锰介导大鼠原代神经元氧化应激及炎症损伤模型的建立  

Establishment of manganese-induced oxidative stress and inflammatory response model of primary neurons in rats

作　　者：李青宴 董宝莲 胡建英 宋超 郭玲 张洪江 LI Qing-yan;DONG Bao-lian;HU Jian-ying;SONG Chao;GUO Lin;ZHANG Hong-jian(Dali University,College of Pharmacy,Dali,Yunnan 671000,China;The Fourth People's Hospital of Kunming and the First People's Hospital of Anning,Anning,Yunnan 650302,China;The Seventh Affiliated Hospital of Dali University,Arning,Yunnan 650302,China)

机构地区：[1]大理大学药学院,云南大理671000 [2]昆明市第四人民医院暨安宁市第一人民医院,云南安宁650302 [3]大理大学第七附属医院,云南安宁650302

出　　处：《阿尔茨海默病及相关病杂志》2022年第1期16-21,共6页Chinese Journal of Alzheimer's Disease and Related Disorders

基　　金：2018年国家自然科学基金项目,地区基金项目(批准编号81760228);昆明市神经退行性疾病防护研究机构(项目编号2020-SW(研)-30);昆明市卫生健康委员会卫生科研课题(项目编号:2020-01-01-001)。

摘　　要：目的:建立氯化锰(manganese chloride,MnCl_(2))介导的SD大鼠大脑皮层原代神经元氧化应激及炎症损伤模型,以进一步探讨锰中毒的细胞机制及其对阿尔茨海默病(Alzheimer's Disease,AD)等神经变性疾病发生发展中的影响。方法:随机将用1~3 d龄SPF级新生SD大鼠大脑皮层培养的原代神经元分为阴性对照组(Control组)、阳性对照组(100 ng/ml LPS)及低、中、高浓度的MnCl_(2)组(250、500、1000 μM),分别采用Griess assay、Western blot法检测一氧化氮(nitric oxide,NO)的浓度及诱导型_氧化氮合酶(inducible nitric oxide synthase,iNOS)、环氧合酶2(cyclooxygenase-2,COX-2)蛋白表达水平;以SD大鼠大脑皮层原代混合胶质细胞作为对照,观察MnCl_(2)对原代神经元分泌NO、iNOS、COX-2的影响。结果:500、1000μM MnCl_(2)组可导致SD大鼠大脑皮层原代神经元分泌的NO浓度明显高于Control组(P<0.05);iNOS和COX-2蛋白表达水平随MnCl_(2)浓度的增加而升高,但与Control组比较差异均无统计学意义(P均〉0.05)。不同浓度的MnCl2对SD大鼠大脑皮层原代混合胶质细胞分泌的NO、iNOS、COX-2均无影响。结论:MnCl_(2)可致使SD大鼠大脑皮层原代神经元分泌的NO浓度及iNOS、COX-2表达水平升高,从而上调NO、iNOS引起的氧化应激反应及COX-2导致的炎症损伤,由此可致神经变性疾病,即锰的暴露可能是AD等神经变性疾病的危险因素。Objective:In order to further understand the cellular mechanisms of the toxicity caused by manganese chloride(MnCl_(2))and its influence on the neurodegenerative diseases such as Alzheimefs Disease(AD),we established a neuronal model of oxidative stress and inflammatoiy response induced by MnCl_(2).Methods:Primary neurons were successfiilly cultured using the cerebral cortex of Sprague Dawley(SD)tats at the age of 1〜3 days old,SPF grade,In the experiments,the primary neurons were exposed to different concentrations of MnCl_(2).The cells in the dishes were randomly divided into 3 groups:negative group(Control group),positive group(100 ng/ml LPS),and MnCl_(2) groups with the low medium,or high concentration(250,500,1000 μM).The production of nitric oxide(NO)was measured by Griess Assay,and the protein expression of inducible nitric oxide synthase(iNOS)or cyclooxygenase-2(COX-2)were tested by Western blot.The effects of MnCl_(2) on NO,iNOS and COX-2 produced by primary neurons were observed.The parallel experiments of the primaiy mixed glia derived from the littermates of SD rats were done as well.Results:To compare to Control,NO was much higher in the MnCl_(2) groups at 500 or 1000 μM(P<0.05),while iNOS or COX-2 were upregulated in the increaseing of concentrations of MnCl_(2) at 24 h.However,there were no significant difference(P>0.05)in the latter Different concentrations ofMnCl_(2) had no effect on the production of NO,iNOS or COX-2 by primary mixed glia.Conclusion:MnCl_(2) could induce the primary neurons to generate oxidative stress response including NO and iNOS upregulation and the inflammatory response such as COX-2 upregulation.Therefore the injury of manganese in the central nervous system could lead to the development of neurodegenerative diseases,that is,manganese exposure may be a risk factor to neurodegenerative diseases inculding AD.

关 键 词：氯化锰 原代神经元 一氧化氮 诱导型一氧化氮合酶 环氧合酶2 

分 类 号：R965[医药卫生—药理学]