目的: 建立氯化锰（manganese chloride, MnCl2）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MnCl2组（250、500、1 000 μΜ）,分别采用Griess assay、Western blot法检测一氧化氮（nitric oxide, NO）的浓度及诱导型一氧化氮合酶（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MnCl2对原代神经元分泌NO、iNOS、COX-2的影响。结果: 500、1 000 μΜ MnCl2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MnCl2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: MnCl2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 (MnCl2) and its influence on the neurodegenerative diseases such as Alzheimer's Disease (AD), we established a neuronal model of oxidative stress and inflammatory response induced by MnCl2. Methods: Primary neurons were successfully cultured using the cerebral cortex of Sprague Dawley (SD) rats at the age of 1~3 days old, SPF grade, In the experiments, the primary neurons were exposed to different concentrations of MnCl2. The cells in the dishes were randomly divided into 3 groups: negative group (Control group), positive group (100 ng/ml LPS), and MnCl2 groups with the low, medium, or high concentration (250, 500, 1 000 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 MnCl2 on NO, iNOS and COX-2 produced by primary neurons were observed. The parallel experiments of the primary mixed glia derived from the littermates of SD rats were done as well. Results: To compare to Control, NO was much higher in the MnCl2 groups at 500 or 1 000 μM (P < 0.05), while iNOS or COX-2 were upregulated in the increaseing of concentrations of MnCl2 at 24 h. However, there were no significant difference (P > 0.05) in the latter. Different concentrations of MnCl2 had no effect on the production of NO, iNOS or COX-2 by primary mixed glia. Conclusion: MnCl2 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.