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

ObjectiveTo 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.MethodsForty-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.ResultsCompared 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 densityIOD)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.ConclusionMn 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.