氧化型α1-抗胰蛋白酶对HBE细胞释放炎症因子的影响

 目的：探讨氧化型α1-抗胰蛋白酶（Ox-AT）对体外培养人支气管上皮(HBE)细胞炎症因子白细胞介素8（IL-8）和单核细胞趋化蛋白1（MCP-1）释放的影响及可能机制。方法：从人血浆中分离纯化获得天然构型AT（N-AT），加入氧化剂获得Ox-AT；用不同浓度N-AT和Ox-AT分别作用于体外培养的HBE细胞，用ELISA方法检测不同时段培养上清液中IL-8和MCP-1的含量，同时观察NF-κB抑制剂Bay11-7082对Ox-AT引起HBE细胞炎症因子释放的影响。结果：Ox-AT可促进HBE细胞释放IL-8和MCP-1，其促进作用与Ox-AT的浓度及作用时间呈正相关；用0.5 g/L Ox-AT孵育HBE细胞4、10和24 h，其促进HBE细胞分泌IL-8和MCP-1的作用与10 μg/L肿瘤坏死因子 α（TNF-α）的作用基本一致，而 N-AT无刺激HBE细胞分泌IL-8和MCP-1的作用；Ox-AT 能显著增加NF-κB活性； Ox-AT的促炎症作用能被NF-κB抑制剂Bay11-7082抑制。结论：Ox-AT是人正常支气管上皮细胞的强致炎因子，机制可能与NF-κB信号通路激活有关。

Oxidized α1-antitrypsin induces IL-8 and MCP-1 production in HBE cells

AIM:To investigate the effect of oxidized α1-antitrypsin (Ox-AT) on interleukin 8 (IL-8) and monocyte chemotactic protein 1(MCP-1) production in cultured human bronchial epithelial (HBE) cells. METHODS:Plasma native α1-antitrypsin (N-AT) was purified from human plasma by 50% and 75% ammonium sulfate fractionation followed by glutathione and anion exchange chromatography. Ox-AT was prepared by incubating N-AT (0.5 g/L) with N-chlorosuccinimide in a 25-fold molar excess to N-AT in PBS at room temperature for 30 min. HBE cells were cultured in the presence of Ox-AT (0.5 g/L) for 4 h, 10 h and 24 h, and the levels of IL-8 and MCP-1 in the supernatant were assayed using respective DuoSet kits. The effect of NF-κB inhibitor Bay11-7082 on the inflammatory cytokine release induced by Ox-AT was also evaluated. RESULTS:Ox-AT concentration-dependently and time-dependently increased the production of IL-8 and MCP-1 in HBE cells. The concentrations of IL-8 and MCP-1 in HBE cells induced by 0.5 g/L Ox-AT at 4 h, 10 h and 24 h were significantly higher than those in blank control and N-AT groups. Ox-AT increased the activity of NF-κB in a dose-dependent manner. The proinflammatory effect of by Ox-AT was inhibited by NF-κB inhibitor Bay11-7082. CONCLUSION: Ox-AT is a strong proinflammatory factor for HBE cells. The mechanism is related to NF-κB signaling pathway activation.