血小板激活因子受体拮抗剂SY0916抑制巨噬细胞血管生成作用的研究

Anti-Angiogenesis Effects of Platelet Activating Factor Receptor Antagonist SY0916 on Macrophage

目的考察血小板激活因子(platelet activating factor,PAF)受体拮抗剂SY0916对巨噬细胞释放血管内皮生成相关因子的影响,探讨SY0916抗血管生成的相关分子机制。方法采用Boyden Chamber法检测小鼠腹腔巨噬细胞的趋化反应;放射性免疫分析法检测巨噬细胞U937上清中白细胞介素(IL)-1β含量;L929生物测定法分析肿瘤坏死因子(TNF)-α分泌;ELISA法测定血管内皮细胞生长因子(VEGF)含量;明胶酶谱法检测间质金属蛋白酶(MMP)-9活性;Western Blot法检测MMP-9蛋白表达;RT-PCR法观察IL-1β、TNF-α、VEGF mRNA的表达;凝胶电泳迁移率变更法(EMSA)分析核转录因子(NF)-κB活性。结果 SY0916对PAF诱导的小鼠腹腔巨噬细胞趋化反应具有显著的抑制作用;SY0916可呈剂量依赖性地抑制PMA刺激人U937巨噬细胞引起的IL-1β、TNF-α、VEGF的生成以及IL-1β、TNF-α、VEGF的mRNA表达;SY0916能显著抑制U937细胞MMP-9的活性及蛋白表达;SY0916明显抑制NF-κB的活化。结论 PAF受体拮抗剂SY0916可能通过抑制巨噬细胞NF-κB的活性而下调促血管生成因子和MMP的表达发挥抗血管生成的作用。

ABSTRACT： OBJECTIVE To investigate the in vitro anti-angiogenesis effect of platelet activating factor（PAF） receptor antagonist SY0916 on macrophage stimulated with PMA, and to analyze the anti-angiogenesis molecular mechanism of SY0916. METHODS The chemotaxis of primary mouse peritoneal macrophages induced by PAF and the inhibitory effect of SY0916 on macrophage was investigated using Boyden Chamber assay. The contents of IL-1β, TNF-αand VEGF induced by PMA in U937 macrophage cell supernatant were measured in turn by radio immunoassay, bioassay and ELISA, and the activity and amount of MMP-9 were determined by zymography and Western Blot. The mRNA expressions of IL-113,TNF-α and VEGF were detected by RT-PCR,and the effect of SY0916 on DNA binding activity of NF-κB was analyzed by EMSA respectively. RESULTS It was shown that SY0916 had significant inhibitory effect on the chemotaxis of mouse peritoneal macrophage induced by PAF. The protein and mRNA expressions of IL-β,TNF-α, VEGF and MMP-9 were beth inhibited by SY0916 in a dose-dependent manner. And the activity of NF-KB was also inhibited by SY0916. CONCLUSION The anti-angiogenesis effect of SY0916 on macrophage may be related to down-regulating the expressions of IL-β,TNF-α VEGF and MMP-9 through blocking the DNA binding activitv of NF-κB in inflammatory condition.