【作者】 蒋建华；

【导师】 顾振纶； 秦正红；

【作者基本信息】 苏州大学 ， 药理学， 2013， 博士

【摘要】 【目的】帕金森病(Parkinson’s disease, PD)是中枢神经系统变性疾病，线粒体氧化应激和神经元凋亡在PD的病理机制中发挥关键的作用。雷帕霉素(rapamycin)为雷帕霉素靶蛋白(Target of rapamycin, TOR)信号通路特异性抑制剂，在一系列神经系统变性疾病的病理机制及治疗中具有重要的意义。本研究通过雷帕霉素预处理PD大鼠及细胞模型，揭示其在PD中的神经保护作用，并探讨其机制。【方法】动物实验：实验SD大鼠随机分为假手术组、PD模型组、雷帕霉素低剂量组(0.05mg/kg/d)、中剂量组(0.5mg/kg/d)及高剂量组(5mg/kg/d)进行雷帕霉素灌胃预处理。7天后，向模型组和所有雷帕霉素预处理组大鼠脑部右侧纹状体两点定向注射6-羟基多巴(6-hydroxydopamine，6-OHDA)(4μg/μl,2μl/注射点)诱导建立PD大鼠模型，假手术组大鼠注射生理盐水作为对照。术后第25天各组大鼠均用阿扑吗啡(0.5mg/kg)诱导旋转试验，检测行为学指标；在造模后第28天，各组大鼠经心脏灌注固定，取脑组织进行黑质-纹状体酪氨酸羟化酶（Tyrosine hydroxylase，TH）免疫组化染色及透射电子显微镜观察；检测各组SD大鼠中脑丙二醛(malondialdehyde, MDA)含量及超氧化物歧化酶(superoxide dismutase, SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-PX)活性；提取脑组织总RNA后，采用RT-PCR方法检测黑质-纹状体内Bax、Bcl-2和p47phox等基因mRNA的表达；同时采用western blot方法检测黑质-纹状体内Bax、Bcl-2、细胞色素c以及cleavedcaspase-9蛋白质的表达。体外实验：采用不同浓度6-OHDA诱导24h后检测PC12细胞存活率，建立PD细胞模型。不同浓度雷帕霉素预处理PC12细胞3h、6h、12h和24h，100μM浓度6-OHDA诱导24h后，使用CCK-8细胞计数试剂盒检测PC12细胞不同时间点的存活率。采用3μM浓度雷帕霉素预处理6h，100μM浓度6-OHDA诱导24h后，使用丙啶碘化物(Propidium iodide, PI)单染试剂盒检测雷帕霉素对损伤PC12细胞周期的影响。同时使用AnnexinⅤ-FITC和PI双染试剂盒，通过流式细胞仪检测各处理组PC12细胞凋亡的变化。雷帕霉素预处理6h，6-OHDA诱导1h后，使用含DCFH-DA荧光探针的试剂盒检测各处理组PC12细胞中活性氧(reactive oxygenspecies, ROS)的生成。收集各组PC12细胞，提取细胞总RNA，通过real-time RT-PCR检测各处理组PC12细胞内P47phox、TH、血红素加氧酶-1(hemo oxygenase-1, HO-1)和转录因子NF-E2相关因子2(transcriptional factor NF-E2-related factor2，Nrf2)的mRNA表达。【结果】一、动物实验结果：(1)与模型组相比，各剂量雷帕霉素预处理组大鼠的旋转行为减轻(p<0.05)。(2)与假手术组相比，PD模型组损伤侧黑质部位中TH+细胞数量明显减少(p<0.001)；与模型组比较，各雷帕霉素预处理组大鼠损伤侧TH+细胞数量则明显增加(p<0.001)。(3)模型组黑质-纹状体细胞器结构不清，细胞核皱缩，染色质异常聚集；线粒体体积增大，呈空泡样变，线粒体嵴溶解。而不同剂量的雷帕霉素预处理则明显减轻细胞超微结构的异常。(4)与假手术组比较，PD模型组MDA含量明显升高(p<0.001)，而SOD和GSH-PX的活性明显降低(p<0.001)。与PD模型组比较，经雷帕霉素预处理后，各剂量组MDA的含量均显著降低(p<0.01)，而SOD和GSH-PX的活性较PD模型组升高(p<0.05)。(5)与假手术组相比，PD模型组Bcl-2mRNA水平(p=0.001)明显降低而Bax mRNA水平(p<0.001)明显升高。与PD模型组比较，雷帕霉素预处理升高Bcl-2mRNA水平(p<0.01)，同时降低BaxmRNA的表达(p<0.05)，同时Bcl-2和Bax蛋白的表达与RT-PCR的检测结果相似。(6)与假手术组比较，PD模型组P47phoxmRNA的表达明显升高(p<0.001)，而雷帕霉素预处理显著下调PD大鼠模型中P47phoxmRNA的表达(p<0.01)。在PD模型组中活性caspase-9的表达和细胞色素c的释放比假手术组均明显升高(p<0.001)。与PD模型组比较，不同剂量雷帕霉素预处理均可降低PD大鼠活性caspase-9的表达和细胞色素c的释放(p<0.05)。然而，在雷帕霉素三个剂量组之间，所有研究结果包括旋转行为、TH+细胞数量、氧化应激指标及Bcl-2、Bax、P47phox、细胞色素c和caspase-9的表达均无显著统计学差异。二、细胞实验结果：(1)100μM浓度的6-OHDA处理24小时后，PC12细胞接近半数死亡，其存活率为57.67±9.84%。(2)0.5μM-15μM浓度的雷帕霉素单独作用24h，PC12细胞存活率与空白对照组相比均无统计学差异(p>0.05)，而雷帕霉素单独作用48h和72h则出现抑制PC12细胞存活率的现象。(3)与6-OHDA组相比，当0.5μM-3μM浓度的雷帕霉素预处理PC12细胞3h-12h，明显升高损伤细胞的存活率(p<0.05)。其中，3μM雷帕霉素预处理6h对损伤PC12细胞存活率的改善作用最显著(p<0.01)。(4)与空白对照组相比，6-OHDA使PC12细胞中处于G0/G1期的细胞明显增多(p<0.001)，S期(p=0.001)和G2/M期(p=0.009)细胞则明显减少。而3μM雷帕霉素的预处理后6h后，G0/G1期细胞与6-OHDA组比较显著减少(p=0.018)，S期细胞明显增多(p=0.028)，但G2/M期细胞数量无统计学差异。(5)单加雷帕霉素对PC12细胞的凋亡无明显影响，6-OHDA处理后细胞凋亡较正常细胞明显增多(p<0.001)，而3μM雷帕霉素预处理6h，则明显减少6-OHDA所诱导的细胞凋亡(p<0.05)。(6)单加雷帕霉素对PC12细胞内ROS的水平无明显影响，6-OHDA的诱导使PC12细胞内ROS含量明显增多(p<0.01)，而雷帕霉素预处理则抑制损伤细胞中ROS的生成(p<0.05)。