【作者】 刘佳；

【导师】 娄海燕；

【作者基本信息】 山东大学 ， 药理学， 2021， 硕士

【摘要】 [研究背景]帕金森病(Parkinson’s disease,PD)是全球第二大神经退行性疾病。其临床表现为运动迟缓、姿势步态障碍等运动症状和一系列非运动症状,包括便秘等。帕金森病主要的病理改变是黑质致密部多巴胺能神经元进行性丢失并伴有纹状体多巴胺水平的下降,已有的研究显示,纹状体多巴胺含量显著下降与帕金森病运动症状的出现密切相关。另一方面,帕金森病的确切发病机制至今尚未明确,可能涉及到了线粒体功能障碍、年龄老化及遗传与环境因素相互作用等多个方面。线粒体生物发生是一种机体在病理生理状态下维持线粒体稳态的机制,该过程通过提高线粒体DNA(mitochondrial DNA,mtDNA)的拷见数,增加ATP的产量,进而维持线粒体功能正常。最近的一项对于Parkin(一种已被证明的与PD发病高度相关的基因)缺陷型人多巴胺能神经元的研究表明,虽然Parkin缺陷的人多巴胺能神经元存在明显的线粒体自噬缺陷,但线粒体功能障碍主要是线粒体生物发生缺陷导致的结果。因此,深入探讨线粒体生物发生的机制并进一步寻找潜在治疗靶点对PD的防治具有重要的临床意义。在线粒体生物发生中,SIRT1/PGC-1α/TFAM轴发挥着至关重要的作用。沉默信息调节因子1(silent information regulator 1,SIRT1)是一种依赖于烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD+)的组蛋白/非组蛋白去乙酰化酶,随着NAD+/NADH比值的增加,SIRT1去乙酰化激活下游过氧化物酶增殖受体γ共激活因子-1α(peroxisome proliferator-activated receptor gamma coactivator-1 α,PGC-1α),最终激活线粒体转录因子 A(mitochondrial transcription factor A,TFAM)前体蛋白的合成。该前体蛋白合成后经一系列转运和加工后结合mtDNA,调节其复制和转录,促进线粒体生物发生。因此,SIRT1/PGC-1α/TFAM通路在线粒体生物发生中发挥着核心作用。尿石素A(Urolithin A,UA)是石榴、浆果、坚果等富含鞣花单宁和鞣花酸的食物在肠道内的主要代谢产物,具有抗炎、抗氧化、抗衰老以及维持和改善线粒体功能等多种药理作用。已有文献表明,UA可通过激活线粒体自噬对阿尔茨海默症(Alzheimer’s disease,AD)发挥神经保护作用,另外也有文献表明UA可上调BV2小胶质细胞中SIRT1的表达。但是,UA是否对PD发挥保护作用目前尚未见报道。因此,本论文将探讨UA对PD的保护作用,并初步探究其是否通过促进线粒体生物发生参与到PD神经保护的调控中,从而为寻找PD的潜在治疗药物和药物靶点提供理论基础。[研究目的]1.明确UA对PD的神经保护作用。2.阐明UA促进线粒体生物发生发挥PD保护作用的分子机制。[研究方法]1.体内实验1.1明确UA对6-OHDA所致PD小鼠的神经保护作用:C57BL/6小鼠腹腔注射给予UA 10 mg/kg 7天后,采用脑立体定位注射6-OHDA建立PD小鼠模型。分别于造模后1周、2周、3周进行体重测量、粪便重量测量和行为学实验(包括爬杆实验、悬挂实验、转棒实验、阿扑吗啡诱导的旋转实验);造模后3周取小鼠脑组织进行冰冻切片,随后进行尼氏染色及TH免疫组化实验检测UA对多巴胺神经元的保护作用;GFAP免疫荧光检测UA对星形胶质细胞活化的影响。另外同时取黑质和纹状体,Western blot实验检测UA对TH蛋白表达的影响。1.2明确UA对6-OHDA所致PD小鼠脑内ROS水平及线粒体形态的影响:DCFH-DA实验检测小鼠脑组织ROS水平;Western blot实验检测4-HNE表达;透射电镜实验检测UA对黑质多巴胺神经元线粒体形态的影响。1.3探究UA在体内对SIRT1/PGC-1 α/TFAM信号通路的影响:Western blot实验检测UA对PD小鼠黑质部位SIRT1、PGC-1α、TFAM蛋白表达的影响。2.体外实验2.1明确UA对PC-12细胞的保护作用:CCK-8实验检测不同浓度UA对细胞存活率的影响及6-OHDA造模条件下不同浓度UA对细胞存活率的影响;TUNEL染色及FITC Annexin V/PI流式双染实验检测6-OHDA造模条件下不同浓度UA对细胞凋亡率的影响。2.2明确UA对PC-12细胞ROS水平及线粒体活性和形态的影响:DCFH-DA流式实验检测6-OHDA造模条件下不同浓度UA对细胞ROS的影响;线粒体活性染色实验检测6-OHDA造模条件下不同浓度UA对线粒体活性的影响;透射电镜观察6-OHDA造模条件下不同浓度UA对线粒体形态的影响。2.3检测UA对6-OHDA所致PC-12细胞线粒体功能障碍的改善作用:Western blot实验检测6-OHDA造模条件下不同浓度UA对线粒体内、外膜蛋白的影响;JC-1流式及荧光染色检测6-OHDA造模条件下不同浓度UA对线粒体膜电位的影响;检测6-OHDA造模条件下不同浓度UA对细胞ATP水平的影响;qPCR实验检测6-OHDA造模条件下不同浓度UA对线粒体DNA的影响。2.4明确UA是否通过激活SIRT1介导PGC-1 α/TFAM信号通路促进线粒体生物发生减轻6-OHDA所致PC-12细胞线粒体功能障碍:Western blot实验检测6-OHDA造模条件下不同浓度UA对SIRT1、PGC-1α、TFAM蛋白表达的影响;利用工具药EX527抑制SIRT1活性,检测UA对细胞存活率、细胞凋亡率、线粒体膜电位及线粒体DNA的影响,明确UA是否通过SIRT1/PGC-1α/TFAM信号通路促进线粒体生物发生对PC-12细胞发挥保护作用。3.生物信息学使用生物信息学手段分析公共数据库,明确PD的发病机制与线粒体功能障碍的相关性,并为SIRT1是UA的药物靶点提供理论依据。[研究结果]1.UA可缓解6-OHDA所致的PD小鼠体重降低及排便量减少;改善6-OHDA所致的PD小鼠行为学障碍,减轻6-OHDA所致的黑质纹状体多巴胺神经元死亡及TH蛋白水平的下降;同时可减少6-OHDA所致的黑质星形胶质细胞活化。2.UA可降低6-OHDA所致PD小鼠脑内ROS水平的上升;并可维持6-OHDA病理条件下线粒体形态正常。3.UA在体内可激活SIRT1/PGC-11α/TFAM信号通路。4.UA可拮抗6-OHDA所致的PC-12细胞存活率下降及细胞凋亡率的上升。5.UA可降低6-OHDA所致的PC-12细胞ROS水平上升;改善6-OHDA所致的PC-12细胞线粒体活性及形态异常。6.UA可减轻6-OHDA所致的PC-12细胞线粒体外膜、内膜标志性蛋白损伤;拮抗6-OHDA导致的线粒体膜电位去极化;改善6-OHDA导致的ATP生成量及mtDNA拷贝数的降低。7.UA在体外可提高6-OHDA病理状态下SIRT1、PGC-1α和TFAM的蛋白表达水平,并可促进线粒体生物发生。SIRT1特异性抑制剂EX527明显减弱了UA对SIRT1/PGC-1α/TFAM信号通路的激活。在SIRT1特异性抑制剂EX527存在的条件下,UA对6-OHDA损伤的PC-12细胞的保护作用、改善线粒体膜电位作用及提高mtDNA拷贝数的能力均显著下降。8.生物信息学角度佐证了 SIRT1是UA发挥PD神经保护作用的靶点,且与维持线粒体功能正常密切相关。[研究结论]1.UA对6-OHDA所致的PD细胞及动物模型具有神经保护作用。2.UA通过激活SIRT1介导PGC-1α/TFAM信号通路促进线粒体生物发生对PD发挥神经保护作用。[创新性与局限性]1.首次明确了 UA对于PD的神经保护作用,为寻找PD的潜在治疗药物提供了新思路。2.首次明确了 UA通过激活SIRT1/PGC-1α/TFAM信号通路介导线粒体生物发生对PD发挥保护作用,为寻找PD的潜在药靶提供了理论基础。3.并未找到UA与SIRT1结合的活性位点。4.衰老是目前热门的研究领域,SIRT1作为“与衰老密切相关的基因”已被广泛报道,而PD是一种老龄化疾病。本研究通过UA将SIRT1和PD联系起来,但并未完全阐明UA以及SIRT1在“老化”过程中的作用。更多还原

