脱氧雪腐镰刀菌烯醇对小鼠巨噬细胞氧化应激及自噬的影响

Deoxynivalenol Induces Oxidative Stress and Autophagy in Mouse Macrophages

为测定脱氧雪腐镰刀菌烯醇(DON)对巨噬细胞氧化应激和自噬的影响,本试验选用小鼠单核巨噬细胞RAW264.7进行试验,使用CCK-8法、荧光染色法和Western blot分析不同浓度DON对RAW264.7细胞活性、活性氧产生水平、线粒体膜电位、抗氧化物活性及自噬和氧化应激通路相关蛋白表达的影响。结果显示,DON可呈时间和剂量依赖性抑制RAW264.7细胞增殖,并诱导活性氧含量增多,极显著抑制抗氧化酶活性(P<0.01);DON可引起线粒体膜电位缺失,Nrf2、HO-1蛋白表达极显著增多(P<0.01),自噬蛋白LC3II/LC3I、Beclin-1表达极显著增多(P<0.01),p-AKT、p-mTOR表达量极显著减少(P<0.01)。由此可见,DON可刺激Nrf2/HO-1信号通路诱导RAW264.7细胞氧化应激,使细胞的线粒体功能异常,并抑制AKT/mTOR信号通路诱导细胞自噬。

To determine the effect of deoxynivalenol(DON) on the oxidative stress and autophagy of macrophages, this study treated RAW264.7 cells with different concentrations of DON, and measured its cell activity, reactive oxygen species(ROS) production level, mitochondrial membrane potential, antioxidant activity and expression of autophagy and oxidative stress pathway related proteins by using CCK-8, fluorescence staining and Western blot. The results showed that DON inhibited cell proliferation in a time-and dose-dependent manner, increased the production of ROS and significantly inhibited the activity of antioxidant enzymes(P<0.01). DON induced mitochondrial membrane potential decrease, significantly increased Nrf2 and HO-1 protein(P<0.01), and increased expression of autophagy protein LC3 II/LC3 I, Beclin-1(P<0.01), and decreased expression of p-AKT and p-mTOR(P<0.01). Thus, DON can stimulate Nrf2/HO-1 signaling pathway to induce oxidative stress, abnormal mitochondrial function, and inhibit AKT/mTOR signaling pathway to induce autophagy in RAW264.7 cells.