(7)单用3μM雷帕霉素处理正常PC12细胞6h-12h，HO-1和Nrf2mRNA的表达明显升高(p<0.05)，而在处理24h-48h两者的表达又回落至接近0h水平；(8)与正常PC12细胞比较，6-OHDA显著升高PC12细胞中P47phox而降低TH mRNA的表达(p<0.05)；与6-OHDA组相比，3μM雷帕霉素预处理6h后，P47phoxmRNA的表达下降而TH mRNA的表达明显升高(p<0.05)。与正常对照组比较，6-OHDA可升高PC12细胞中HO-1和Nrf2mRNA的表达(p<0.05)，与6-OHDA组相比，3μM雷帕霉素预处理6h后，则进一步上调HO-1和Nrf2mRNA的表达(p<0.05)。【结论】雷帕霉素可有效改善PD大鼠模型的行为学症状、降低黑质多巴胺能神经元的丢失和促凋亡分子的表达。在PD细胞模型中证实，雷帕霉素具有提高损伤细胞的生存能力和减少细胞凋亡的生物学作用。细胞周期的改变和抗氧化应激能力的增强可能是其神经保护作用的重要机制。由此，恢复氧化应激中调控信号的平衡可能为PD的药物治疗拓展新的途径。更多还原

【Abstract】 ObjectiveParkinson’s disease (PD) is a neurodegenerative disease of central nervous system,in which oxidative stress and neuronal apoptosis play a key role in its pathogenesis.Rapamycin, a widely used immunosuppressant, is involved in some pathophysiologicalprocesses by inactivating the target of rapamycin (TOR). In a series ofneurodegenerative diseases, rapamycin displayed crucial significance in theirtherapeutic pathogenesis. However, role of rapamycin in PD therapy is not well known.In view of this, this study pre-treated6-hydroxydopamine (6-OHDA)-induced cell andrat models of PD with rapamycin to explore its roles and mechanisms inneuroprotections.MethodsIn vivo, for the induction of PD,6-OHDA (4μg/μl,2μl/site) was injected into2different sites within the right striatum of female Sprague-Dawley (SD) rats. SD ratswere randomly divided into the following5groups: Sham-operated (group S); PDmodel (group P); Pre-treated with a low (group L-R,0.05mg/kg/d), moderate (groupM-R,0.5mg/kg/d) or high (group H-R,5mg/kg/d) dose of rapamycin. Different doses ofrapamycin or vehicle were intragastrically administered once daily to the rats from day7before the induction of PD. On day25all animals underwent rotational testing byusing apomorphine (0.5mg/kg) to evaluate the motor asymmetry caused by the unilateral nigrostriatal lesion. On day28, the rats were sacrificed by cardiac perfusionand the brains were removed. Tyrosine hydroxylase (TH)++neurons in the substantianigra were evaluated by immunohistochemistry. Neuronal and mitochondrialultrastructures were detected by transmission electron microscope (TEM). Peroxidelevels and antioxidant activities in the midbrain were measured by assay kits, includingmalondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase(GSH-PX). After total RNA was extracted, semi-quantitative RT-PCR was performedto examine the mRNA levels of Bcl-2, Bax and P47phox. The protein expressions ofBcl-2, Bax, cytochrome c and cleaved caspase-9were detected by western blot.In vitro, cell PD model was induced by various concentrations of6-OHDA for24hin PC12cells. The damaged PC12cells induced by6-OHDA were pre-treated withvarious concentrations of rapamycin at different time points. The viability of damagedPC12cells was determined by cell counting kit-8(CCK-8). After selection of optimalconcentration, the damaged PC12cells induced by6-OHDA were