【作者】 薛莉君；

【导师】 祝慧凤；

【作者基本信息】 西南大学 ， 微生物与生化药学， 2012， 硕士

【摘要】 背景与目的：脑血管疾病是目前世界公认的人类三大致死疾病之一。脑缺血患者存在神经功能缺失,偏瘫感觉减退和认知功能损伤等症状,影响日常活动或运动功能康复,严重的可引起死亡,危害人类的健康。临床上目前缺医少药,缺乏既能促进神经功能恢复,又能改善认知功能的药物。梓醇是中药地黄的主要活性成分之一,具有神经保护、促进缺血脑区血管新生、改善记忆等药理作用,因此可以作为一个潜在的新药物来治疗中风和提高记忆能力。本实验分别选择目前临床用于治疗中风(依达拉奉,edaravone)和促智的药物(奥拉西坦,oxiracetam)为阳性对照,研究梓醇能否治疗脑缺血,并改善学习记忆功能,旨在与现有临床药物比较,探讨梓醇治疗脑相关疾病的优势。在此基础上探讨梓醇改善记忆的可能机制。方法：第一部分：梓醇与依达拉奉、奥拉西坦治疗小鼠脑缺血的比较研究(1)模型制备与给药小鼠分为5组：假手术组,模型组,奥拉西坦组,依达拉奉组,梓醇治疗组。采用经颈内动脉线栓法制备小鼠左侧的大脑中动脉闭塞脑缺血模型,于术后24 h开始尾静脉注射药物,连续3天。(2)神经行为学评分小鼠术后1、2、3天参照Zea longa的5分制标准进行神经行为学评分,评价各组神经功能恢复状况。(3)激光多普勒血流仪(LDF)检测小鼠局部脑血流量术后第3天采用LDF检测小鼠局部脑血流量(rCBF).(4)小鼠大脑梗死面积测定每组5只小鼠脑切片置于0.5% 2,3,5-氯代三苯基四氮唑(triphenyltetrazolium chloride, TTC)中染色测定脑梗死面积。(5)大脑缺血侧海马组织HE和尼氏染色每组5只小鼠大脑进行冰冻切片,HE和尼氏染色,镜下观察大脑缺血侧海马组织神经元形态结构。第二部分：梓醇与依达拉奉、奥拉西坦治疗小鼠学习记忆障碍的比较研究(1)记忆障碍模型制备与药物干预小鼠分为7组：正常对照组,模型组,奥拉西坦组,依达拉奉组,梓醇低、中、高剂量组。造模前每组尾静脉注射药物(正常对照组和模型组给予等体积生理盐水),连续3天后,进行Morris水迷宫,每次测试前腹腔注射氢溴酸东莨菪碱2 mg/(kg·次),制备记忆障碍模型。(2)Morris水迷宫测试水迷宫测试分为定位航行实验和空间探索实验,前3天为定位航行实验,第4天为空间探索实验。第三部分：梓醇改善小鼠学习记忆障碍的机制研究(1)水迷宫测试结束后,将各组小鼠眼眶取血,分离大脑海马待用。(2)比色法检测小鼠的乙酰胆碱酯酶(AChE)和乙酰胆碱转移酶(ChAT)的活性；ELISA定量检测乙酰胆碱(ACh)和脑源性神经营养因子(BDNF)含量.(3) Western blot半定量检测小鼠大脑海马的胆碱受体M1、M2、ChAT和AChE的蛋白表达。结果：(1)梓醇对脑缺血小鼠的神经行为学的影响采用Zea longa神经功能评分评定脑缺血后1、2和3天各实验组神经缺失功能恢复状况,证实梓醇对脑缺血后功能恢复有促进作用。在脑缺血后第3天,梓醇高剂量组和依达拉奉组的Zea longa神经功能分数分别为2.10±0.32和2.00±0.47,与模型组(2.50±0.53)比较差异有显著性(P<0.01)；而奥拉西坦组(2.22±0.44)与模型组差异无显著性(P>0.05)。提示梓醇和依达拉奉可促进脑缺血小鼠神经功能恢复,而奥拉西坦不明显。(2)梓醇对脑缺血小鼠脑血流值变化率的影响在脑缺血后第3天测量小鼠局部脑血流值,与模型组小鼠的脑血流值变化率(88.59%±0.55%)相比,依达拉奉组(72.56%±3.24%,P<0.05)和梓醇高剂量组(60.99%±1.99%,P<0.05)明显降低,而与奥拉西坦组(89.83%4.50%)差异无显著性(P>0.05)。提示梓醇和依达拉奉明显改善脑缺血后小鼠脑血流值,而奥拉西坦不明显。(3)梓醇对小鼠脑梗死面积的影响脑缺血后第3天测量脑梗死面积,与假手术组(0±0)比较,其他各实验组的梗死面积明显增大(P<0.01)。与模型组(18.20%±0.42%)相比,依达拉奉组(10.65%±0.58%)和梓醇高剂量组(11.14%±0.5%)梗死面积均明显降低(P<0.05),而奥拉西坦组(18.19%±0.41%)差异无显著性(P>0.05)。提示梓醇治疗明显减少小鼠脑梗死面积,而奥拉西坦不明显。(4)梓醇对小鼠大脑缺血侧的海马组织神经元形态学的影响HE染色：假手术组海马神经元细胞排列致密、整齐,结构正常,胞体较大,染色质分布均匀,核仁明显；模型组神经细胞变性坏死,周围间隙扩大,胞浆和胞核皱缩、染色加深,细胞体积缩小,细胞数量减少；奥拉西坦组神经细胞较模型组整齐,但也可见神经元变性坏死；与模型组和奥拉西坦组比较,依达拉奉组和梓醇组的海马神经细胞明显整齐,排列致密,形态较正常,胞体较大,染色质分布均匀,核仁比较明显。