Nrf2诱导EAATs和Xc-系统调控GSH合成在锰神经毒性中的作用及机制

环境锰暴露致脑内GSH耗竭是其诱发神经毒性的原因之一，但锰对GSH合成的影响及机制尚不明确。本项目在前期对锰致纹状体GSH耗竭、EAAT1、2转运Glu（GSH合成前体）障碍及Nrf2表达异常的研究基础上，提出锰可能是通过干扰Nrf2信号通路导致EAATs和Xc-系统调控GSH合成异常而产生神经毒性的科学假设。本项目拟用体内、外实验，观察不同浓度/时间锰暴露的神经毒性，评价其对GSH合成、EAATs和Xc-系统、Nrf2信号通路的影响及剂量/时间规律；给予PDC、DHK、MeβCD和SPAP以抑制EAAT1-3和xCT，明确这几种亚型对GSH合成的调控作用；检测Nrf2蛋白与EAAT1-3和xCT启动子DNA的结合，并用商品化Nrf2敲基因小鼠及Nrf2基因沉默的神经细胞，分析Nrf2通路对EAATs和Xc-系统的诱导作用。旨在阐明锰诱发神经毒性的机制，为锰中毒防治提供新的理论和实验依据。

In the environment, excessive manganese (Mn) exposure would caused glutathione (GSH) depletion, which was a factor in pathogeny of Mn neurotoxicity. However, the effect of Mn exposure on GSH synthesis and its related mechanism were unclear. Basing on the results of our previous study that Mn induced depletion of GSH, dysfunction of EAAT 1, 2 on transporttiong glutamate (Glu, the precursor of GSH synthesis), and disregulation expression of Nrf2 in striatum, we gave the hypothesis that Mn caused neurotoxicity through Nrf2-triggered EAATs and system Xc- to modulate GSH synthesis. The mice models in vivo and co-cultured neurons and astrocytes models in vitro will be used. In the Mn exposure model, the mice will be inhaled 0.2, 0.4, 0.8, 1.6, and 3.2 MnCl2/m3 for 1-4w. The co-cultured neurons and astrocytes will be exposed to 50-400 μmol/L MnCl2 for 0-72 h. After that, locomotor activity of mice, levels of Mn, histomorphology and ultrastructure of striatum, cell viability, LDH leakage, levels of GSH, Glu, cysteine (Cys), cystine (Cys2), glycine (Gly), and activities of γ-glutamyl cysteine synthetase (γ-GCS) and glutathione synthetase (GSS) will be measured. In order to find the roles of subtypes of EAATs and systems Xc- in modulation of GSH systhesis, PDC, DHK, MeβCD, and SPAP (the inhibitors of EAAT1-3, and xCT) will be given. Then, presursors of GSH synthesis, the related enzymes, the mRNA expression and protein levels of EAAT1-3 and xCT will be determined by Real-time PCR and Western Blotting. The uptake of [3H]-Glu, [52S]-Cys and [14C]-Cys2 will be measured. To observe the effect of Nrf2 pathway on EAATs and systems Xc-, the Nrf2 knocked-out mice and Nrf2 gene silenced co-cultured neurons and astrocytes will be used. The mRNA expression and protein levels of Nrf2, Keap1, and ARE, and their location and expression will be observed. Nrf2 protein binds to the ARE-related sequence of EAAT1-3 and xCT promoter on their DNA will be analyzed by CHIP methods. Meanwhile, precursors of GSH synthesis, the related enzymes, and expression and function of EAAT1-3, and xCT are going to be measured according the above methods. This study will clarify the mechanism of Mn neurotoxicity and provide theoretical and experimental basis on the prevention and therapy of manganism.