pre-treated with3μMrapamycin for6h. The cycles of damaged PC12cells were analyzed by propidiumiodide (PI) staining kit in a flow cytometer, which was also employed to detect theapoptosis of PC12cells by Annexin Ⅴ-FITC and PI double staining kit. Afterrapamycin-pretreated PC12cells were induced by6-OHDA for1h, reactive oxygenspecies (ROS) were measured by an assay kit containing a DCFH-DA probe. Followingextraction of total RNA in treated PC12cells, real-time reversetranscriptional-polymerase chain reaction (RT-PCR) was performed to determine geneexpressions of P47phox, TH, hemo oxygenase-1(HO-1) and transcriptional factorNF-E2-related factor2(Nrf2).ResultsIn vivo experiment:(1) Pre-treatment with various doses of rapamycin significantlyreduced the number of contralateral rotations as compared with those in group P(p<0.05).(2) The number of remaining TH+neurons in the substantia nigra from groupP rats was significantly lower as compared with that from group S rats (p<0.001). Pre-treatment with rapamycin significantly increased the number of TH+neurons in thesubstantia nigra as compared to group P (p<0.001).(3) TEM detections demonstratedcondensed chromatin in shrunken nuclei in the neurons of PD model rats. Themitochondria were evidently swelled and vacuolated, and the cristae were lessened,distorted or even disappeared. However, in the rats pre-treated with rapamycin，ultrastructual injuries were significantly alleviated.(4) In group P rats, MDA levelswere elevated but SOD and GSH-PX activities were decreased as compared with thosein group S rats (p<0.001). Following pre-treatment with rapamycin, a reduction in MDAlevels and an increase in SOD and GSH-PX activities were observed as compared withthose in group P (p<0.05).(5) In group P rats, RT-PCR analyses showed lower Bcl-2but higher Bax mRNA levels than those in group S rats (p<0.01). Pre-treatment withrapamycin significantly increased Bcl-2and decreased Bax mRNA levels as comparedto pre-treatment with the vehicle (group P)(p<0.01). Western blot analyses revealedsimilar results to RT-PCR detection in protein expression of Bcl-2and Bax (p<0.05).(6)In group P rats, RT-PCR analyses showed that the P47phoxmRNA levels weresignificant elevated as compared with those in group S rats (p<0.001). Pre-treatmentwith rapamycin significantly reduced P47phoxmRNA expression as compared topre-treatment with vehicle (group P)(p<0.01). Western blot analyses demonstrated thatthe release of cytochrome c and the expression of cleaved caspase-9were increased ingroup P rats, as compared with those in group S rats (p<0.001). Pre-treatment withrapamycin significantly suppressed cytochrome c release and caspase-9expression ascompared with pre-treatment with the vehicle (group P)(p<0.05). In addition, all theresults including behavioral rotations, remaining TH+neurons, oxidative stressindicators, as well as expressions of Bcl-2, Bax, P47phox, cytochrome c and cleavedcaspase-9showed no statistical differences among all doses of rapamycin pre-treatedgroups.In vitro experiment:(1) PD cell models were successfully induced by100μMconcentration of6-OHDA for24h in PC12cells, which resulted in57.67±9.84%of cellviability.