尼氏染色结果同时显示,假手术组神经元结构完整,胞体体积较大,细胞轮廓清楚,尼氏小体呈深蓝色斑块；模型组神经元,胞体变小胞核固缩、胞浆减少,神经元数量较假手术组比较明显减少；奥拉西坦组同模型组相似,神经元减少,排列紊乱疏松；依达拉奉组和梓醇组的神经元的存活数量较模型组比较明显增加,整齐形态规整,胞浆均匀。提示梓醇和依达拉奉能明显改善小鼠大脑海马组织神经细胞的损伤程度,而奥拉西坦不明显。(5)梓醇对小鼠水迷宫的影响定位航行实验测试结果显示,模型组小鼠逃避潜伏期(108.40 s±11.34 s)与正常对照组(102.47 s±20.62s)比较差异有显著性(P<0.05)；第3天,与模型组(102.07 s±13.93 s)比较,奥拉西坦组(74.36 s±8.29s)和梓醇中高剂量组(76.66s±19.27s和66.20s±13.20s)均显著缩短小鼠找到隐匿平台时间(P<0.05),但模型组与依达拉奉组(89.63 s±20.86s)无明显差异(P>0.05)。空间探索实验测试结果显示,模型组小鼠跨越原平台次数(0.40±0.70)与正常对照组(3.89±1.97)比较差异显著(P<0.01),奥拉西坦组(3.30±1.16)和梓醇各剂量组(2.33±0.86、3.40±0.70、4.70±0.67)均能显著增加跨越平台次数(P<0.05)。提示梓醇具有改善学习记忆的能力。(6)比色法检测梓醇对乙酰胆碱酯酶和乙酰胆碱转移酶活性的影响与模型组(0.454±0.202)比较,奥拉西坦组的小鼠海马中的AChE的蛋白活性(0.293±0.063)明显降低(P<0.05),而其它各实验组间无显著性差异(P>0.05)。与正常对照组(0.322±0.039)比较,模型组的小鼠海马的ChAT的蛋白活性(0.173±0.024)明显降低(P<0.05)；与模型组相比,奥拉西坦组(0.284±0.017)和梓醇中、高剂量组(0.279±0.043和0.319±0.067)明显升高(P<0.05)；而与依达拉奉组(0.367±0.081)比较无显著差异(P>0.05)。提示梓醇和奥拉西坦可明显降低海马中ChAT的蛋白活力,而依达拉奉却不明显。(7) ELISA检测梓醇对ACh及BDNF影响与模型组比较,梓醇中、高剂量组和奥拉西坦组的小鼠大脑海马ACh和BDNF含量都明显增加(P<0.01)；且梓醇中、高剂量组和奥拉西坦组小鼠大脑海马ACh及BDNF含量均超过正常对照组,梓醇治疗组和奥拉西坦组ACh含量与BDNF含量组间比较差异无显著性(P>0.05)。BDNF与ACh的含量进行线性回归分析显示二者有较强相关性(r=0.859,P<0.05)。提示梓醇能改善丁溴东莨菪碱诱导的学习记忆机能障碍,明显上调脑海马ACh和BDNF水平。(8) Western blot检测梓醇对大脑海马的胆碱受体M1、M2、ChAT和AChE的影响与正常对照组(M1：0.98±0.05,M2：0.49±0.01)比较,模型组的小鼠海马的M1、M2受体的相对蛋白条带光密度值(0.24±0.00和0.24±0.00)均显著增加(P<0.01)。与模型组相比,梓醇低、中、高剂量组和奥拉西坦组的小鼠大脑海马M1受体相对蛋白条带光密度值(0.35±0.00、0.56±0.02、0.73±0.01和0.45±0.02)、M2受体相对蛋白条带光密度值(0.33±0.01、0.42±0.01、0.62±0.03和0.32±0.02)明显增加(P<0.01),依达拉奉组的M1受体(0.46±0.01)蛋白表达也明显增加(P<0.01)。提示梓醇能明显上调Ml、M2受体的蛋白表达。与正常对照组(0.19±0.00)比较,模型组的小鼠海马的AChE的蛋白条带光密度值(0.22±0.01)显著降低(P<0.01)。与模型组比较,梓醇低、中、高剂量组(0.12±0.00、0.08±0.00和0.19±0.01)和奥拉西坦组(0.15±0.00)的小鼠大脑海马AChE的蛋白条带光密度值明显降低(P)<0.01),而依达拉奉组的AChE的蛋白条带光密度值(0.29±0.02)却明显增加(P<0.01)。提示梓醇能明显降低AChE的蛋白表达。与正常对照组(0.83±0.03)比较,模型组的小鼠海马的ChAT的蛋白条带光密度值(0.53±0.03)显著降低(P<0.01)。与模型组比较,梓醇高剂量组(0.62±0.02),奥拉西坦组(1.06±0.01)和依达拉奉组(0.60±0.03)的小鼠大脑海马ChAT蛋白条带光密度值表达明显增加(P<0.01)。提示梓醇能明显上调ChAT的蛋白表达。结论：与临床现有抗脑缺血药物依达拉奉比较,梓醇和依达拉奉均可治疗脑缺血,但梓醇具有增强学习记忆能力的优势；与奥拉西坦比较,梓醇不仅显著促进学习记忆,而且具有显著的治疗脑缺血前景；梓醇既可以抗脑缺血,同时可以改善学习记忆障碍,其机制与胆碱能系统和BDNF蛋白表达上调有关。更多还原

