【作者】 王芳；

【导师】 孙圣刚；

【作者基本信息】 华中科技大学 ， 神经病学， 2011， 博士

【摘要】 第一部分阿司匹林对脂多糖诱导帕金森病模型多巴胺神经元的保护作用目的：探讨阿司匹林（aspirin. ASA）对脂多糖（lipopolysaccharide, LPS）诱导的帕金森病细胞模型中多巴胺能神经元损伤是否具有保护作用,并初步探讨其可能的作用机制。方法：采用胚胎大鼠中脑原代细胞培养法,分别建立原代中脑神经元-胶质细胞,神经元细胞,神经元-星型胶质细胞和神经元-小胶质细胞培养体系。在原代中脑神经元-胶质细胞中给予不同浓度ASA（0.01、0.1、和1mmol/L）预处理1h后,给予脂多糖（10ng/ml）,培养七天后,免疫细胞化学法检测酪氨酸羟化酶阳性神经元的数目,western-blot法检测酪氨酸羟化酶（Tvrosine Hvdroxvlase. TH）在蛋白水平的表达：在原代中脑神经元细胞,神经元-星型胶质细胞和神经元-小胶质细胞体系中ASA（lmmol/L）预处理1h后,LPS （1Ong/ml）作用七天,采用免疫细胞化学法检测空白对照组,ASA组,ASA+LPS组和LPS组中酪氨酸羟化酶阳性神经元数目。结果：在LPS诱导的中脑原代多巴胺能神经元损伤中,LPS组中TH阳性细胞数明显低于ASA预处理组和空白对照组（P<0.05）,并且LPS组中TH阳性神经元突起与其他组相比明显减少甚至消失。ASA（0.01-1mmol/L）预处理能显著改变这一变化,且呈剂量依赖性；在原代中脑神经元细胞培养体系中,空白对照组和各实验组中TH阳性细胞数并无显著性差异（P>0.05）；在原代神经元-星型胶质细胞系中,LPS组与对照组相比,TH阳性细胞明显减少（P<0.05）,但是LPS+ASA组与LPS组相比较,TH阳性细胞数无显著性差异（P>0.05）；在原代神经元-小胶质细胞培养体系中,与空白对照组相比,LPS作用后可显著减少TH阳性细胞数（P<0.05）,并且ASA预处理后可显著减轻LPS对多巴胺能神经的损伤作用（P<0.05）,提示ASA对多巴胺能神经元的保护作用是通过小胶质细胞介导的。结论：ASA对脂多糖诱导的中脑多巴胺能神经元损伤具有保护作用,并且此作用是由小胶质细胞介导的。第二部分阿司匹林对脂多糖诱导的帕金森病细胞模型中炎症因子的调节作用目的：本实验采用胚胎大鼠中脑原代神经元-胶质细胞混合培养体系,应用脂多糖建立多巴胺能神经元损伤的炎症机制模型,研究ASA对炎症因子表达的影响,探讨其神经保护机制。方法：在原代培养中脑神经元-胶质细胞中,加入不同浓度ASA（0.01-1mmol/L）预处理1h后,给予LPS（10ng/ml）共培养七天后,检测促炎症因子,包括肿瘤坏死因子-α（TNF-a） （ELISA kit）,一氧化氮（NO） （Griess法）,超氧化物（WST-1法）,和胞内活性氧（reactive oxygen species, ROS） （DCFH-DA）;抗炎症因子,包括IL-10（ELISA kit）和TGF-β1 （ELISA kit）的表达变化。结果：0.01、0.1、1mmol/L ASA+LPS组中NO, TNF-α,超氧化物和胞内ROS的浓度均较LPS组明显降低（P<0.05）；同时,0.01、0.1、1mmol/L ASA+LPS组中IL-10和TGF-β1的浓度与LPS组相比较均有显著增高（P<0.05）。结论：ASA能够抑制LPS诱导的PD细胞模型中促炎症因子表达和促进抗炎症因子生成,这可能是ASA保护多巴胺能神经元的机制之一。第三部分阿司匹林对脂多糖诱导的帕金森病模型中NADPH氧化酶的调节作用目的：本实验主要研究阿司匹林对脂多糖诱导的帕金森病细胞模型中NADPH氧化酶的影响,进一步探讨其神经保护作用机制。方法：采用胚胎大鼠中脑原代细胞培养法,建立原代中脑神经元-胶质细胞混合培养体系。ASA （1mmol/L）或NADPH氧化酶抑制剂Apocynin （APO,0.25mmol/L）预处理1h后,给予LPS（10ng/ml）培养七天后,免疫细胞化学法检测酪氨酸羟化酶阳性神经元的数目,western-blot法检测胞膜中P47^phox在蛋白水平的表达。结果：TH免疫细胞化学法显示LPS作用后可显著减少TH阳性细胞数（与空白对照组相比,P<0.05）,ASA或APO预处理后,能显著增加TH阳性细胞数,ASA+LPS组,APO+LPS与LPS组相比较,均有显著性差异（P<0.05）。这一结果提示ASA和APO都具有保护多巴胺能神经元的作用,已知APO能阻止NADPH氧化酶胞浆亚单位(包括P47^phox与胞膜亚单位结合形成有活性的NADPH氧化酶复合体,抑制NADPH氧化酶活性。因此我们进一步采用Western-blot法检测空白对照组和各实验组中胞膜蛋白中P47^phox表达变化,研究结果提示LPS可增加P47^phox在胞膜的表达,ASA+LPS组,APO+LPS组与LPS组相比较,均显著减少P47^phox在胞膜中的表达水平（P<0.05）,从而抑制了NADPH氧化酶的活性。结论：阿司匹林能显著抑制NADPH氧化酶胞浆亚单位P47^phox在胞膜中的表达,从而抑制了NADPH氧化酶的活性。我们的研究率先表明在脂多糖诱导的原代中脑混合细胞PD模型中,阿司匹林可以通过抑制NADPH氧化酶活性,对抗过度炎症反应的毒性作用,从而保护多巴胺能神经元,这为探讨阿司匹林的神经保护作用机制提供了一个新的视角。更多还原

