRNA and pretein levels means the correctness of the experimental results. Limited by the experimental techniques and conditions, the aPL used in our research is the purified IgG from APS patients, the purified IgG from normal person was used as normal control to exclude the possible effects of the normal types of IgG.The paper is divided into three parts:the first part was to investigate the effects of various titers of aPL on endothelial cells. Culturing human umbilical vein endothelial cells (HUVECs) in vitro, stimulating them with purified IgG proteins from APS or normal non-pregnant women. Endothelial cell secretion function was assessed by measuring mRNA and protein levels of E-selectin by the means of real-time fluorescent quantitative-polymerase chain reaction (RT-PCR) and flow cytometry (FACS), the protein levels of MCP-1, IL-6and IL-8were measured by mean of enzyme-linked immunosorbent assay (ELISA), TLR2and TLR4 were measured by Western blot. The second part was to study the effect of TNF-α and the role of TLR2and TLR4in activation of the endothelial cells by aPL. By blocking TLR2or (and) TLR4signal transduction way, we study the role of TLR2/4directly. The third part was about investigating the effects of BCG polysaccharides nucleic acid (BCG-PSN) in activation of endothelial cells by aPL, to explore whether BCG-PSN can be used in APS. Chapter1Secretion of E-selectin, IL-6, IL-8and MCP-1by endothelial cell stimulated by various titers of antiphospholipid antibodyObjective:To study the expression of E-selectin, IL-6, IL-8, MCP-1on endothelial cell when stimulated by various titers of aPLs.Methods:HUVECs were cultured in vitro, and stimulated with aPLs from APS (5%,10%,20%) or normal non-pregnant women. Endothelial cell activation was assessed by measuring mRNA and protein levels of E-selectin by the means of RT-PCR and FACS, the protein levels of MCP-1, IL-6and IL-8were measured by mean of ELISA, Western blot analysis was used to measure the protein levels of TLR2and TLR4.Results:Stimulated by aPL showed a significant increase in SELE, IL-6, IL-8, MCP-1expression, comparing every two groups of blank, normal control or aPL (5%,10%,20%) groups, p<0.01.There are no significant difference on TLR2expression between the groups of blank, normal and5%aPL(p>0.05), TLR2expression in10%and20%aPL groups were significantly increased compared with the blank and normal control groups or with each other (p<0.05). aPL has no effect on the expression of TLR4(p>0.05)Conclusions:Expression of E-selectin, IL-6, IL-8and MCP-1on endothelial cell increased when stimulated by aPL, the higher the antibody concentration, the higher the expression. Chapter2The role of TNF-a, TLR2and TLR4in activation of endothelial cell by antiphospholipid antibodiyObjective:To investigate the role of TNF-a, TLR2/4in secretion function of endothelial cell when stimulated by antiphospholipid antibodiy.Methods:(1) HUVECs were cultured in vitro, part of them were stimulated with100ng/ml TNF-a for24hours followed by an8h wash-out, collecting some TNF-pretreated and non-pretreated cells, using Western blot analysis to measure the protein levels of TLR2and TLR4. The rest cells were stimulated with LTA or aPLs from APS women. Endothelial cell activation was assessed by measuring mRNA and protein levels of E-selectin by the means of RT-PCR and FACS, the protein levels of MCP-1, IL-6and IL-8were measured by mean of ELISA;(2) HUVECs were cultured in vitro and stimulated with100ng/ml TNF-a for24hours followed by an8h wash-out, then treated with aPL, Anti-TLR2antibodies or/and anti-TLR4antibodies were used before adding aPL to block TLR2or/and TLR4signal conduct pathways. Endothelial cell activation was assessed by measuring mRNA and protein levels of E-selectin by the means of RT-PCR and FACS, the protein levels of MCP-1, IL-6and IL-8 were measured by mean of ELISA.Results:After pre-stimulation by TNF-a, the TLR2expression on HUVEC was significantly increased compared with the non-pretreated group (p<0.01), the TLR4expression had no change (p>0.05). stimulated by LTA, the specific agonist of TLR2, the expression of SELE, IL-6, IL-8, MCP-1were significantly increased in TNF-apretreated group compared with the non-pretreated group (p<0.01). Under the same concentration of aPL, the expression of SELE, IL-6, IL-8, MCP-1in TNF-pretreated group were significantly higher than non-pretreated group (p<0.01).Blocking TLR2signal conduct pathways significantly attenuated the expression of SELE, IL-6, IL-8, MCP-1expression on HUVEC stimulated by aPL (p<0.01). blocking TLR4did not have significant effect on the expression of SELE, IL-6, IL-8, MCP-1stimulated by aPL HUVEC (p>0.05).Conclusions:(1) TNF-apre-treated endothelial cell secretes more SELE, IL-6, IL-8, MCP-1than non-pre-treated cell when stimulated by aPL.(2) Antiphospholipid antibodies activate endothelial cell via TLR2signal transduction pathways. Chapter3The impact of BCG-polysaccharides nucleic acid on E-selectin, IL-6, IL-8and MCP-1expression on endothelial cell stimulated by antiphospholipid antibodyObjective:To investigate the impact of BCG-polysaccharides nucleic acid (BCG-PSN) on secretion function of endothelial cell stimulated by antiphospholipid antibody.Methods:HUVECs were cultured in vitro, and stimulated with100ng/ml TNF-a for24hours followed by an8h wash-out, then treated with20%of APS aPLs, APS aPLs treated groups were also treated with different concentrations of BCG-PSN (0,25,50and100μg/ml). Endothelial cell activation was assessed by measuring mRNA and protein levels of E-selectin by the means of RT-PCR and FACS, the protein levels of MCP-1, IL-6and IL-8were measured by mean of ELISA.Results:APS IgG induced endothelial cells’ SELE, IL-6, IL-8, MCP-1expression increasing, while the intervention of BCG-PSN attenuated APS IgG-induced SELE, IL-6, IL-8, MCP-1expression, compared any two of BCG-PSN groups (0,25,50and100μg/ml), the differences were statistically significant (p<0.01). Conclusion:Antiphospholipid antibody induced secretion of E-selection, IL-6, IL-8, MCP-1on endothelial cell decreased when interfered by BCG-polysaccharides nucleic acid, suggesting that it may be useful for the treatment of APS.