银杏平颤方对PD鼠脑氧应激作用的机制研究

【作者】 张军；

【导师】 白丽敏；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2004， 博士

【摘要】 帕金森病( Parkinson’s disease, PD)是一种多发于中老年人的慢性神经系统退行性疾病,其病理特征为黑质致密部多巴胺能神经元(dopaminergic neuron)变性、坏死,继而引起纹状体多巴胺(dopamine, DA)含量降低。最近的研究认为 DA 能神经元的损伤与氧化应激有关。抗氧化治疗是目前公认的确实有效的治疗 PD 的方案,也是今后的发展趋势。因此,寻找一种毒副作用小、效果明显的抗氧化药物将具有重要的临床意义。 我们在预实验中发现银杏平颤方具有明显的抗氧化和清除自由基的作用,同时还具有调节脑铁代谢的作用,而氧化应激和铁均参与了 PD 的发生发展过程,提示银杏平颤方可能具有治疗 PD 的作用。本研究以 1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP)诱发 PD 小鼠模型,研究银杏平颤方及其拆方对PD鼠的抗氧化应激作用,以探讨中医药治疗PD的机理。 本研究选取 C57BL/6J 品系雄性小鼠,随机分为六组:正常对照组(N)、模型对照组(M)、全方治疗组(G)、解毒治疗组(J)、平肝治疗组(P)和化瘀治疗组(H)。其中正常对照组不作任何处理,模型对照组和中药治疗组以 MPTP(30mg/kg,i.p)连续 5 天给药制备 PD 小鼠模型,从造模第一天起模型组给予生理盐水灌胃,中药治疗各组给予银杏平颤方及其拆方中药提取液每日两次灌胃给药,同时进行爬杆实验,分别于治疗 14 天和 28 天后处死、取材。应用高效液相色谱法(high peformance liquid chromatography, HPLC)测定小鼠前脑内多巴胺(dopamine, DA)和高香草酸 (homovanillic acid, HVA)的含量;应用光电比色法检测小鼠前脑纹状体内 SOD、MDA 和 GSH-Px 含量;应用免疫组织化学方法检测黑质和纹状体神经元型一氧化氮合酶(nNOS)、硝基化酪氨酸(NT)、铁蛋白(Fn)的表达;原位杂交技术观察nNOSmRNA的分布和表达。本实验分为六个部分,其主要结果如下:1. 银杏平颤方及其拆方对 PD 小鼠行为学的影响 爬杆实验结果显示,模型对照组(Model control,MC)小鼠均出现不同程度的行为障碍和自发性活动减少,评分结果显示模型对照组较正常对照组(Normal control,NC)和中药银杏平颤方及其拆方治疗组明显升高;中药治疗各组得分接近正常对照组,以 28d 组尤为明显,经统计学处理差异显著(P<0.05);但 14d、28d 各治疗组之间比较无统计学意义。提示银杏平颤方及其拆方具有改善PD小鼠行为障碍的作用2. 银杏平颤方及其拆方对 PD 小鼠 DA 神经元和 DA 及其代谢产物的影响 HE 染色结果显示:与正常组比较,模型组小鼠中脑黑质致密部 DA 神经元明显减少;而中药治疗各组(G、J、P、H组)与模型组相比神经元数目明显增多并接近正常组。 HPLC 结果显示:模型组小鼠纹状体内 DA、DOPAC 和 HVA 含量较正常组降低约 70%以上,HVA/DA 比值增高;中药各治疗组(G、J、P、H 组:14d,28d)与模型组比较小鼠纹状体内DA、DOPAC和HVA含量明显增高 (14天组P<0.05;28天组P<0.01=,HVA/DA比值降至正常。 以上结果提示银杏平颤方及其拆方具有保护黑质致密部DA能神经元的作用。3.银杏平颤方及其拆方对帕金森病(PD)模型小鼠前脑纹状体 SOD、MDA 和 GSH-Px 含量的影响<WP=5>2 银杏平颤方对PD鼠脑氧应激作用的机制研究 实验结果显示:模型组 SOD、GSH-Px 活力明显低于正常组(P<0.001),而 MDA 含量显著高于正常组(P<0.001); 14 天、28 天各中药治疗组脑组织中 SOD、GSH-Px 活力明显高于模型组(P<0.001),而 MDA 含量低于模型组(P<0.001)。提示银杏平颤方及其拆方能提高抗氧化酶活性,减轻脂质过氧化损伤。4.银杏平颤方及其拆方对 PD 小鼠黑质、纹状体 nNOS 和 NT 表达的影响4.1 各组小鼠脑组织纹状体、黑质 nNOS 的表达 结果显示:①模型组 14d 纹状体、黑质 DA 能神经元胞质 nNOS 表达量较正常组 14d 显著升高( P <0.01) ;全方组14d纹状体和黑质与正常组比较差异均不显著(P>0.05),与模型组比较nNOS表达明显降低且差异显著(P<0.05)。②28d各组统计结果同上。4.2 各组小鼠脑组织纹状体、黑质 NT 的表达 结果显示:①模型组 14d 纹状体、黑质 DA 能神经元胞质 NT 表达量较正常组显著升高(P < 0.01) ;全方组 14d 纹状体 NT 表达与模型组 14d 相比降低不明显,差异不显著(P>0.05),与正常组相比差异显著(P<0.05);全方组 14d 黑质 NT 表达较模型组下降,但与正常组和模型组比较均有显著性差异(P <0.01)。②模型组 28d 纹状体 NT 表达与正常组比较亦升高(P<0.05),而黑质 NT 表达较正常组升高,且有显著性差异(P<0.01)。全方组 28d 纹状体 NT 表达较模型组 28d 明显降低,接近正常组,与模型组比较差异较显著(P<0.05),与正常组比较差异不显著(P>0.05);全方组 28d 黑质与正常组 28d 比较虽有统计学意义(P<0.05),但较模型组28d NT表达明显降低且差异较显著(P<0.01)。 以上结果提示银杏平颤方及其拆方具有抑制 PD 小鼠黑质、纹状体 nNOS 和 NT 表达的作用 5.银杏平颤方对 PD 小鼠脑内铁离子和铁蛋白含量的影响 铁组化染色显示:模型组(14d,28d)在纹状体和黑质部位的阳性细胞平均光密更多还原

