紫檀芪通过激活自噬缓解脊髓神经元细胞氧化应激损伤的作用及其机制  

Pterostilbene Inhibits ROS Production in Primary Spinal Cord Neurons by Activating Autophagy

作　　者：贺靖澜[1] 李宗虎[1] 董晓辉[1] 王小英[1] 刘月梅[1] 申娜[1] 

机构地区：[1]河北工程大学附属医院,邯郸056002

出　　处：《中国细胞生物学学报》2017年第8期991-999,共9页Chinese Journal of Cell Biology

基　　金：邯郸市科技局(批准号:1523108076-21)资助的课题~~

摘　　要：自噬在保护脊髓神经元细胞氧化应激损伤中具有重要的作用。紫檀芪(pterostilbene,PTE)是具有抗氧化作用的天然植物的提取物,但其对神经元细胞的作用及其机制尚不清楚。该文采用CCK-8分析PTE对大鼠原代脊髓神经元细胞的细胞毒性;不同浓度PTE作用神经元细胞24 h和48 h,透射电镜和Western blot检测微管相关蛋白轻链3-II(microtubule-associated protein 1 light chain3-II,MAP1LC3-II)、Beclin-1和P62蛋白质水平并分析自噬水平。PTE处理H2O2作用下的神经元细胞24 h,Western blot检测LC3-II水平,GFP-LC3转染观察自噬的数量。2′,7′-二氯二氢荧光素乙酰乙酸(2′,7′-dichloro-djhydrofl uorescein diacetate,DCFDA)和Mito SOX染色分析细胞活性氧(reactive oxygen species,ROS)水平,自噬相关基因5(autophagy related gene 5,ATG5)si RNA转染分析自噬在其中的作用。结果显示,20μmmol/L PTE对于神经元细胞无细胞毒性,PTE作用下神经元细胞中LC3-II、Beclin-1蛋白质水平呈剂量依懒性升高,而P62则呈剂量依懒性下降(P<0.05)。PTE增加H2O2作用下的神经元细胞中LC3-II蛋白质水平(P<0.05),自噬体数量增加,PTE可提高神经元细胞中自噬体数量。PTE明显降低神经元细胞中ROS水平,但ATG5 si RNA转染抑制自噬后显著逆转PTE的保护作用。该研究结果提示,PTE可能通过提高氧化应激状态下的脊髓神经元细胞自噬水平来抑制细胞ROS的产生。Autophagy is an important self-adaptive mechanism by inhibiting ROS（reactive oxygen species） in primary spinal cord neurons. Pterostilbene, a natural plant extract, has an antioxidant effect; however whether pterostilbene could protect spinal cord neurons from oxidative stress remains unclear. In the present study, primary spinal cord neurons of Sprague Dawley rats were seperated and cultured. CCK-8 analysis was used to detect the cytotoxicity. Spinal cord neurons were treated with pterostilbene in different doses for 24 h and 48 h, then LC3-II,Beclin-1 and P62 levels were determined by Western blot and the autophagy level was determined by transmission electron microscope. Primary spinal cord neurons were treated with H2O2 and pterostilbene for 24 h, then LC3-II level was determined by Western blot and the number of autophagosomes by GFP-LC3 analysis. DCFDA and Mito SOX Red staining were used to detect the ROS production in cells, and ATG5 si RNA transfection was used to analyze the involvement of autophagy. The results showed that there was a dose-dependent change in the levels of LC3-II, Beclin-1 and P62, and increased autophagosome number were observed in the pterostilbene-treated neurons（P〈0.05）. In addition, pterostilbene increased the level of LC3-II in cells treated with H2O2（P〈0.05）, and GFPLC3 analysis demonstrated the increased number of autophagosomes in pterostilbene-treated cells. Compared with that in the cells treated with H2O2, pterostilbene signifi cantly inhibited the ROS production; however, ATG5 si RNA transfection signifi cantly reversed the protection of pterostilbene. These results indicate that pterostilbene inhibits the ROS production in spinal cord neurons by activating autophagy.

关 键 词：脊髓神经元细胞 紫檀芪 自噬 氧化应激 

分 类 号：R651.2[医药卫生—外科学]