【Abstract】 BackgroundParkinson’s disease(PD)is the second most common neurodegenerative disease in the world.Its clinical manifestations include motor symptoms such as motor retardation,postural and gait disorders,and a series of non-motor symptoms,including constipation.The main pathological change of PD is the progressive loss of dopaminergic neurons in the pars compacta of substantia nigra accompanied by a decrease in the level of dopamine in the striatum.Previous studies have shown that the significant decrease in dopamine content in the striatum is closely related to the occurrence of motor symptoms of PD.On the other hand,the exact pathogenesis of PD is not clear,which may involve mitochondrial dysfunction,aging,and the interaction between genetic and environmental factors.Mitochondrial biogenesis is a mechanism to maintain mitochondrial homeostasis under pathophysiological conditions.This process maintains normal mitochondrial function by increasing the copy number of mitochondrial DNA(mtDNA)and increasing the production of ATP.A recent study showed that in Parkin-deficient human dopaminergic neurons,mitochondrial dysfunction was mainly the result of mitochondrial biogenic defects,although the neurons have obvious mitophagy defects.Therefore,targeting mitochondrial biogenesis may become a new strategy for the prevention and treatment of PD.SIRT1/PGC-1α/TFAM axis plays an important role in mitochondrial biogenesis.The silent information regulator 1(SIRT1)is a histone/non-histone deacetylase dependent on nicotinamide adenine dinucleotide(NAD+).With the increase of NAD+/NADH ratio,SIRT1 deacetylation activates downstream peroxisome proliferator y coactivator-1α(PGC-1α),resulting in activation of mitochondrial transcription factor A(TFAM)precursor protein synthesis.After a series of transport and processing,the precursor protein is combined with mtDNA to regulate its replication and transcription and promote mitochondrial biogenesis.Therefore,SIRT1/PGC-1α/TFAM pathway plays a central role in mitochondrial biogenesis.Urolithin A(UA)is the main intestinal metabolite of foods that are rich in ellagic tannins and ellagic acids,such as pomegranate,berries,and nuts.It has a variety of pharmacological effects,such as anti-inflammation,anti-oxidation,anti-aging,maintaining,and improving mitochondrial function.It has been shown that UA can play a neuroprotective effect on Alzheimer’s disease(AD)by activating mitophagy.Another study also revealed that UA can up-regulate the expression of SIRT1 in BV2 microglia.However,whether UA plays a protective role in PD is unknown.Therefore,in the present study,we investigated whether UA is protective in the experimental model of PD by promoting mitochondrial biogenesis.The results of this study will be helpful for finding potential therapeutic drugs and drug targets for PDObjectives1.To determine the neuroprotective effect of UA on PD.2.To elucidate the molecular mechanism that UA promotes mitochondrial biogenesis and plays a protective role of PD.Methods1.In