Inflammatory activation of BV2 cells induced by manganese involving with mitophagy

Objective To investigate whether manganese-induced inflammatory activation in BV2 cells is associated with mitophagy.

Methods Mouse microglia cells BV2 were exposed to different concentrations of manganese (0 μmol/L, 50 μmol/L and 100 μmol/L) for 12 h, and 1 μg/mL LPS was used as a positive control group. Cell vialibility was detected by Alarmarblue assay a and changes of lysosomal number and fluorescence intensity were detected by fluorescent probe; autophagic changes was observed by transmission electron microscopy; the extent of lysosomal and mitochondrial fluorescence co-localization was observed by confocal 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 verified the effect of manganese exposure on autophagic flow and the aforementioned inflammatory hallmarks.

Results When BV2 cells were exposed to manganese at concentrations greater than 50 μmol/L, the BV2 cell survival rate was decreased, NLRP3 expression was increased (P＜0.01), and the number of lysosomes 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 manganese-exposed group (P＜0.05). Bafilomycin A1 pretreatment significantly reversed the autophagy marker proteins except p62 (P＜0.05).

Conclusion Manganese induces inflammatory activation in BV2 cells and increases mitophagy at 50-100 μmol/L exposure dose. Inhibition of mitophagy by Bafilomycin A1 can promote manganese exposure-induced inflammatory activation in BV2 cells.