EGCG And Fluvastatin Inhibit Activation Of THP-1Cells Induced By Anti-β2GPI/β2GPI Complex

Objective:Anti-β2-glycoproteinI/β2-glycoproteinI (anti-β2GPI/β2GPI) complex could activate endothelial cells and monocytes upon binding to the surface membrane of the cells, enhancing the expression and secretion of many cytokines, thereby increasing the risk of thrombosis, which are beneficial to the thrombus formation of antiphospholipid syndrome (APS). Our previous study reveals that the Toll-like receptor4(TLR4) and its signal transduction pathway can be induced to express and participate in cellular activation, and the effects of TLR4with annexinA2(ANX2) are tightly cooperative in process of anti-β2GPI/β2GPI-stimulated tissue factor (TF) and tumor necrosis factor-a (TNF-a) expression in the human monocytic cell line THP-1. These may be responsible for the thrombotic mechanisms in APS. In the present study, we investigated the effects of EGCG (epigallocatechin-3-gallate) and fluvastatin on anti-β2GPI/β2GPI complex-induced THP-1cells activation, targeting to the expression of TF and TNF-a, as well as the possible mechanisms involved in this process. The study may provide a new way for prevention and treatment of thrombotic issues in APS.Methods:(1) The THP-1cells were treated with anti-β2GPI/β2GPI complex. The TF and TNF-a mRNA expression on cells were detected by Real-time quantitative PCR (RT-qPCR), the TF activity and TNF-α protein level were estimated by commercial kits respectively.(2) Pretreated with different concentration of EGCG or fluvastatin, the cell proliferation ability was detected by MTT assay. (3) Pretreated with different concentration of EGCG or fluvastatin, the levels of TF and TNF-a induced by anti-β2GPI/β2GPI complex were examined by RT-qPCR and commercial kits respectively. The dose course and specific effects of EGCG or fluvastatin on THP-1cells were also investigated.(4) EGCG and fluvastatin were used to investigate whether the TLR4expression (mRNA and protein) in cells induced by anti-β2GPI/β2GPI could be intervened.(5) The phosphorylation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in THP-1cells induced by anti-β2GPI/β2GPI complex were investigated by western blotting. The time course of anti-β2GPI/β2GPI complex in cells was also investigated.(6) EGCG and fluvastatin were used to investigate whether the phosphorylation of NF-κB and MAPKs in cells induced by anti-β2GPI/β2GPI could be decreased by western blot assays, the dose course in THP-1cells was also investigated.Results:(1) The TF and TNF-α expression in THP-1cells were significantly up-regulated with treatment of anti-β2GPI/β2GPI complex, compared with untreated cells (p<0.05).(2) High concentration of EGCG (100μg/ml) or fluvastatin (50μg/ml) could inhibit cell proliferation in THP-1cells.(3) EGCG and fluvastatin could dose-dependently decrease TF and TNF-a level in THP-1cells stimulated with anti-β2GPI/β2GPI complex.(4) The expression of TLR4was significantly increased with stimulation of anti-β2GPI/β2GPI complex. EGCG and fluvastatin reduced anti-β2GPI/β2GPI complex-enhanced TLR4mRNA and its protein levels.(5) The phosphorylation of NF-κB and MAPKs in THP-1cells were also significantly increased with stimulation of anti-β2GPI/β2GPI complex, in a manner of time-dependence. EGCG and fluvastatin dose-dependently decrease phosphorylation of NF-κB and MAPKs in the presence of anti-β2GPI/β2GPI (p<0.05).Conclusions:(1) The TF and TNF-a expression in THP-1cells can be significantly up-regulated with treatment of anti-β2GPI/β2GPI complex.(2) EGCG and fluvastatin can decrease anti-β2GPI/β2GPI-induced TF and TNF-a expression in THP-1cells.(3) EGCG and fluvastatin can reduce the expression of TLR4, phosphorylation of NF-κB and MAPKs in the presence of anti-β2GPI/β2GPI complex.(4) EGCG and fluvastatin attenuate anti-β2GPI/β2GPI-induced activation in THP-1cells through inhibiting the intracellular signal transduction pathway of TLR4-MAPKs-NF-κB axis and may serve as preventive and therapeutic agents for APS.