(2) As compared with blank control, mono-treatment with0.5μM-15μM rapamycin for24h exhibited no impact on the viability of normal PC12cells (p>0.05).However, mono-treatment with rapamycin for48h and72h showed inhibition of theviability of PC12cells.(3) Pre-treatment with0.5μM-3μM rapamycin for3h-12hsignificantly elevated the viability of damaged PC12cells induced by6-OHDA(p<0.05), of which pre-treatment with3μM rapamycin for6h offered an optimalcondition (p<0.01).(4) Cycle analyses demonstrated that6-OHDA remarkablyincreased the percentage of PC12cells in G0/G1phase (p<0.001), and decreased thepercentage of PC12cells in S and G2/M phase (p<0.01). Following pre-treatment with3μM rapamycin for6h, in damaged PC12cells, the percentage of G0/G1phase wasobviously reduced (p=0.018) and the percentage of S phase was elevated (p=0.028) ascompared with6-OHDA group.(5) Mono-treatment with rapamycin had no effect onapoptosis of normal PC12cells. However,6-OHDA induction significantly promotedapoptosis of PC12cells (p<0.001), which could be remarkably alleviated bypre-treatment with3μM rapamycin for6h (p<0.05).(6) There was no differencebetween normal and rapamycin mono-treated cells on ROS levels, and6-OHDAsignificantly promoted the production of ROS in damaged PC12cells (p<0.01).However, pre-treatment of rapamycin resulted in a reduction of ROS levels in6-OHDA-induced PC12cells (p<0.05).(7) After mono-treatment with3μM rapamycinfor6h-12h, mRNA expressions of HO-1and Nrf2were higher than in normal PC12cells (p<0.05), which fell to baseline levels in24h-48h.(8)6-OHDA inductionobviously increase P47phoxbut reduced TH mRNA expressions in PC12cells (p<0.05),which were reversed by pre-treatment with3μM rapamycin for6h (p<0.05). HO-1andNrf2mRNA expressions in damaged PC12cells induced by6-OHDA were higher thanthose in normal PC12cells (p<0.05), which were further up-regulated by pre-treatmentwith rapamycin (p<0.05).ConclusionsRapamycin could provide behavioral improvements, protect against the loss ofdopaminergic neurons and reduce expressions of pro-apoptotic markers in PD rats, which was consistent with enhanced viability and reduced apoptosis of damaged PC12cells in rapamycin-pretreated PD cell models. In PD, enhanced capabilities againstoxidative stress and changes in cell cycles may play a key role in the neuroprotectivemechanisms of rapamycin. In view of this, recovery of equilibrium between pro-andanti-oxidative stress signals may provide a promising strategy in PD therapy.更多还原