| Objective:Antiphospholipid syndrome (APS) is a kind of non organ specific autoimmune disease. Trombosis is not only the major pathological basis and the most prominent clinical manifestations, but also the primary cause of death in APS. The majority of studies have demonstrated that high titers of serum antiphospholipid antibodies (aPL), especially anit-β2GPI antibodies, play a pivotal role in the pathological mechanism of the thrombus formation of APS, but the mechanisms of thrombosis are not yet elucidated. Our previous results have illuminated that nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) colud be activated in the process of anti-β2GPI/β2GPI-induced expression of tissue factor(TF) and inflammatory cytokines in monocytes, but there are still insufficient experiments in vivo to elucidate the role of NF-κB and AP-1 in aPL/anti-β2GPI-induced thrombosis in vivo. In addition, a recent study revealed that the mammalian target of rapamycin complex (mTORC)pathway have a crucial role in activating inflammation among endothelial vessel wall causing vascular lesions in APS. In the present study, we investigated the role of NF-κB/AP-1 and mTOR in the pathogenic effects of aPL/anti-(32GPI-induced thrombosis and expression of effector molecules in experimental antiphospholipid antibody syndrome (EAPS)mice model and monocytes (THP-1 cells and Human peripheral blood mononuclear cells), respectively. We hope to provide new ideas for prevention and treatment of thrombotic issues in APS.Methods:(1) The experimental antiphospholipid antibody syndrome (EAPS) mice model were established using the male BALB/c mice by intraperitoneal injection (i.p.) with 500 μg of anti-β2GPI or IgG-APS purified from the APS patients at 0 h and 48 h, respectively. The peritoneal macrophages in each animal were collected at 72 h. The cells were homogenized by sonication and the phosphorylation of NF-B p65 and c-Jun/AP-1 was detected by Western blotting. This study aimed to validate whether anti-β2GPI antibody can induce the activation of NF-κB and AP-1 in vivo.(2) BALB/c mice were pretreated with NF-κB inhibitor PDTC (100 mg/kg, once a day) by i.p. or AP-1 inhibitor Curcumin (50 mg/kg,once a day) by oral gavage for 3 consecutive days at 2 h before anti-β2GPI injections in subsequent experiments. In order to investigate the effects of PDTC and curcumin on the activation of anti-β2GPI-induced NF-κB and AP-1, aorta and peritoneal macrophages were harvested from experimental animals, and the phosphorylation of NF-κB p65 and c-Jun/AP-1 was measured by Western blotting.(3) In order to further analyze the effects of NF-κB and AP-1 activation on expression of anti-β2GPI-induced TF and proinflammatory molecules in vivo, peritoneal macrophages, bilateral carotid artery and aorta were collected from EAPS mice. The mRNA and protein expression of TF, E-selectin, ICAM-1, VCAM-1, IL-1p, IL-6 and TNF-α were detected by real-time quantitative PCR (RT-qPCR) and Western blotting. TF activity was examined through Xa generation method. The expressions of TF, IL-1β, IL-6 and TNF-α in peritoneal macrophages were measured by immunofluorescence.(4) The THP-1 cells or human peripheral blood mononuclear cells were treated with anti-β2GPI (10 μg/mL)/β2GPI (100 μg/mL) or APS-IgG(250 μg/mL)/β2GPI (100 μg/mL) complex for a certain time. In order to observe the effects of anti-(32GPI/(32GPI or APS-IgG/β2GPI on TF and IL-8 expression and mTOR activation in monocytes, the total proteins and RNA of the cells and supernatant were collected. The mRNA expression of TF and IL-8 was detected by RT-qPCR. TF activity was examined through Xa generation method. The protein expression of IL-8 in supernatant was measured by enzyme linked immunosorbent assay (ELISA). The phosphorylation of mTOR was evaluated by Western blotting.