【作者】 于嵩；

【导师】 王占友；

【作者基本信息】 中国医科大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 前言帕金森病(Parkinson’s disease, PD)是一种以黑质中多巴胺(dopamine, DA)能神经元变性坏死为主要病理特征的退行性中枢神经系统疾病,其主要临床表现为静止性震颤、肌强直、运动迟缓及姿态反射障碍。PD黑质细胞死亡的原因尚不清楚,普遍认为与环境毒素、遗传因素、基因突变、氧化应激、免疫异常、铁离子聚集和神经兴奋性毒性等诸多机制有关。阿尔茨海默病(Alzheimer’s disease,AD)是一种发生于老年及老年前期的以进行性痴呆为主要特征的神经系统退行性疾病,其主要临床特征是进行性记忆丧失、认知缺陷及严重的精神障碍。AD的主要病理学特征是脑部出现β-淀粉样蛋白(amyloid-p, Ap)沉积即老年斑(senil plaque, SP)和神经纤维缠结(nerve fiber tangles, NFTs)。随着人们生活水平的提高和人口老龄化的加剧,以PD和AD为代表的大脑神经退行性疾病己成为仅次于心血管疾病、恶性肿瘤和中风之后的主要致死疾病,给患者和社会造成严重的负担。因此,解析神经退行性疾病的发病机理以及寻找此类疾病的治疗策略已经成为神经科学研究领域的热点之姜黄素为常用传统中药,来源于姜科姜黄素属植物的干燥根茎,也是咖哩、芥末中的主要黄色色素,有着重要的经济价值和广泛的药理作用。联合国粮农组织专家委员会和世界卫生组织已批准姜黄素为天然的食用色素。研究发现姜黄素作为天然色素不仅可用于食品添加剂和香料成分,还对多种疾病具有预防和治疗功能。由于姜黄素安全性高且毒副作用小,其药理作用已经逐渐被医学科学工作者所关注。自从1937年第一篇关于姜黄素治疗人类疾病的论文发表在The Lancet期刊以来,至今已有2600多篇关于姜黄素的研究论文发表在英语杂志上。姜黄素具有多方面的药理作用机制,在基因和细胞信号通路多水平上具有抗炎、抗氧化应激、抗癌、免疫调节、凋亡调节以及鳌合金属离子等方面的复合特性。近年来的研究表明,姜黄素可用于神经退行性疾病如PD和AD的治疗。应用MPP+诱导的PC12细胞凋亡作为PD的细胞模型,体外研究证实姜黄素能够保护MPP+诱导的细胞凋亡；PD动物模型的研究表明姜黄素能够保护6-OHDA诱导的多巴胺能神经元死亡；此外,姜黄素能够减轻AD转基因鼠大脑氧化应激水平、抑制Ap聚集从而减少老年斑形成；姜黄素还可以保护大鼠大脑中动脉闭塞诱导脑缺血引起的神经元死亡。本研究采用PD细胞和小鼠模型以及AD转基因小鼠模型,应用行为学、形态学和分子生物学等手段,探讨姜黄素对神经元的保护作用及其机制,为进一步将姜黄素在神经退行性疾病治疗中的应用提供可靠的实验依据。本研究采用PD细胞和小鼠模型以及AD转基因小鼠模型,应用行为学、形态学和分子生物学等手段,探讨姜黄素对神经元的保护作用及其机制,为进一步将姜黄素在神经退行性疾病治疗中的应用提供可靠的实验依据。实验方法采用C57BL／6小鼠腹腔注射MPTP制备的PD小鼠模型、MPP+诱导SH-SY5Y细胞制备的PD体外模型、双转染人APP695swe基因和人早老素1(presenilin,PS-1)突变基因的AD模型小鼠(APP/PS1小鼠)为研究对象,分别给予姜黄素处理；应用旷场实验和牵引实验检测PD小鼠的行为学变化；应用免疫组织化学技术、体视学方法及Western blot技术检测姜黄素对PD模型的细胞保护作用及其机制；采用PCR技术对APP／PS1转基因小鼠进行鉴定,应用Morris水迷宫及跳台实验检测AD小鼠的记忆和学习能力变化；免疫组织化学技术、Western blot技术检测姜黄素对APP/PS1转基因小鼠大脑神经元的保护作用。实验结果一、姜黄素对PD多巴胺能神经元存活的影响1、姜黄素减弱MPTP诱导的小鼠行为学异常旷场实验结果：与正常对照组小鼠相比,MPTP模型组小鼠的水平运动和垂直运动评分(即爬格数和直立数)显著降低。姜黄素给药7天后,与模型组小鼠比较,姜黄素治疗组小鼠的水平运动和垂直运动评分显著增加。牵引实验：正常对照组小鼠四肢都能抓住电线,第12天时模型组小鼠仅能用前爪抓住电线,姜黄素治疗组小鼠能用一只或两只后爪抓住电线,与模型组相比牵引评分显著增高。2、姜黄素保护MPTP诱导的多巴胺能神经元的退行性变免疫组织化学染色(IHC)结果显示：与正常对照组小鼠相比,MPTP模型组小鼠黑质和纹状体多巴胺能神经元和神经纤维显著减少,然而姜黄素治疗组小鼠黑质和纹状体内的神经元和神经纤维的数量明显增加。体视学和光密度分析结果显示：MPTP模型组小鼠黑质的酪氨酸羟化酶(TH)阳性细胞丢失达43%,纹状体的多巴胺转运体(DAT)免疫阳性产物的光密度值(代表多巴胺能神经纤维)减少了91%,然而姜黄素给药组小鼠脑内的TH阳性细胞增加到82%和DAT标记的阳性纤维光密度增加到36%3、姜黄素抑制星形胶质细胞的激活IHC结果显示：MPTP模型组小鼠黑质内活化的星形胶质细胞的数量比正常对照组小鼠高出2.5倍。姜黄素治疗后明显减少了由MPTP激活的星形胶质细胞的数量。4、姜黄素对MPTP诱导的JNK, c-Jun和caspase-3激活的抑制作用免疫印迹(Western blot)结果显示：MPTP诱发了小鼠大脑黑质磷酸化JNK1和JNK2的明显增高,而给予姜黄素(50 mg/kg i.p.q.d. for 7 days)治疗后，MPTP诱发的磷酸化JNK1／2的增高得到显著抑制。同时姜黄素给药还可以抑制MPTP诱导的小鼠脑内c-Jun和caspase-3的活性。5、姜黄素减弱MPP+诱导的SH-SY5Y细胞死亡MTT结果显示：0-5 mM的MPP+处理SH-SY5Y细胞,细胞活性呈现剂量依赖性和时间依赖性的降低。3 mM MPP+处理细胞18h,细胞活性的抑制率达到50%。0-5μM的姜黄素以及0-10μM的SP600125对SH-SY5Y细胞没有明显的毒性作用。当使用1μM姜黄素或5μM SP600125处理细胞2h,再给予3 mM MPP+处理细胞18h,细胞活性显著增高。Hoechst33258染色结果显示：MPP+处理后,SH-SY5Y细胞的细胞核出现明显核固缩。使用姜黄素处理细胞可以减轻MPP+对细胞的损伤。6、姜黄素抑制SH-SY5Y细胞中JNK通路的激活Western blot结果显示：MPP+处理后,SH-SY5Y细胞的JNK1和JNK2活性显著增高。使用1μM姜黄素或5μM SP600125处理,可以明显抑制细胞JNK1和JNK2的活性。