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

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.

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。

环境锰暴露致可神经毒性已得到普遍公认。谷胱甘肽（GSH）耗竭是其诱发神经毒性的重要因素，而锰对GSH合成的影响及机制尚不明确。在中枢神经系统中，神经元和星形胶质细胞均可以利用前体物质谷氨酸（Glu）、半胱氨酸（Cys）及甘氨酸（Gly）在γ-谷氨酰半胱氨酸合成酶（γ-GCS）和谷胱甘肽合成酶（GSS）的催化下合成GSH。兴奋性氨基酸转运体（Excitatory amino acid transporters, EAATs）和Xc-系统（system Xc-）因其直接或间接具有转运Glu和Cys的功能，在促进GSH合成方面发挥重要作用。一类具有抗氧化特征的转录因子即核转录因子NF-E2相关因子2（nuclear factor-erythroid 2 related factor 2, Nrf2）可能会对EAATs和Xc-系统产生影响。本项目从Nrf2信号通路导致EAATs和Xc-系统调控GSH合成异常角度研究锰产生神经毒性的分子机制。本项目通过体内外实验相结合的方式，发现锰可致小鼠自主活动、转棒活动、纹状体组织形态损伤、神经元超微结构损坏、神经元退行性变性和神经细胞凋亡，并造成体外培养的神经元和星形胶质细胞活力下降和形态损伤。研究证实锰可使GSH水平下降、其合成前体物质Glu、Cys和Gly发生变化及合成相关酶r-GCS和GSS表达下降。并发现锰所致Glu和Cys的转运体EAAT1, 2, 3和xCT表达异常。应用PDC、DHK、MeβCD和SPAP证实EAATs和xCT与锰造成的GSH含量降低有关，证明EAATs和xCT在锰致GSH合成障碍过程中发挥重要作用。发现锰可改变Nrf2信号通路，且其变化趋势与锰致EAAT3和xCT的改变一致。而后，通过给予小鼠Nrf2激动剂SFN和拮抗剂INH，基因沉默神经元和星形胶质细胞的Nrf2表达，进一步证实锰可以通过干扰Nrf2对EAAT3和xCT的调控造成GSH的合成障碍。为阐明锰神经毒性机制及其防治提供依据。