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目的探讨不同浓度锰暴露后小鼠脑黑质内活性氧(ROS)变化及其对Nrf2信号通路影响及相关机制。方法小鼠48只,随机分为4组,即对照组、低、中、高剂量染锰组,分别给予腹腔注射生理盐水及12.5、25和50 mg/kg MnCl2,连续2周。用流式细胞仪测定小鼠脑黑质细胞内ROS,免疫组化法检测核转录因子NF-E2相关因子2(Nrf2),蛋白伴侣分子Keap1,血红素氧化酶-1(HO-1)和醌氧化还原酶1(NQO1)表达,Western blotting检测Nrf2,Keap1,HO-1和NQO1的蛋白水平。结果与对照组比较,低、中、高剂量染锰组小鼠脑黑质细胞中ROS含量明显升高(P<0.05);免疫组化结果显示,与对照组比较,低剂量染锰组小鼠脑黑质Nrf2、HO-1、NQO1表达[分别为(4.46±0.83)、(10.10±2.33)、(8.15±0.24)]明显升高(P<0.05),Keap1表达(6.22±0.58)降低(P<0.05),高剂量染锰组结果与低剂量呈相反趋势;Western blotting结果显示,与对照组比较,低剂量染锰组小鼠脑黑质Nrf2、HO-1、NQO1蛋白表达量[分别为(0.85±0.13)、(0.76±0.12)、(0.86±0.14)]明显升高(P<0.01),Keap1蛋白表达(0.48±0.05)下降(P<0.05),高剂量染锰组结果与低剂量呈相反趋势。结论锰暴露可导致小鼠脑黑质细胞内ROS含量增加,低浓度锰能激活Nrf2,增加HO-1和NQO1表达,高浓度锰则会抑制Nrf2通路。
Objective To investigate the changes of reactive oxygen species (ROS) in brain substantia nigra of rats exposed to different concentrations of manganese and their effects on the Nrf2 signaling pathway and its related mechanisms. Methods Forty-eight mice were randomly divided into 4 groups: control group, low, middle and high dose manganese group, intraperitoneal injection of normal saline and 12.5, 25 and 50 mg / kg MnCl2 respectively for 2 weeks. The ROS in brain substantia nigra of mice were determined by flow cytometry. The expression of nuclear factor-kappa B (NF-2), Keap1, HO-1 and The quinone oxidoreductase 1 (NQO1) expression was detected by Western blotting. The protein levels of Nrf2, Keap1, HO-1 and NQO1 were detected by Western blotting. Results Compared with the control group, ROS levels in brain substantia nigra of low, medium and high dose MnO2 groups were significantly increased (P <0.05). The results of immunohistochemistry showed that compared with the control group, The expression of Nrf2, HO-1 and NQO1 in rat brain substantia nigra ([4.46 ± 0.83], (10.10 ± 2.33), (8.15 ± 0.24)] and Keap1 expression (6.22 ± 0.58) (P <0.05). The results of high-dose Mn-Zn treatment showed the opposite trend with low-dose MnO2. Western blotting showed that compared with the control group, the expression of Nrf2, HO-1 and NQO1 protein in brain substantia nigra of low- (0.85 ± 0.13), (0.76 ± 0.12) and (0.86 ± 0.14), respectively (P <0.01), while Keap1 protein expression (0.48 ± 0.05) decreased significantly (P <0.05) Contrary to the low dose. Conclusion Manganese exposure can increase ROS content in brain substantia nigra cells. Low concentrations of Mn can activate Nrf2, increase the expression of HO-1 and NQO1, while high concentrations of Mn can inhibit Nrf2 pathway.