JNK蛋白一个上游调控因子MKK4,其磷酸化水平改变与JNK活性蛋白水平变化相似,即姜黄素也能抑制MPP+诱导的SH-SY5Y细胞中MKK4的活性。同时姜黄素也抑制了JNK下游c-Jun的活性。二、姜黄素对AD模型小鼠神经元的保护作用1、姜黄素对APP／PS1转基因小鼠学习记忆能力的影响Morris水迷宫实验：4天定位航行实验中,野生型、对照组、姜黄素喂养组三组小鼠搜索平台的潜伏期呈逐渐下降趋势。统计结果显示,姜黄素喂养组小鼠在第2、4天的平均潜伏期分别为30.65+3.15 s和28.42+6.57 s,显著低于对照组小鼠的潜伏期,但却高于野生型小鼠,表明姜黄素喂养组小鼠的空间学习能力得到改善但尚未达到野生型小鼠的水平。在第5天的探索实验中,1min内姜黄素喂养组小鼠经过相应平台位置的次数明显高于对照组小鼠,这表明姜黄素喂养组小鼠的空间记忆能力得到改善。跳台实验：与野生型小鼠的学习记忆成绩相比,对照组小鼠的潜伏期延长,错误次数增加。与对照组小鼠比较,姜黄素喂养组小鼠的潜伏期缩短,学习能力提高,同时,错误次数减少,记忆成绩也得以改善。2、姜黄素减少APP／PS1转基因小鼠的老年斑的形成IHC结果显示：对照组和姜黄素喂养组小鼠的大脑皮质及海马等区域均出现Aβ阳性神经元及阳性斑块(SP),说明这两组小鼠脑组织神经元内及神经元间出现Aβ的沉积。对照组小鼠脑内的SP体积大,数量多；而姜黄素喂养组小鼠脑内SP体积小,数量明显减少。图像分析定量结果显示：姜黄素喂养可以降低APP／PS1转基因小鼠海马和皮层的SP的数量,老年斑的数量分别减少34%和24%。3、姜黄素对APP／PS1转基因小鼠脑内APP和Tau蛋白的影响Western blot结果显示：姜黄素喂养组与对照组小鼠脑内的APP表达没有显著差异,均高于野生型小鼠。姜黄素喂养组小鼠脑内p-Tau蛋白水平低于对照组小鼠而高于野生型小鼠。结论1、姜黄素腹腔注射给药可明显改善MPTP模型小鼠的运动能力,使小鼠黑质纹状体多巴胺能神经元及纤维数量增加,黑质内MKK4、JNK、c-Jun、caspase-3的活性减弱,GFAP数目减少,表明姜黄素通过抑制JNK通路起到抗凋亡作用从而缓解了多巴胺能神经元的损伤。2、MPP+作为内源性毒素可诱发SH-SY5Y细胞凋亡。姜黄素通过抑制JNK通路蛋白的活性,延缓SH-SY5Y细胞凋亡的发生,而且其抑制JNK的效果同已知的特异性JNK抑制剂SP600125相似,从而进一步证实姜黄素通过抑制JNK通路起到保护多巴胺能神经元的作用。3、姜黄素口服给药可以提高APP/PS1转基因小鼠的学习能力和记忆能力,减少小鼠脑内SP的形成,同时也降低小鼠脑内p-Tau蛋白的含量,提示姜黄素可能通过抑制SP的形成和Tau蛋白的磷酸化对AD小鼠脑内神经元起保护作用。更多还原

【Abstract】 PrefaceParkinson’s Disease (PD) is a common age-related neurodegenerative disorder characterized by a progressive dopaminergic neuronal cell loss in the substantia nigra (SN), resulting in extrapyramidal motor dysfunction, including tremor, rigidity, and bradykinesia. Although the underlying mechanism of PD neurodegeneration is currently unclear, many studies have suggested that nigral dopaminergic cell apoptosis may play a critical role in the neurodegenerative processes in PD. Alzheimer’s disease (AD) is a disease clinically characterized by progressive intellectual deterioration. AD is pathologically characterized by senile plaques (SP) formed by pathological deposition of (3-amyloid (Aβ), neurofibrillary tangles (NFT). With the gradual ageing of the population, the morbidity of PD and AD increased year by year. It had brought heavy burden to the society and family and become one of the fatal disease that hazard to human health. Now, how to find the causes of brain diseases, and to explore ways to treat these diseases have become the focus of attention worldwide.The natural phenolic compound curcumin, isolated from the roots of Curcuma longa (Zingiberaceae) and commonly used as a spice, is well documented for its medicinal properties in the traditional Indian and Chinese systems of medicine. Since 1937 the first paper published on The Lancet, there were 2600 papers which about curcumin cure human diseases have published on English Journals. Several epidemiological, clinical, and animal studies have shown that curcumin has a variety of pharmacological