青蒿琥酯通过活性氧自噬途径增加小鼠巨噬细胞杀菌活性

目的 研究青蒿琥酯(artesunate,AS)通过增加小鼠巨噬细胞的自噬水平进而上调其杀菌活性的药理作用及机制.方法 分离及体外分化培养获得原代小鼠骨髓巨噬细胞.细菌计数实验分为大肠埃希菌(E.coli,EC)组、EC+ AS组和EC+AS+抑制剂组(3-MA和Ly294002),建立EC感染细胞模型,给予AS和抑制剂处理,采用平板培养法检测胞内活菌数量.自噬和活性氧检测实验分为medium组、AS组、AS+自噬抑制剂组和AS+活性氧清除剂组还原型谷胱甘肽(GSH)、N-乙酰半胱氨酸(NAC)],RT-PCR和Western blot法检测自噬体标志物的表达,荧光探针法检测胞内活性氧水平.结果 经定向分化后,获得形态良好的原代巨噬细胞.细菌计数实验结果显示,与EC组比较,EC+ AS组细胞胞内EC数量显著减少(P＜0.01),且具有剂量和时间效应关系.EC+ AS+自噬抑制剂组和EC+AS+活性氧清除剂组细胞胞内细菌较EC+ AS组显著升高(P＜0.01).自噬和活性氧检测结果显示,sAS组较medium组自噬基因LC3B和Atg5的mRNA和蛋白表达均明显升高,且具有剂量效应关系(P＜0.01).AS+自噬抑制剂组或AS+活性氧清除剂组自噬蛋白的表达较AS组明显降低,AS+活性氧清除剂组ROS水平较AS组也显著降低(P＜0.05).结论 青蒿琥酯能够增加巨噬细胞的杀菌活性,且该作用与其上调细胞活性氧水平,进而激活细胞自噬效应有关.

Artesunate enhances bactericidal activity in murine macrophages by activating ROS-autophagy pathway

Objective To investigate the pharmacological roles and mechanisms of artesunate (AS) in enhancing autophagy and promoting the bactericidal activity in murine macrophages.Methods Murine bone marrow derived macrophages (BMDMs) was isolated and cultured primarily.In the bactericidal study,BMDMs were divided into groups of E.coli (EC),EC + AS and EC + AS + autophagy inhibitor (3-MA and Ly294002s).The EC infection cell model was set up in the cultured BMDMs.Then AS and/or autophagy inhibitors were added to treat cells.Bacterial counts were detected by plate culture assay.For the detection of autophagy and reactive oxygen species (ROS),BMDMs were divided into control,AS,AS + autophagy inhibitors and AS + ROS scavengers reduced glutathione (GSH) and N-acetylcysteine (NAC)] groups.Then RT-PCR and Western blotting were used to detect the expression of autophagic markers at mRNA and protein levels,respectively.Fluorescence probe was employed to detect intracellular ROS levels.Results The obtained BMDMs were in good morphology after isolation and differentiation.In the bactericidal study,the EC + AS group demonstrated markedly less bacterial counts in EC infected BMDMs in time and dose dependent manners (P ＜ 0.01).The treatments of AS + autophagy inhibitors and AS + ROS scavengers resulted in significantly elevated bacterial counts,as compared with the treatment of AS alone (P ＜ 0.01).In the detection of autophagy and ROS levels,AS upregulated the mRNA and protein levels of LC3B and Atg5 in a dose dependent manner (P ＜ 0.01).While their expression levels were obviously decreased in the groups of AS + autophagy inhibitors and AS + ROS scavengers when compared to the AS group.What's more,AS + ROS scavengers also decreased ROS levels (P ＜ 0.05).Conclusion AS enhances the bactericidal activity in murine macrophages,which may be mediated by its increasing cellular ROS levels and thus activating cellular autophagy.