vivo experiments1.1 To determine the neuroprotective effect of UA on 6-OHDA-induced PD mice model:After intraperitoneal injection of UA 10 mg/kg for 7 days in C57BL/6 mice,the PD mouse model was established by stereotactic injection of 6-OHDA.Weight measurement,fecal weight measurement,and behavioral experiments were performed at 1 week,2 weeks,and 3 weeks after 6-OHDA injection,and the mice brain tissues were taken for frozen section 3 weeks after 6-OHDA injection.Nissl staining and TH immunohistochemical experiments were performed to detect the protective effect of UA on dopaminergic neurons.GFAP immunofluorescence was carried out to detect the effect of UA on the activation of astrocytes.Besides,the substantial nigra and striatum were taken at the same time,and the effect of UA on the expression of TH protein was detected by western blot assay.1.2 To determine the effect of UA on the level of ROS and the morphology of mitochondria in the brain of PD mice induced by 6-OHDA:DCFH-DA test was used to measure the production of ROS under pathological conditions;western blot analysis was performed to detect the expression of 4-HNE;transmission electron microscope experiment was carried out to detect the effect of UA on dopaminergic neurons mitochondrial morphology in the substantia nigra.1.3 To explore the effect of UA on the SIRT1-mediated PGC-1α/TFAM signaling pathway in vivo:western blot assay was used to detect the effect of UA on SIRT1,PGC-1α,and TFAM protein expression in the substantia nigra of PD mice.2.In vitro experiments2.1 To determine the protective effect of UA on PC-12 cells:CCK-8 test was used to detect the effect of different concentrations of UA on cell viability after exposure to 6-OHDA;TUNEL staining and FITC Annexin V/PI flow double staining experiment were used to test the effect of different concentrations of UA on cell apoptosis after exposure to 6-OHDA.2.2 To determine the effect of UA on ROS level and mitochondrial activity and morphology of PC-12 cells:Cellular ROS level was assessed using DCFH-DA;mitochondrial activity staining test was used to detect the effect of different concentrations of UA on mitochondrial activity;transmission electron microscope was used to observe the effect of different concentrations of UA on mitochondrial morphology.2.3 To detect the protective effect of UA on 6-OHDA-induced mitochondrial dysfunction in PC-12 cells:the effects of different concentrations of UA on mitochondrial membrane proteins were detected by Western blot analysis;the effects of different concentrations of UA on mitochondrial membrane potential were detected by JC-1 flow cytometry analysis and fluorescence staining,and the effects of different concentrations of UA on cellular ATP level were detected.qPCR was used to detect the effect of different concentrations of UA on mitochondrial DNA content.2.4 To determine whether UA promotes mitochondrial biogenesis and alleviates 6-OHDA-induced mitochondrial dysfunction in PC-12 cells by activating the SIRT1-mediated PGC-1α/TFAM signaling pathway:western blot assay was used to detect the effects of different concentrations of UA on the expression of SIRT1,PGC-1α,and TEAM protein levels.EX527 was used to inhibit the activity of SIRT1,and the effects of UA on cell survival