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

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.目的： 观察低浓度百草枯（PQ）对小鼠小胶质细胞（BV2）的激活情况，探讨PQ对BV2细胞M1/M2表型变化的影响。 方法： 以BV2细胞为模型，用终浓度0、0.015、0.03、0.06、0.12、0.24、0.48 μmol/L PQ及0.05 μmol/L 1-甲基-4-苯基吡啶离子（MPP(+)）处理细胞24 h，CCK8法测定细胞存活率；用终浓度0、0.015、0.03、0.06、0.12 μmol/L PQ及0.05 μmol/L MPP(+)处理细胞24 h，酶联免疫吸附（ELISA）法测定细胞培养上清液中肿瘤坏死因-α（TNF-α）、白细胞介素（IL）-6、IL-1β含量，Transwell小室法测定细胞迁移能力，免疫荧光法和流式细胞仪测定细胞吞噬能力；用终浓度0、0.03、0.06、0.12 μmol/L PQ及0.05 μmol/L MPP(+)处理细胞24 h，蛋白免疫印迹（Western blot）法测定M1型BV2细胞标志物TNF-α、IL-6、IL-1β、一氧化氮合酶（iNOS）、CD86及M2型BV2细胞标志物Ⅰ型精氨酸酶（Arg-1）、甘露糖受体（CD206）的蛋白表达水平。 结果： 与0 μmol/L PQ组比较，0.03~0.12 μmol/L PQ组BV2细胞增殖活性均明显升高，0.48 μmol/L PQ组增殖活性明显抑制，差异均有统计学意义（P<0.05）；0.03 μmol/L PQ处理24 h的BV2细胞增殖活性明显升高（P<0.05）；ELISA结果显示，与0 μmol/L PQ组比较，0.03、0.06 μmol/L PQ组BV2细胞上清液中TNF-α、IL-6、IL-1β含量明显升高（P<0.05）；免疫荧光和流式细胞仪测定结果显示，与0 μmol/L PQ组比较，0.015、0.03、0.06 μmol/L PQ组细胞吞噬能力明显增强（P<0.05）；Transwell结果显示，0.03、0.06、0.12 μmol/L PQ组细胞侵袭能力较0 μmol/L PQ组明显升高（P<0.05）；Western blot结果显示，与0 μmol/L PQ组比较，0.03、0.06 μmol/L PQ组的M1型标志物TNF-α、IL-6、IL-1β、iNOS和CD86蛋白表达水平明显升高，0.06、0.12 μmol/L PQ组M2型标志物Arg-1、CD206蛋白表达水平明显降低（P<0.05）。 结论： 低浓度PQ可以异常激活BV2细胞，使得BV2细胞向促炎型M1型转化而抑制其向抗炎型M2型转化。.