activities, including anti-inflammatory, antioxidant, anticarcinogenic, and wound-healing effects. In recent years, the potential therapeutic value of curcumin for neurodegenerative diseases, such as AD and PD, has been increasingly recognized. For example, curcumin has been found to inhibit the formation of amyloid b oligomers and fibrils and reduce oxidative damage and amyloid burden in AD transgenic mouse brain. Curcumin treatment protects against neuronal death in middle cerebral artery occlusion-induced focal cerebral ischemia in rats. An in vitro study has shown that curcumin protects PC 12 cells against MPP+-induced apoptosis. Furthermore, curcumin has been demonstrated to protect against nigral dopaminergic cell death from 6-OHDA neurotoxicity.Therefore, our study systematic and comprehensive reveals curcumin’s protective effect on the PD mice cell models, and AD double-transgenic mouse model from a new perspective, which is an experimental basis in the treatment of neurodegenerative diseases.MethodsC57BL/6 mice IP MPTP, MPP+induced SH-SY5Y cells prepare model of PD, APP/PSl transgenic mice as the model of AD were used for the present study. Applicated open-field test and traction test in mice models of PD to detect and identify of behavior change of mice model. Immunohistochemical techniques, Stereology and Western blot were used for detected protection mechanisms of curcumin on the model of PD in vivo and in vitro. PCR technique was used for assaying AD transgenic mice, Morris water maze and Step Down Test, immunohistochemistry, Western blot were used for detected protective effect of curcumin on the APP/PS1 transgenic model of AD.Results1、The protective effect of curcumin to dopaminergic neuron of the mice model and SH-SY5Y model(1) Curcumin alleviates MPTP-induced mouse behavioral symptomsOpen-field test is shown 1. The MPTP+vehicle group exhibited a significant decrease in locomotion frequencies and rearing frequencies, compared with the vehicle control group on day 5 and 12. On day 12, the MPTP+curcumin group mice showed a significant increase in locomotion frequencies and rearing frequencies compared with the MPTP+vehicle group.In the traction test, the vehicle control mice gripped the wire with all four paws, while on day 12, in the MPTP+vehicle treated group, mice gripped the wire only by their forepaws. MPTP+curcumin group mice gripped the wire by one or two hindpaws, and the traction scores were increased significantly compared with the MPTP+vehicle group.