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目的从人肺泡上皮细胞甲酰肽受体激动剂——膜联蛋白A1(Annexin A1,AnxA1)的表达变化,探讨植物雌激素影响缺氧肺部炎症浸润的机制。方法人肺泡Ⅱ型上皮细胞(A549)接种于60 mm培养皿中常规培养,待培养皿中细胞长满约70%,根据是否用药分为:对照组(溶剂DMSO对照)、染料木黄酮(genistein,Gen)组、大豆苷元(daidzein,DD)组,每组细胞再根据是否缺氧再分为常氧组(N)、缺氧组(H)。Gen组、DD组于缺氧处理前分别加入Gen及DD,终浓度均为15μmol/L。缺氧组细胞置于缺氧培养箱(1%O2)24 h。取细胞培养上清及细胞裂解离心上清,结合甲酰肽受体特异性抑制剂tBoc2,通过趋化实验,检测其甲酰肽受体激动活性。用Western blot分析AnxA1、缺氧诱导因子-1α(HIF-1α)、核因子-κB(NF-κB)在蛋白水平的变化,用RT2-PCR法分析其mRNA水平的变化。结果缺氧后的细胞冻融上清及培养上清趋化活性显著高于常氧组(P<0.05),而植物雌激素处理后,两种上清的趋化活性均显著高于对照组(P<0.05)。Western blot与RT2-PCR检测结果显示,缺氧组AnxA1在蛋白水平和mRNA水平均高于常氧组(P<0.05),两种植物雌激素不同程度地削弱了缺氧对AnxA1的上调效果,同时对HIF-1α、NF-κB的蛋白水平也产生同样的影响。结论植物雌激素削弱了缺氧对AnxA1基因表达的上调效应,其机制与HIF-1α、NF-κB信号途径有关。
OBJECTIVE: To study the mechanism of phytoestrogen affecting inflammatory infiltration in hypoxic lungs from the change of human alveolar epithelial-derived formyl peptide receptor agonist, Annexin A1 (AnxA1). Methods Human alveolar epithelial cells type Ⅱ (A549) were inoculated into 60 mm culture dishes. When the culture dish grew to about 70%, the cells were divided into control group (solvent DMSO control), genistein , Gen group and daidzein (DD) group. The cells in each group were further divided into normoxia group and hypoxia group according to whether they were hypoxic or not. Gen and DD groups were added Gen and DD respectively before hypoxia treatment, and the final concentration was 15μmol / L. Hypoxia group cells were placed in hypoxia incubator (1% O2) 24 h. Cell culture supernatants and cell lysate supernatants were harvested, combined with tBoc2, a specific inhibitor of formyl peptide receptor, to determine the agonist activity of formyl peptide receptor by chemotaxis assay. Western blot was used to analyze the changes of AnxA1, HIF-1α and NF-κB in the protein level, and RT2-PCR was used to analyze the mRNA level of AnxA1. Results The chemotactic activity of supernatants and culture supernatants after hypoxia were significantly higher than that of normoxia group (P <0.05). After phytoestrogen treatment, the chemotactic activities of both supernatants were significantly higher than those of control group (P <0.05). The results of Western blot and RT2-PCR showed that the level of AnxA1 in hypoxia group was higher than that in normoxia group (P <0.05). The two phytoestrogens reduced the effect of hypoxia on AnxA1 up-regulation, At the same time on the HIF-1α, NF-κB protein levels have the same impact. Conclusion Phytoestrogen attenuates the up-regulation of AnxA1 gene expression induced by hypoxia, and its mechanism is related to the signaling pathway of HIF-1α and NF-κB.