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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-PAGE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。
To explore the role of Pinellia ternate lectin (PPL) in stimulating macrophage-induced inflammation and inflammatory body NLRP3. The purity of PLG was determined by gel permeation chromatography (SDS-PAGE) and the purity of PLG was analyzed by SDS-PAGE. The effect of IL-1β on the release of inflammatory cytokines by PPL was measured by ELISA. Fluorescent probe DCFH-DA The level of reactive oxygen species (ROS) in macrophages stimulated by PPL was detected by fluorescence microplate reader. Macrophages were pretreated with ROS inhibitor NAC (N-acetylcysteine), and the overproduction of ROS and IL- 1β release in large numbers. The expression of Caspase-1 p20, NLRP3, TXNIP, ASC and other proteins related to the formation of inflammasome were detected by Western Blot. The results showed that purified PPL reached electrophoretic purity; PPL stimulated macrophages after excessive release of ROS, causing strong oxidative stress response, intracellular inflammatory factor IL-1β content was significantly increased; NAC can inhibit PPL-induced ROS over-production , And IL-1β release was also significantly reduced. At the same time PPL can induce intracellular Caspase-1 p20, NLRP3, ASC protein expression increased, TXNIP expression was significantly reduced. The results show that PPL stimulation of macrophages can lead to a strong oxidative stress response, while activating the inflammasome NLRP3, leading to a large number of release of IL-1β, PPL can be promoted through the ROS-TXNIP-NLRP3-IL-1β signaling pathway The maturation and secretion of IL-1β results in an inflammatory cascade.