(2) Curcumin protects against dopaminergic neuronal degenerationAs shown, compared with the vehicle control mice, a significant loss of the dopaminergic neurons and nerve fibers were seen in the SN and striatum after MPTP+ vehicle treatment. In contrast, those losses in the MPTP+curcumin group were clearly reduced.Unbiased stereological and optical density (O.D.) quantification showed that MPTP+vehicle produced a 43% loss of TH positive neurons in the SN and a 91% reduction of DAT-IR fibers, whereas curcumin administered once a day for 7 days improved the TH positive neuronal in the SN and DAT-IR fibers in the striatum to 82% and 36% of MPTP+curcumin-treatedmice, respectively.(3) Curcumin inhibits astrocyte activation in miceImmunoreactivity of GFAP results showed that MPTP+vehicle-treated mice exhibited 2.5 times more activated astrocytes than the vehicle control mice. MPTP+ curcumin treatment significantly attenuated the MPTP-induced increase in the number of activated astrocytes.(4) Inhibitory effects of curcumin on MPTP-induced activation of JNK, c-Jun and caspase-3 in mouse substantia nigraOur immunoBlotting results showed that MPTP induced a robust increase in phosphorylated forms of both JNK1 and JNK2, the key molecules in the JNK pathways, in the SN. Interestingly, curcumin treatment (50 mg/kg i.p.q.d. for 7 days) significantly inhibited MPTP-induced JNK1/2 phosphorylation、the activation of c-Jun and cleaved caspase-3.(5) Curcumin attenuates MPP+-induced SH-SY5Y cell deathMTT assay shown:SH-SY5Y cells treated with MPP+(0-5 mM) showed a dose-and time-dependent reduction in the levels of cell viability a 50% reduction in cell viability was obtained when cells were treated with 3 mM MPP+for 18 h. The MTT assay also showed that curcumin (0-5μM) and SP600125 (0-10μM) did not exhibit significant toxic effects on the cells. When the cells were pretreated with 1μM curcumin and 5μM SP600125 for 2 h prior to the addition of MPP+, the cell viability was significantly increased.Hoechst 33258 staining showed that distinct nuclear condensation was observed in MPP+-treated cells. This phenomenon was inhibited when curcumin was added prior to MPP+treatment.(6) Curcumin inhibits JNK pathway activation in SH-SY5Y cellsOur immunoBlotting results showed that the phosphorylated forms of both JNK1 and JNK2 were increased significantly in response to MPP+stimuli. The expression levels of JNK1 and JNK2 phosphorylation were inhibited by pretreatment with 1μM curcumin, and 5μM SP600125. The changes in the expression levels of MKK4 phosphorylation, an upstream molecule of JNK protein, were similar to that of JNK protein, i.e. curcumin could also inhibit MPP+-induced MKK4 activation in SH-SY5Y cells. Coincident with the result of JNK phosphorylation, MPP+-induced c-Jun phosphorylation was inhibited by curcumin in SH-SY5Y cells, which is similar to the effect of SP600125 on c-Jun activation.2、The protective effect of curcumin to the neuron of APP/PS1 transgenic mice model(1) Curcumin change behavior abnormalities in