rate,apoptosis rate,mitochondrial membrane potential,and mitochondrial DNA were detected as previously described,to determine whether UA protects PC-12 cells by promoting mitochondrial biogenesis through SIRT1/PGC-1α/TFAM signal pathway.3.BioinformaticsBioinformatics was used to analyze the public database to clarify the correlation between the pathogenesis of PD and mitochondrial dysfunction and to provide a theoretical basis for SIRT1 as a drug target for UA.Results1.UA could alleviate the weight loss and the constipation of PD mice caused by 6-OHDA;UA could alleviate 6-OHDA-induced behavioral disorder and dopaminergic neuronal loss in PD mice;UA could alleviate the neuronal inflammation in the brain caused by 6-OHDA.2.UA could reduce the 6-OHDA-induced increase of ROS level in the brain of PD mice,and maintain the normal morphology of mitochondria under the pathological condition of 6-OHDA.3.UA could activate the SIRT1/PGC-1α/TFAM signal pathway in vivo.4.UA could protect PC-12 cells against 6-OHDA-induced cell death.5.UA could reduce the increase of total ROS level in PC-12 cells induced by 6-OHDA,and ameliorate the morphological abnormality induced by 6-OHDA in PC-12 cells.6.UA could alleviate 6-OHDA-induced mitochondrial dysfunction in PC-12 cells:UA could increase the levels of the mitochondrial outer membrane and intimal marker protein in 6-OHDA-treated PC-12 cells,decrease 6-OHDA-induced mitochondrial membrane potential depolarization,increase ATP production and mtDNA copy number in PC-12 cells.7.UA could increase the protein expression of SIRT1,PGC-1α,and TFAM and promote mitochondrial biogenesis in PC-12 cells exposed to 6-OHDA.The protective effect of UA on PC-12 cells was partly abolished in the presence of EX527,a specific inhibitor of SIRT1,and the protective effect of UA on mitochondrial membrane potential and the increase in the copy number of mtDNA caused by UA in PC-12 cells were also attenuated by EX527.EX527 also inhibited the activation of SIRT1/PGC-1 a/TFAM signal pathway induced by UA.8.The results of bioinformatics analysis further support that SIRT1 may be the target of UA to exhibit its neuroprotective effects in PDConclusions1.UA protects PC-12 cells against 6-OHDA-induced injury and attenuates dopaminergic neurotoxicity in the 6-OHDA-induced PD mice model.2.UA exhibits its neuroprotective effects in PD by promoting mitochondrial biogenesis through activating the SIRT1-mediated PGC-1α/TFAM signal pathway.Innovations and limitations1.The neuroprotective effect of UA on PD is clarified for the first time in our study.The results of our study provide a new strategy for finding potential therapeutic drugs for PD.2.Our study revealed for the first time that UA mediates the protective effect of mitochondrial biogenesis on PD by activating the SIRT1/PGC-1α/TFAM signal pathway,which provides a theoretical basis for finding potential drug targets of PD.3.The active site of UA binding to SIRT1 was not found.4.Aging is currently one of the hottest fields in medical sciences and SIRT1 has been widely reported as a "senescence gene" while PD is also an aging neurodegenerative disease.This study connects SIRT1 and PD through UA,but does not fully clarify the role of UA and SIRT1 in the process of "aging".更多还原