【Abstract】 Parkinson’s disease(PD) is a chronic neurodegenerative disease of nervoussystem.which is mainly characterized by a progressive degeneration and necrosis ofdopaminergic neurons in substantia nigra(SN), as well as the decrease of dopamine(DA)content in striatum. The theory on the oxidative stress-free radicals play avery important role in the death of dopaminergic neurons in PD, and anti-oxidanttherapyis currently regared as an effective strategy to halt the progression ofPD.Therefore, it is Clinically significant to explore the effective anti-oxidantdrug with mild toxicity.Ginkgo Biloba Pingchan Recipe ( GBPR ) is a complex mixture constituted ofpolypeptides that exhibiting different pharmacological activities. Our previous workfound that Ginkgo Biloba Pingchan Recipe had the effect of clearing out of theoxygen-derived free radicals by in vitro experiments. In addition, Ginkgo BilobaPingchan Recipe resisted to the toxicity of Fe. Whereas, oxidative stress andneurotoxicity of Fe are both involved in the progression of PD. This indicates thatGinkgo Biloba Pingchan Recipe possibly possesses the therapeutic effect on PD. Inthis study, the protective effect of Ginkgo Biloba Pingchan Recipe on dopaminergicneurons in SN and CPu was investigated by the PD model mice treated with l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP). Meanwhile, the cytomolecular mechanism ofGinkgo Biloba Pingchan Recipe on therapeutic was also studied. In experimental research,male C57BL/6J mice were intraperitoneal injected withMPTP(30mg/kg/day, i.s.c.)for 5days to establish PD model. The modeled mice arecalled Parkinsonian mice.The experimental animals is divided randomly into sixgroups:(1)The normal control group(NC),(2)The model contral group(MC),(3)GinkgoBiloba Pingchan Recipe group(GBPR),(4)Jiedu Recipe group(JDR),(5)Pinggan RecipeGroup(PGR),(6)Huayu Recipe group(HYR).The doses of Ginkgo Biloba Pingchan Recipe andits three similar prescriptions were respectively given for 2 weeks and 4 weeks inthe treatment groups.The animals were killed respectively on the 14th and the 28th dayafter first injecting with MPTP.Animals were examined behaviorally with the poletest consecutively. The brains were then removed immediately and the striatum weredissected out. Contents of Dopamine (DA), dihydroxy-phenyl acetic acid (DOPAC) andhomovanillic acid(HVA)were measured by high performance liquid chromatography withelectrochemical detection (HPLC-EC), MDA, SOD, GSH-Px were measured in forebrainstriatum of model mouse for PD in spectrophotometer. The remained tissue of brainswere fixed with 10% formaldehyde and the coronally 6um-thick sections from<WP=8>英文摘要 5mesencephalon were prepared for immunocytochemistry(ICC).Tyrosine hydroxylase(TH),constitutive nitric oxide synthase (nNOS) , nitrotyrosine (NT)and ferritin(Fn) wereobserved by ICC. The expression of nNOSmRNA were detected by in situ hybridization(ISH). The results are as followings: 1.Ginkgo Biloba Pingchan Recipe(GBPR) and its three similar prescriptionsimproved the dyskinesia of PD miceAll the mice intra peritoneal injected with MPTP showed different disability inpole the test and decrease of spontaneous motor activity, the model group(MPTP+NS ) mice demonstrated dyskinesia, scores obtained from the behavioral test wereincreased significantly compared with Normal control group(NS+Ns), but there wasno different change in Normal contral(NC) group in pole test. While the scores intreatment groups (Ginkgo Biloba Pingchan Recipe(GBPR) and its three similarprescriptions)showed a marked decrease to some degree. Chinese medicine groupsimproved disability in pole test and increased spontaneous motor activity(allP<0.05). But there was no difference among the Chinese medicine groups(P>0.05).2.Ginkgo Biloba Pingchan Recipe(GBPR) and its three similar prescriptionsprotected DAergic neurons in substantia nigra compacta(SNpc)The IC更多还原