Effect of low concentration paraquat on activation of mouse microglia M1/M2 phenotypes

Abstract

Objective: To observe the effect of low concentration paraquat (PQ) on activation and phenotypic M1/M2 polarization of mouse microglia cells (BV2) . Methods: BV2 cells were used as model, and cultured in vitro were exposed to paraquat at designed concentrations of 0, 0.015, 0.03, 0.06, 0.12, 0.24, 0.48 μmol/L and 0.05 μmol/L 1-methyl-4-phenylpyridinium (MPP(+)) for 24 h, and cell viability was determined by CCK8 assay. After induced by 0, 0.015, 0.03, 0.06, 0.12 μmol/L PQ and 0.05 μmol/L MPP(+) for 24 h, the contents of tumor necrosis factor-α (TNF-α) , interleukin-6 (IL-6) and IL-1β in cell culture supernatant were determined by enzyme-linked inmunosorbent assay (ELISA) . Cell migration ability was determined by transwell. Immunofluorescence (IF) and flow cytometry were used to determine the phagocytic capacity of cells. Designed concentrations of 0, 0.03, 0.06, 0.12 μmol/L PQ and 0.05 μmol/L MPP(+) for 24 h, the protein expressions of M1 markers of BV2 (TNF-α, IL-6, IL-1β, Nitric oxide synthase-iNOS, CD86) and M2 markers of BV2 (Arginase type-1 Arg-1 and Mannose recepteor-CD206) were determined by Western Blot after PQ expourse (0, 0.03, 0.06, 0.12 μmol/L) and 0.05 μmol/L MPP(+) induction. Results: Compared with 0 μmol/L PQ group, proliferation activity of BV2 cells was significantly increased by 0.03~0.12 μmol/L PQ while inhibited by 0.48 μmol/L PQ (P<0.05) . The cell proliferation activity of cells treated with 0.03 μmol/L PQ was significantly increased in 24 hours (P<0.05) . ELISA showed that TNF-α, IL-6 and IL-1β contents in the cell supernatant of the PQ group were significantly higher than those of 0 μmol/L PQ group, especially in 0.03 and 0.06 μmol/L PQ exposed group (P<0.05) . The results of IF and flow cytometry showed that phagocytic capacity of 0.015, 0.03 and 0.06 μmol/L PQ group was significantly enhanced compared with 0 μmol/L PQ group (P<0.05) . Transwell showed that the cell invasion ability of 0.03, 0.06, 0.12 μmol/L PQ was significantly higher than that of 0 μmol/L PQ group (P<0.05) . Western blot showed that compared with 0 μmol/L PQ group, the expression levels of M1 markers TNF-α, IL-6, IL-1β, iNOS and CD86 were significantly increased in 0.03 and 0.06 μmol/L PQ exposed group, while the expression levels of M2 markers Arg-1 and CD206 protein were decreased in 0.06 and 0.12 μmol/L PQ exposed group (P<0.05) . Conclusion: Low concentration PQ can abnormally activate BV2 cell, making the transformation of BV2 cell into pro-inflammatory M1 type and inhibiting its transformation into anti-inflammatory M2 type.