【Abstract】 Part IAspirin protects rat primary dopaminergic neurons induced by lipopolysaccharidesObjective:To investigate the effects of aspirin （ASA） on the protection of rat primary dopaminergic neurons induced by lipopolysaccharides （LPS）. and to explore the possible underlying mechanisms.Methods:We co-cultured the primary neuron-glia cells. The co-cultured cells were divided into 5 groups:control group, LPS group （lOng/ml）. LPS+ASA （0.01.0.1. 1mmol/L） group. The positive cells of tyrosine hydroxylase （TH） and the TH protein expression were detected by immunohistochemistry and western-blot. Further, we developed different primary cell cultures:neuron-enriched cultures, neuron-astrocyte cultures and neuron-enriched cultures supplemented by 10% microglia. They were randomly divided into 4 groups:the control group, ASA （lmmol/L） group, ASA （1mmol/L）+LPS （1Ong/ml） gruoup, LPS （lOng/ml） group. The number of the survival TH positive cells was evaluated by immunocytochemistry.Results:The number of TH positive neurons in the group of LPS was less than that in control group; the adding of different dose of ASA significantly reversed these changes caused by LPS. Morphological observation showed relatively short neuronal dendrites, and 1mmol/L ASA successfully reversed the change. Western-blot showed that the protein of TH in ASA+LPS group was more than that in LPS group （p<0.05）. In the three different cell component cultures, we counting the number of TH positive neurons after immunostanning. it showed that LPS did not affect the DA neuron survival in neuron-enriched cultures; in the neuron-microglia cultures, The number of TH positive neurons in the group of LPS was less than that in control group, and lmmol/L ASA successfully reversed the change.Conclusion:ASA can protect embryonic rat mesencephalic DA-ergic neurons against LPS induced neurotoxicity in a dose-dependent manner, and this effect is likely to mediate through microglia activation.Part IIEffect of ASA on LPS-induced production and release of reactive oxygen species and proinflammatory and anti-inflammatory factorsObjective:To investigate the regulation of ASA on the production of inflammatory factors and reactive oxygen species （ROS） in primary neuron-glia co-cultures, and to explore the mechanisms underlying the neuroprotective role.Methods:Primary cultured cells were dissociated from embryonic rat mesencephalon and the cell cultures were treated by addition of ASA （0.01-lmmol/L） and LPS （10ng/ml）. The production of superoxide was determined by measuring the SOD-inhibitable reduction of WST-1, intracellular ROS were determined by using a DCFH-DA assay. The production of nitric oxide （NO） was assessed as the accumulation of nitrite in the supernatants using Griess reagent; TNF-a. IL-10 and TGF-β1 concentrations in cell supernatants were measured using a commercial ELISA kit.Results:The production of ROS, NO, TNF-a significantly increased after stimulated by LPS, pretreatment with 0.01-1 mmol/L ASA significantly reversed these changes caused by LPS （p<0.05）. The amounts of IL-10 and TGF-β1 were prominently increased after addition of ASA.Concludion:ASA acted on microglia principally by down-regulating the production of ROS and pro-inflammatory factors and up-regulating the production of anti-inflammatory cytokines to exert its neuroprotective role.Part IIINADPH oxidase plays an important role in ASA-mediated protection against LPS-induced neurodegenerationObjective:To investigate the effects of ASA on the activity of nicotinamide adenine dinucleotide phosphate oxidase （NADPH oxidase, PHOX） in the LPS induced neurotoxicity.Methods:We developed the embryonic rat mesencephalic neuron-glia culture, and the cultured cells were randomized into groups of control, LPS （10ng/ml）, ASA（1mmol/L）+LPS（10ng/ml）, apocynin（APO,0.25mmol/L）+LPS（10ng/ml）. The survival number of TH positive cells was examined by immunocytochemistry. The western-blot was used to detect the levels of P47^phox on the cytomembrane.Results:The numbers of TH positive cells in ASA+LPS group and APO+LPS group were more than that in LPS group （p<0.05）. Western-blot showed that the cytomembrane protein of P47^phox in ASA+LPS group and APO+LPS group were lower than that in LPS group.Conclusion:Pretreatment with ASA could protect rat primary DA neurons induced by LPS. The effect may be contributed to ASA-blocked activity of PHOX induced by LPS.更多还原