APP/PS1 transgenic miceMorris water maze test:In hidden platform tests, the escape latency has a downward trend in three groups of mice. Movement trail of Wild-type mice and mice of fed curcumin focused on the appropriate platform location, while control APP/PS1 mice almost along the pool wall and away from the platform location. Results showed, the escape latency on the second and fourth day of the hidden platform test was shorter than control mice, suggesting that curcumin-treated improved spatial learning and memory ability, but not reach normal levels. In the probe trial on the fifth day, the curcumin-treated APP/PS1 mice traveled into the third quadrant, where the hidden platform was previously placed, significantly more times than controls.Step Down Test:compared to wild-type mice, control transgenic mice reaction time, error frequency increased; The curcumin fed group compared with control group, curcumin can shorten the reaction time, improved learning ability, at the same time reduce the number of errors, improve memory performance.(2) Curcumin protect the burden of senile plaques in APP/PS1 transgenic miceIHC results showed that senile plaque formation was significantly decreased and the area of senile plaques was also significantly reduced in the brain of curcumin fedding mice.Quantification showed that curcumin treatment reduced senile plaque number in the hippocampus and cortex to 34% and 24% respectively.(3) Curcumin effect APP and Tau protein in APP/PS1 transgenic mice brainWestern blot results showed that APP has not significant difference between curcumin fed and control transgenic mice. p-Tau protein levels in the brain of curcumin feeding group mice lower than control transgenic mice but higher than wild-type mice.Conclusion1. The administration of intraperitoneal injection curcumin can significantly improve behavior of mice, while the number of nigrostriatal dopaminergic neurons and fibers increased, the active ingredient of MKK4, JNK, c-Jun, caspase-3 reduced in substantia nigra, the number of GFAP reduction which indicates that cucumin play a role in anti-apoptosis and thus alleviate neuronal damage by inhibiting the JNK pathway2. MPP+as an endogenous toxin can induce apoptosis in SH-SY5Y cells, while curcumin could inhibit the active ingredient of JNK to delay the occurrence of apoptosis, and its inhibitory effect is similar to the specific JNK inhibitor SP600125, which further confirmed that curcumin protected neurons by inhibiting the JNK pathway.3. Oral curcumin can improve memory and learning ability of transgenic mice, reduce the burden of senile plaques in brain of transgenic mice, and decrease the content of p-Tau. These results confirm the neuroprotective effect of curcumin in AD transgenic mouse brain.更多还原