掌叶半夏凝集素蛋白刺激巨噬细胞致炎作用研究.docVIP

掌叶半夏凝集素蛋白刺激巨噬细胞致炎作用研究 　　[摘要] 为探索掌叶半夏凝集素蛋白（PPL）刺激巨噬细胞诱导炎症的作用与炎症小体NLRP3的相关性。采用凝胶色谱纯化掌叶半夏凝集素蛋白并采用SDS凝胶电泳分析其纯度；采用ELISA法以IL-1β为指标考察PPL刺激巨噬细胞释放炎症因子的影响；采用荧光探针DCFH-DA荧光酶标仪检测PPL刺激巨噬细胞后活性氧ROS的水平变化；以ROS抑制剂NAC（N-乙酰半胱氨酸）预处理巨噬细胞，考察PPL刺激ROS的过量生成与炎症因子IL-1β大量释放的相关性；采用Western Blot方法检测PPL对炎症小体生成相关的Caspase-1 p20，NLRP3，TXNIP，ASC等蛋白的表达水平变化。结果显示分离纯化的PPL达到电泳纯；PPL刺激巨噬细胞后引起ROS过量释放，引起强烈的氧化应激反应，胞内炎症因子IL-1β含量显著升高；NAC可抑制PPL导致的ROS过量生成，且IL-1β释放也显著降低。同时PPL可诱导胞内Caspase-1 p20，NLRP3，ASC蛋白表达升高，TXNIP表达显著降低。研究结果表明，PPL刺激巨噬细胞可导致强烈的氧化应激反应，同时能够激活炎症小体NLRP3，导致IL-1β大量生成释放，即PPL能够通过ROS-TXNIP-NLRP3-IL-1β信号通路促进IL-1β的成熟和分泌，导致炎症级联反应。 　　[关键词] 掌叶半夏；凝集素；炎症；ROS；NLRP3；IL-1β；NAC 　　[Abstract] To investigate the mechanism of lectin from Pinellia pedatisecta（PPL） on macrophage-induced inflammation and its association with inflammatory corpuscles NLRP3. Lectin from P. pedatisecta was isolated and purified by gel chromatography， and its purity was analyzed by using SDSgel electrophoresis. ELISA was used to investigate the effect of PPL on inflammatory cytokines released by macrophages， with IL-1β as indicators；and fluorescence probe DCFH-DA fluorometer was used to determine changes in active oxygen ROS of macrophages after application of lectin from P. pedatisecta.RAW264.7 cells were pre-treated with ROS inhibitor N-acetylcysteine （NAC） to investigate the effect on ROS and the release of inflammatory factor IL-1β from macrophages to research the relationship between them. The protein levels of NLRP3， Caspase-1 p20， ASC and TXNIP were determined by Western blot.The results showed that isolated and purified PPL could reach electrophoretic purity；PPL stimulated macrophages and induced the excessive release of ROS， leading to strong oxidative stress reaction， and the levels of intracellular inflammatory factorsIL-1β were significantly increased. NAC could inhibit PPL-induced ROS excessive production and significantly reduce the release of IL-1β. In addition， PPL could induce the increase in protein expression levels of Caspase-