摘要
现代社会中,锰元素被广泛用于冶金、电池以及杀虫剂等工农业生产中,特别是作为新一代的汽油防爆剂使用,使得环境中锰的含量逐渐升高,锰的毒性危害也日益受到人们的重视。研究表明,长期接触锰元素能够引起基底神经节和锥体外系统的神经毒性,并且能够增加帕金森疾病的发病几率。
目前为止,锰对神经系统的毒性作用机制尚不十分清楚。以往对锰神经毒性的研究,主要关注锰对神经元的直接损伤和对星形胶质细胞功能的影响上,而中枢神经系统中的小胶质细胞的作用却一直被忽视。小胶质细胞是脑内具有巨噬细胞类似功能的非特异性免疫细胞,在维持中枢神经系统的正常功能中发挥重要作用。过度激活的小胶质细胞释放大量的神经毒性因子,如一氧化氮(NO)、活性氧簇(ROS)、肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)和白介素6(IL-6),以及谷氨酸(Glu)等兴奋性氨基酸,可诱发或参与多种神经退行性疾病的发生。因此,本论文以NO、Glu和胞内ROS为检测指标,考察了氯化锰(MnCl2)单独作用和与细菌脂多糖(LPS)联合作用对小胶质细胞活化的影响,并进行相关的机制探讨；还考察了米诺环素等四种单体化合物对MnCl2诱导活化的小胶质细胞NO释放的影响,为研究锰神经毒性的机制和临床药物干预提供理论依据。
首先,MTT的结果发现,MnCl2(300、1000μM)影响N9小胶质细胞的存活率。实验选用MnCl2(10、30、100μM)进行后期实验。接着,运用Griess法检测MnCl2对N9细胞NO释放的影响,结果显示单独MnCl2不能引起N9细胞NO的释放,但是能显著增加LPS诱导的N9细胞NO释放。说明MnCl2对小胶质细胞NO释放的增加,需要小胶质细胞的活化为前提。10 ng/mL的LPS对N9细胞NO的释放没有影响,但与MnCl2合用后,能够显著刺激N9细胞释放NO,说明MnCl2能够使阈下浓度的LPS诱导N9细胞释放NO。实验还发现,提前给予LPS 3 h不能使单独的MnCl2具有刺激N9细胞释放NO的能力；但是提前给予MnCl23 h,LPS对N9细胞的NO释放比单独LPS刺激的NO释放量高。
采用原代培养的大鼠小胶质细胞验证以上实验结果。结果显示,MnCl2单独作用,不能增加大鼠原代小胶质细胞NO释放,但是能显著增强LPS诱导的小胶质细胞NO的释放。这些结果都与MnCl2作用于N9小胶质细胞系的结果一致。
采用高效液相结合荧光检测器的方法研究了MnCl2和MnCl2+LPS两种模型活化N9细胞释放谷氨酸(Glu)水平的变化。首次发现了MnCl2能够刺激N9细胞释放谷氨酸,此作用可被谷氨酸/胱氨酸反向转运体(Xc转运体)的抑制剂L-a-aminoadipic acid (AAA)显著抑制。并且,MnCl2和LPS联合作用对N9细胞谷氨酸的释放有协同增加作用。本文进一步研究了MnCl2促进N9细胞谷氨酸释放的机制,发现MnCl2作用N9细胞早期,能够显著增加细胞内ROS的含量以及降低细胞内GSH水平。抗氧化剂NAC不但能够抑制细胞内ROS的增加,提高细胞内GSH水平,而且能够明显抑制MnCl2刺激的谷氨酸释放。RT-PCR的结果也证实,MnCl2能够显著增加小胶质细胞Xc转运体mRNA的表达。以上结果说明,小胶质细胞的氧化应激和Xc转运体的激活可能均参与了氯化锰诱导的N9细胞谷氨酸的释放。
最后,本文考察了药物对MnCl2+LPS和LPS刺激N9细胞NO释放的抑制作用。结果表明,米诺环素,姜黄素以及白藜芦醇对两种模型都具有显著的抑制作用,但对两种模型的抑制率存在差异,可能是因为药物抑制小胶质细胞活化的主要分子靶点不同决定的。
综上所述,MnCl2对小胶质细胞NO的释放需要小胶质细胞的活化为前提。本文首次发现氧化应激和Xc转运体的激活参与了MnCl2刺激的小胶质细胞谷氨酸的释放。提示小胶质细胞释放的谷氨酸可能也参与了锰诱导的神经系统兴奋性毒性。此外,米诺环素、白藜芦醇和姜黄素能够抑制MnCl2与LPS联合诱导的N9小胶质细胞的活化。以上结果丰富了锰的神经毒性机制研究,并为寻找相应的防治药物提供了一定的理论依据。
Manganese,(Mn), used in many industries, is becoming a serious environmental contaminant. It has been established that neurons related to motor function can be damaged by Mn accumulation in the globus pallidus and striatum. There are increasing evidences indicating that exposure to excessive levels of Mn participates in numerous disorder diseases of human central nervous system (CNS) such as hyperactivity, chronic Parkinson's disease and manganism.
