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目的探讨锰对小鼠黑质GSH(glutathione)合成相关酶γ-GCS(γ-glutamylcysteine synthetase)和GSS(glutathione synthetase)的影响,为锰中毒机制的研究提供依据。方法小鼠64只,雌雄各半,依体重随机分为4组,每组16只,第1组为对照组,第2~4组分别为低、中、高剂量染锰组。第1组腹腔注射0.9%氯化钠,第2~4组分别腹腔注射12.5、25、50 mg/kg Mn Cl2,注射剂量为5 ml/kg,每天染毒1次,持续2周。最后一次染毒24 h后,将小鼠用水合氯醛麻醉后快速处死,冰浴条件下分离脑组织并取黑质;HE染色和透射电镜观察黑质组织形态及超微结构,Neu N和GFAP荧光双染观察神经元和星形胶质细胞分布及数量变化,试剂盒检测黑质内GSH含量,Western blotting法检测γ-GCS和GSS蛋白水平。结果随着各组染锰浓度升高,黑质组织形态和超微结构损伤逐渐加剧;免疫荧光发现胶质细胞增多,神经元减少;黑质内GSH水平下降,中剂量染锰组下降了53.82%,高剂量染锰组下降了68.36%;γ-GCS和GSS蛋白表达水平下降,其中γ-GCS在中、高剂量染锰组分别下降了7.8%、28.9%,GSS在中、高剂量染锰组分别下降了12.94%、32.94%。结论锰可通过干扰GSH合成相关酶γ-GCS和GSS的表达,影响GSH的合成,产生神经毒性。
Objective To investigate the effects of manganese on γ-glutamylcysteine synthetase (GSH) and glutathione synthetase (GSS) in the substantia nigra of GSH (glutathione synthase) in mice and provide a basis for the study of manganese poisoning mechanism. Methods Sixty-four mice, male and female, were randomly divided into four groups according to body weight, with 16 rats in each group. The first group was control group, and the second to fourth groups were low, middle and high dose of manganese dye. Group 1 received intraperitoneal injection of 0.9% sodium chloride. Groups 2 to 4 received intraperitoneal injections of 12.5, 25 and 50 mg / kg of MnCl 2 at a dose of 5 ml / kg once daily for 2 weeks. The mice were anesthetized with chloral hydrate immediately after the last exposure, and the brain tissue was isolated under ice bath and taken into the substantia nigra. HE staining and transmission electron microscopy were used to observe the morphology and ultrastructure of substantia nigra. Neu N and GFAP double staining was used to observe the distribution and quantity of neurons and astrocytes. The kit was used to detect GSH content in substantia nigra and the protein levels of γ-GCS and GSS were detected by Western blotting. Results As the concentrations of manganese in each group increased, the morphological and ultrastructural damage of substantia nigra increased gradually. Immunofluorescence showed that glial cells increased, neurons decreased, GSH level decreased in substantia nigra and decreased by 53.82 %, High-dose manganese group decreased by 68.36%; γ-GCS and GSS protein expression decreased, of which γ-GCS in medium and high doses of manganese group decreased by 7.8%, 28.9%, GSS in the medium and high doses of dye Manganese group decreased by 12.94% and 32.94% respectively. Conclusion Manganese can affect GSH synthesis and produce neurotoxicity by interfering with the expression of GSH and GSH related enzymes.