锰对小鼠黑质内活性氧及Nrf2信号通路影响

Effects of Mn exposure on ROS levels and Nrf2 signaling pathway in substantia nigra of mice

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 evaluate the change of intracellular reactive oxygen species（ROS）levels in the substantia nigra of mice after exposed to different concentrations of manganese（Mn） and their impact on nuclear factor erythroid 2 related factor（Nrf2）signaling pathway.Methods Forty-eight mice were randomly divided into four groups：control,low-,medium and high-dose MnCl2 groups.The mice in the control group were intraperitoneally injected with 0.9% NaCl and the mice in the dose groups were injected intraperitoneally with 12.5,25 and 50 mg/kg MnCl2,respectively,once a day for two weeks.Intracellular ROS levels were measured by flow cytometry;the expressions of Nrf2,Kelch-like ECH-associated protein 1（Keap1）,heme oxygenase-1（HO-1）,and and NAD（p）dehydrogenase quinine 1 （NQO1）were determined by immunohistochemical analysis and the protein levels of Nrf2,Keap1,HO-1 and NQO1 were determined by Western blot.Results Compared with the control group,ROS levels elevated significantly in low-,medium-and high-dose Mn groups（P〈0.05）.The immunohistochemistry analyses showed that,compared with the control group,in low-dose Mn group,the expressions （integral optical density[IOD]）of Nrf2（4.46±0.83）,HO-1（10.10±2.33）and NQO1（8.15±0.24）in the substantia nigra of mice increased significantly （P〈0.01,P〈0.01,P〈0.05）;the expression （IOD）of Keap1（6.22±0.58）decreased（P〈0.05）,whereas the results of high-dose Mn group showed the opposite trend.The results of Western blot showed that,compared with the control group,the protein levles of Nrf2（0.85±0.13）,HO-1（0.76±0.12）and NQO1（0.86±0.14）increased significantly （P〈0.01 for all）,the expression of Keap1（0.48±0.05）decreased （P〈0.05）,and the results of high-dose Mn group also showed the opposite trend.Conclusion Mn exposure can lead to the elevation of ROS levels in the substantia nigra of mice.Low level Mn exposure can activate Nrf2 pathway and then increase the expression of HO-1 and NQO1,but high level of Mn exposure can inhibit Nrf2 pathway.