【作者】 王姝；

【导师】 吴春福；

【作者基本信息】 沈阳药科大学 ， 药理学， 2008， 硕士

【摘要】 阿尔茨海默病(Alzheimer’s disease,AD)是一种常见的原发性老年痴呆疾病,AD的炎症机制学说逐渐形成并引起了国内外学者的热切关注。作为脑内常驻的免疫和吞噬细胞,小胶质细胞在AD炎症中的作用及其与神经元细胞变性的关系已成为近年来研究的焦点。活化的小胶质细胞可以通过氧化应激损伤、兴奋毒性、细胞因子损伤等多种途径导致神经元的功能失调及死亡。因此,通过研究活化的小胶质细胞对神经元损伤的具体通路可以为寻找安全可靠的防治AD的药物提供理论依据。本文建立了脂多糖(LPS)活化小胶质细胞引起兴奋性氨基酸的释放以及LPS活化小胶质细胞导致神经元DNA损伤的两个模型,并以此模型考察了米诺环素及9种已知能够抑制小胶质细胞活化的天然成分对兴奋性氨基酸释放的抑制作用和对神经元DNA损伤的保护作用。首先,采用高效液相结合荧光检测器的方法研究了LPS活化原代小胶质细胞外液中谷氨酸(Glu)水平的变化,发现活化的小胶质细胞Glu的释放量显著增加,此作用可分别被谷氨酸/胱氨酸反向转运体的抑制剂L-2-aminoadipic acid(AAA)、谷氨酰胺酶抑制剂6-diazo-5-oxo-L-norleucine(DON)显著性抑制,表明异常活化的小胶质细胞主要通过谷氨酰胺酶和胱氨酸/谷氨酸反向转运体(Xc~-)释放Glu,参与对神经元的损伤过程。在评价天然成分对小胶质细胞外液中兴奋性氨基酸浓度的影响时,将天然成分和LPS共同作用于小胶质细胞,结果表明,阳性药米诺环素、人参皂苷Rd、Rg1、PF11显著降低细胞外液中Glu的含量,对天冬氨酸(Asp)和谷氨酰胺(Gln)的含量无显著影响;人参皂苷RT5显著降低细胞外液中Asp、Glu的含量,增加Gln的含量;人参皂苷Re、葡萄提取物Res、Pro、polyphenol显著降低细胞外液中Glu的含量,增加Gln的含量,对Asp的含量无显著影响;人参皂苷Rb2对细胞外液中Asp、Glu的含量无影响,但显著增加Gln的含量。然后,采用单细胞凝胶电泳技术考察了LPS活化的小胶质细胞导致的神经元DNA的损伤。与LPS条件培养液(L-CM)组比较,AAA和DON的条件培养液显著降低大鼠皮质神经元DNA的损伤,表明小胶质细胞释放Glu介导的兴奋性毒性参与到了L-CM对神经元的损伤作用。同时,NMDA受体拮抗剂MK-801本身对神经元DNA没有影响,但显著降低L-CM对神经元DNA的损伤,表明小胶质细胞异常活化释放的Glu,通过作用于神经元上的NMDA受体参与到了对神经元的损伤作用。将天然成分和LPS共同作用原代小胶质细胞后收集细胞外液作用于原代培养大鼠皮质神经元,结果表明,米诺环素及9种天然成分均显著性降低条件培养液造成的DNA损伤。米诺环素、人参皂苷Rd、Rb2、Re、RT5、PF11,葡萄提取物Res、polyphenol、Pro药物本身对正常原代大鼠皮质神经元DNA具有保护作用。为评价9种天然成分的抗氧化活性,考察了天然成分本身对DPPH自由基和丙二醛(MDA)的清除作用。结果表明,米诺环素、葡萄提取物Res、polyphenol、Pro具有较强的DPPH自由基清除能力;人参皂苷Rb2、Rg1、RT5、PF11,葡萄提取物Res、polyphenol、Pro具有一定的脂质过氧化产物MDA的清除作用。综上所述,活化的小胶质细胞能够通过释放的Glu参与到对大鼠皮质神经元DNA的损伤过程,米诺环素及9种天然成分通过不同的作用机制抑制小胶质细胞Glu的释放,对神经元DNA起到保护作用。但小胶质细胞条件培养液对神经元的损伤是多种机制共同作用的结果,其它可能的机制仍有待于进一步的探讨。更多还原

