激活PPAR—γ对缺氧诱导的N9小胶质细胞迁移和炎症因子释放能力的影响

Role of peroxisome proliferator-activated receptor-γ in secretion of inflammatory cytokine and migration of N9 microglia cells induced by hypoxia

目的探讨激活过氧化物酶增殖物激活受体-γ（PPAR-γ）信号通路对缺氧诱导的小胶质细胞的迁移及炎症因子释放能力的调节作用及分子机制。方法将体外培养的N9小胶质细胞分为4组：常氧组、缺氧组、吡格列酮＋缺氧组（应用吡格列酮激活PPAR-γ信号通路）和T0070907（PPAR-γ信号通路抑制剂）＋吡格列酮＋缺氧组。提取各组细胞总蛋白，采用Western blotting检测缺氧诱导因子-1α（HIF—1α）蛋白表达水平。采用Transwell法检测各组细胞的迁移能力。采用免疫荧光染色在倒置显微镜下观察HIF-1α的表达和细胞形态变化。提取各组细胞总RNA，采用RT-PCR检测白介素-1β（IL-1β）、白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）等炎症因子mRNA表达水平。结果与常氧组N9小胶质细胞相比，缺氧组N9小胶质细胞HIF-1α蛋白表达水平明显升高，迁移能力显著增强，IL-1β、1L-6、TNF-α mRNA表达水平升高，差异均有统计学意义（P〈0．05）。与缺氧组N9小胶质细胞相比，吡格列酮＋缺氧组N9小胶质细胞HIF-1α蛋白表达水平明显降低，迁移能力显著减弱，IL-1β、1L-6、TNF-α mRNA表达水平降低，差异均有统计学意义（P〈0．05）。与吡格列酮＋缺氧组N9小胶质细胞相比，T0070907＋吡格列酮＋缺氧组N9小胶质细胞HIF-1α蛋白表达水平明显升高，迁移能力显著增强，IL-1β、1L-6、TNF-α mRNA表达水平升高，差异均有统计学意义（P〈0．05）。结论PPAR-γ信号通路被激活后可以通过下调HIF-1α蛋白表达和IL-1β、1L-6、TNF-α等炎症因子mRNA表达水平，从而抑制缺氧诱导的小胶质细胞的迁移及炎症因子释放能力。

Objective To explore the role of peroxisome prolifcrator-activated receptor （PPAR）-γ in hypoxia-induced secretion of inflammatory cytokine and migration of N9 microglia cells by activating PPAR-γ signaling with pioglitazone and its mechanism. Methods N9 microglia cells cultured in vitro were divided into normoxia group, hypoxia group, pioglitazone＋hypoxia group and T0070907 （PPAR-γ pathway inhibitor）＋pioglitazone＋hypoxia group. Total cell RNA and protein were prepared, Western blotting was employed to detect the protein expressions of hypoxia-inducible factor-1 （HIF-1α）. Real-time PCR was used to detect the interleukin （IL）- 1β, IL-6 and TNF-α mRNA expressions. The migration ability of N9 cells was detected by Transwell assay. The HIF-1α protein expression in N9 cells was detected by immunofluorescence. Results As compared with cells from normoxia group, cells from the hypoxia group had significantly increased HIF-la protein expression, markedly enhanced migration ability and significantly increased mRNA levels of IL-1β, IL-6 and TNF-α （P〈0.05）. As compared with those in the cells from the hypoxia group, the HIF-1α protein expression, migration ability and IL-1β,IL-6 and TNF-α mRNA levels were significantly decreased in the cells from pioglitazone＋hypoxia group （P〈 0.05）. T0070907＋pioglitazone＋hypoxia group had significantly increased HIF-1α protein expression, migration ability and IL-1β, IL-6 and TNF-α mRNA levels as compared with pioglitazone＋hypoxia group （P〈0.05）. Conclusion Activation of PPAR-γ pathway could inhibit the hypoxia-induced secretion of inflammatory cytokine and migration ability of N9 microglia cells via down-regulation of HIF-1α protein and IL-1β, IL-6 and TNF-α mRNA expressions.