(5) The THP-1 cells or human peripheral blood mononuclear cells were pretreated with mTOR inhibitor rapamycin (100 nM) at 20 h before stimulation with anti-β32GPI/β32GPI or APS-IgG/(32GPI complex for a certain time. To investigate the effects of rapamycin on anti-β2GPI/β2GPI or APS-IgG/(32GPI-induced expression of TF and IL-8 and activation of mTOR, p38, ERK1/2, JNK and NF-κB p65 in monocytes, the total proteins and RNA of the cells and supernatant were collected. The mRNA expression of TF and IL-8 was detected by RT-qPCR. TF activity was examined through Xa generation method. The protein expression of IL-8 in supernatant was measured by ELISA. The phosphorylation of mTOR, p38, ERK1/2, JNK and NF-κB p65 was evaluated by Western blotting.(6) The THP-1 cells were pretreated with p38 inhibitor SB203580 (10μM) or ERK1/2 inhibitor U0126 (5 μ,M) or JNK 1/2 inhibitor SP600125 (90 nM) or NF-κB inhibitor PDTC (20 μM) or TLR4 inhibitor TAK-242 (5 μM) at 2 h before stimulation with anti-β2GPI/β2GPI or APS-IgGβ2GPI complex for a certain time. To investigate the effects of rapamycin on anti-β2GPI/β2GPI or APS-IgG/β2GPI-induced mTOR activation in THP-1 cells, the total proteins of the cells were collected, and the phosphorylation of mTOR was analyzed by Western blotting.Results:(1) The phosphorylation of NF-κB p65 and c-Jun/AP-1 was markely elevated in the peritoneal macrophages from BALB/c mice after anti-β2GPI or IgG-APS treatment, compared to control mice injected with NR-IgG (p<0.05).(2) PDTC and Curcumin could markedly attenuate anti-β2GPI-induced activation of NF-κB p65 and c-Jun/AP-1 in the aorta and peritoneal macrophages respectively (p<0.05), but combination of two inhibitors had no synergistic effect. Curcumin treatment also obviously decreased the anti-β2GPI-induced phosphorylation of NF-κB p65 in peritoneal macrophages (p<0.05), and slightly decreased but not statistically significant in the aorta (p>0.05).(3) PDTC and/or Curcumin could significantly inhibit anti-β2GPI-enhanced TF activity in homogenates of carotid arteries and peritoneal macrophages, the mRNA and protein expression of TF,VCAM-1, ICAM-1 and E-selectin in the aorta and the mRNA and protein expression of TF, IL-1(3, IL-6 and TNF-a in peritoneal macrophages (p<0.05), in which PDTC showed the strongest inhibitory effect, but combination of two inhibitors had no synergistic effect.(4) The treatment of THP-1 cells or human peripheral blood mononuclear cells with anti-β2GPI/P2GPI or APS-IgG/β2GPIcomplex could markedly induce TF and IL-8 expression, as well as mTOR activation in a manner of time-dependence, with the maximal levels at 45 min(THP-1 cells) and 60 min (human peripheral blood mononuclear cells)of stimulation (p<0.05).(5) The mTOR inhibitor rapamycin (100 nM, 20 h) could attenuate TF and IL-8 expression, the levels of phosphorylated p38 (p-p38),p-ERK1/2 and p-NF-KB p65 stimulated by anti-β2GPI/β2GPI or APS-IgG/(32GPI complex in THP-1 cells or human peripheral blood mononuclear cells (p<0.05), but had no effect on p-JNK (p>0.05).(6) The levels of phosphorylation of mTOR caused by anti-β2GPI/β2GPI or APS-IgG/β2GPI complex in THP-1 cells were downregulated through inhibition of p38 (10 μM, SB203580) or ERK1/2 (5 μM,U0126) or TLR4 (TAK-242, 5 μM) rather than inhibition of JNK (90 nM, SP600125) or NF-κB (20 μM, PDTC).Conclusions:(1) NF-κB and c-Jun/AP-1 are involved in regulating anti-β2GPI-induced expression of TF, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and inflammatory factors (IL-1β, IL-6 and TNF-α) in EAPS mice, indicating that NF-κB and c-Jun/AP-1 play an important role in aPL induced prothrombotic and proinflammatory phenotype.(2) In vitro studies found that mTOR are involved in regulating anti-β2GPI/β2GPI- or APS-IgG/(32GPI complex-induced TF and IL-8 expression in monocytes. TLR4, p38, and ERK1/2 can modulate mTOR activation induced by anti-β2GPI/(32GPI- and APS-IgG/β2GPI complex in monocytes. Hence, these results indicate that mTOR play crucial roles in the pathogenic mechanism of APS.(3) The inhibitors of NF-κB or c-Jun/AP-1 or mTOR can decrease anti-β2GPI-induced expression of prothrombotic and proinflammatory molecule, indicating NF-κB, c-Jun/AP-1 and mTOR can be used as new targets for therapy as well as prevention of thrombotic events in APS. |
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