【Abstract】 Alzheimer’s disease(AD)is the most common type of primary demenia.Recent studies suggest that the appearance of activated-microglia is another characteristic of AD.Microglia, the main immune cell of the central nervous system(CNS),plays the critical role as resident immunocompetent and phagocytic cells in CNS.During the inflammatory processes in CNS, activated microlia can directly and indirectly induce the dysfunction and death of neurons through the oxidative stress,exitotoxicity,cytokine and so on.Thus,inhibition of microglia activation will be an effective therapeutic approach to alleviate the progression of diseases such as AD.According to the present study,models of the release of excitatory amino acids and the DNA damage of rat cortical neurons induced by lipopolysaccharides(LPS)-activated microglia were established.Then,we evaluated the effects of the 9 natural compounds which were extracted from ginseng or grape seed on the models,as well as the mechanisms of action of the active compounds.Firstly,the conditioned medium collected from normal or LPS-activated microglia(C-CM and L-CM,respectively)was used to estimate whether the release of excitatory amino acids (EAA)was related to the damage of rat cortical neurons.The concentration of EAA was determined by HPLC with fluorescence detector.The time-effect curve of the release of glutamate indicated that primary microglia treated with 1μg/ml LPS for 24 h increased the release of glutamate significantly.The release of glutamate from malactivated microglia was mainly conveyed by the Xc exchange sysrem.LPS-induced glutamate synthesis in microglia was primarily through the glutaminase pathway.In our study,the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine(DON)and the Xc exchange system inhibitor L-2-aminoadipic acid (AAA)suppressed LPS-induced glutamate release significantly. We estimated the effect of minocycline and 9 natural compounds on the model of EAA release.Compared with the LPS-treated group,minocycline,ginsenosides Rd,Rg1,PF11 attenuated microglial glutamate release and had no effect on the concentration of aspartate and glutamine.Ginsenoside RT5 depressed the aspartate and glutamate release and increased the content of glutamine.Ginsenosides Re,grape extracts resveratrol,procyanidine and polyphenol depressed the glutamate release,increased the content of glutamine and had no effect on the concentration of aspartate;Ginsenoside Rb2 increased the concentration of glutamine and had no effect on the aspartate and glutamate release.Then,we investigated the DNA damage of rat cortical neuron induced by malactivated microglia using the single cell gel electrophoresis(Comet assay),a simple and sensitive technique for genotoxicity studies.First,the LPS treated primary microglia-conditioned medium was collected with or without AAA,DON and natural compounds for 48 h,which was referred to as "NC-CM".Compared with the neurons treated with L-CM,the microglia-conditioned medium with AAA and DON depressed the DNA damage of rat cortical neuron.Meanwhile, MK-801,an antagonist of NMDA receptor,depressed the DNA damage of rat cortical neuron induced by L-CM.The result indicated that neuronal damage by LPS-activated microglia was likely to be excitoneurotoxicity through NMDA receptor signaling.We estimated the neuroprotective role of the 9 natural compounds on the DNA damage of rat cortical neuron induced by malactivated microglia.The results showed that the neuronal injury by L-CM can be protected by treating with the NC-CM of minocycline and 9 natural compounds.In addition,minocycline,ginsenosides Rd,Rb2,Re,RT5,PF11 and grape exteacts resveratrol,polyphenol,procyanidine had the protection on the DNA level of primary rat cortical neuron.Next,We detected the 1,1-diphenyl-2-picrylhydrazyl(DPPH-)radical and MDA scavenging effect of minocycline and 9 natural compounds.The result showed that minocycline, grape exteacts resveratrol,polyphenol and procyanidine had strong radical scavenging activity towards DPPH.Ginsenosides Rb2,Rg1,RT5,PF11,grape exteacts resveratrol,polyphenol and procyanidine had a certain lipid peroxidation scavenging activity towards MDA.In conclusion,the release of glutamate from malactivated microglia involved in the DNA damage of primary rat cortical neurons.The neuroprotective effects of Minocycline and 9 natural compounds against neuronal DNA damage induced by the LPS-activated microglia were confirmed.Both the glutaminase inhibitor and Xc exchange system inhibitors might be a novel effective strategy for the treatment of AD with minimum adverse side effects.更多还原