锰诱导BV2细胞炎症活化与线粒体自噬有关

Inflammatory activation of BV2 cells induced by manganese involving with mitophagy

目的:探究锰诱导的BV2细胞炎症活化是否与线粒体自噬有关。方法:小鼠小胶质细胞BV2予以不同浓度锰暴露(0μmol/L、50μmol/L、100μmol/L)12 h,并以1μg/mL脂多糖(LPS)为阳性对照组;刃天青法检测细胞活性;荧光探针检测溶酶体数量及荧光强度变化;透射电镜观察自噬变化;共聚焦显微镜观察溶酶体和线粒体荧光共定位的程度;蛋白免疫印迹(western blotting)实验检测不同浓度锰暴露12 h后细胞炎症蛋白NLRP3、自噬相关蛋白(p62、LC3-Ⅱ/Ⅰ)以及线粒体外膜蛋白VDAC1的表达水平;线粒体自噬抑制剂巴弗洛霉素A1预处理验证锰暴露对自噬流及上述炎症标志蛋白表达的影响。结果:BV2细胞染锰浓度≥50μmol/L时,BV2细胞存活率下降,NLRP3表达上调(P<0.01),溶酶体数量及与线粒体共定位显著增加(P<0.05);锰暴露组VDAC1和自噬标志蛋白LC3-Ⅱ/Ⅰ的蛋白表达水平下降,p62的蛋白表达水平升高(P<0.05);巴弗洛霉素A1预处理后,显著逆转除p62外的其他自噬标记蛋白表达(P<0.05)。结论:50~100μmol/L染毒剂量下,锰诱导BV2细胞炎症活化和线粒体自噬增加;巴弗洛霉素A1抑制线粒体自噬可促进锰暴露诱导的BV2细胞炎症活化。

Objective:To investigate whether manganese-induced inflammatory activation in BV2 cells is associ-ated with mitophagy.Methods:Mouse microglia cells BV2 were exposed to different concentrations of manga-nese(0μmol/L,50μmol/L and 100μmol/L)for 12 h,and 1μg/mL lipopolysaccharide(LPS)was used as a posi-tive control group.Cell vialibility was detected by Alarmarblue assay and changes of lysosomal number and fluo-rescence intensity were detected by fluorescent probe;autophagic changes were observed by transmission elec-tron microscopy;the extent of lysosomal and mitochondrial fluorescence co-localization was observed by confo-cal microscopy;western blotting was performed to detect the expression levels of cellular inflammatory proteins NLRP3,autophagy-related proteins(p62,LC3-II/I),and mitochondrial outer membrane protein VDAC1 after 12 h of exposure to different concentrations of manganese.Mitophagy inhibitor Bafilomycin A1 pretreatment veri-fied the effect of manganese exposure on autophagic flow and the aforementioned inflammatory hallmarks pro-tein expression.Results:When BV2 cells were exposed to manganese at concentrations greater than 50μmol/L,the BV2 cell survival rate was decreased,NLRP3 expression was increased(P<0.01),and the number of lyso-somes and their co-localization with mitochondria significantly were increased(P<0.05).The protein expression levels of VDAC1 and autophagy marker protein LC3-II/I were decreased and p62 was increased in the manga-nese-exposed group(P<0.05).Bafilomycin A1 pretreatment significantly reversed the autophagy marker protein expression except p62(P<0.05).Conclusion:Manganese induces inflammatory activation in BV2 cells and in-creases mitophagy at 50-100μmol/L exposure dose.Inhibition of mitophagy by Bafilomycin A1 can promote manganese exposure-induced inflammatory activation in BV2 cells.