However, the underlying mechanism of Mn-induced neurotoxicity remains poorly understood. In past, the study on the toxicity of manganese used to focus on the neuron and astrocyte injury, while the microglial effects on the nerve toxicity by manganes were always neglected. Microglia are resident monocytes in the CNS, functionally similar to macrophages. They play a major role in homeostatic and reparative functions but unregulated response or over-activation of microglia can have disastrous neurotoxic consequences. This might be mediated by the microglial release of NO, ROS, TNF-a, IL-1β, IL-6 and glutamate (Glu), which is associated with several neurodegenerative diseases. The present dissertation evaluated the NO、ROS and Glu production in MnCl2-activated microglia, and studied on the mechanism of MnCl2-activated microglia. Then, we evaluated the effects of Curcumin; Resveratrol; Pseudoginsenoside-PF11 and Minocycline on the models above.
Firstly, the effect of MnCl2 on microglial cells viability was assessed using MTT assays. The result showed that 300 and 1000μM MnCl2 groups can significantly reduce the viability of N9 microglia cells, so the final MnCl2 concentration was 0-100μM for next experiments. Our results showed MnCl2 can potentiate NO production in LPS-activated microglia, which indicated that manganese could significantly increase NO production by activated microglial cells. Furthermore, MnCl2 with LPS in subthreshold concentration can induce the NO production in microglia. Exposure to LPS 3 h before MnCl2 activation, No significant nitrite production was observed, but exposure to MnCl2 3 h before LPS activation, compared to LPS lonely activation, the nitrite production was significant higher.
Besides the N9 microglial cell line, we next determined the effect of MnCl2 on the release of NO in primary rat microglia. Although, MnCl2 did not result in significant production of NO as measure, it can potentiate the NO production in LPS-activated microglia, which is similar to observation for the N9 microglial cell line.
The release of glutamate from MnCl2-stimulated N9 cells was examined by HPLC. we reported, for the first time that manganese chloride (MnCl2) significantly increases Glu release in a time-and concentration-dependent manner in N9 cells, which may be mediated by intracelluar ROS, mainly via xCT.
Finally, the effects of Curcumin, Resveratrol, Pseudoginsenoside-PF11 and Minocycline on the NO production in LPS/MnCl2+LPS-activated microglial cells were assessed by Griess reaction. It was found that the inhibition of Curcumin, Resveratrol and Minocycline were different in two models, which could be due to the different molecular pharmacological targets.
In conclusion, we first demonstrate that MnCl2 can induce sustained release of Glu in microglia which may be mediated by intracelluar ROS, mainly via xCT. Curcumin, Resveratrol and Minocycline could inhibit the activation of MnCl2-induced microglia. They may have therapeutic potential in the treatment of neurodegenerative diseases accompanied by microglial activation. These results may provide a novel insight into the potential mechanism of action for manganese-induced neurotoxicity.
引文
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