【Abstract】 Background and Purpose:As one of the big three human deadly disease in the world, stroke has disability, neurological deficits, cognitive impairments, and so on. But clinic is lack of drug not only to improve cognitive impairments but also to promote the neurobehavior function recovery. Catalpol is an iridoid from Rehmannia glutinosa, which is a widely used as traditional Chinese medicine for Neuroprotective or neurodegenerative diseases. So catalpol is a potential new drug to treat stroke and improve memeory.This study was performed to determine advantage of catalpol compared with a free radical scavenger and a neuroprotectives as edaravone, and memory improvement drug as oxiracetam at first, and then to investegate the mechanisms especialy there are differences between edaravone and catalpol.Methods:Part One:The comparative study of catalpol, edaravone and oxiracetam on the cerebral ischemic(1) The pMCAO model and drug administrationExcept the sham group, all mouse were underwent the left side of permanent middle cerebral artery occlusion. Animals were divided into 5 groups:sham, model, oxiracetam, edaravone, catalpol as treatment group. Drugs were injected into the tail vein for three days.(2) Zea Longa’s ScoreThe neurological function of all mouse was evaluated daily with a 5-point scale after 1.2 and 3 day stroke.(3) Cerebral blood flow ratiosThree days later, we measured blood flow ratios in the difference between normal (right) and ischemic (left) brain by LDF.(4) TTC stainingFive mouse were taken respectively from each of the group to make TTC staining after 3 days.(5) HE and Nissl stainingThe ischemic (left) brain of hippocampus was displayed by HE and Nissl staining. Part Two:The comparative study of catalpol, edaravone and oxiracetam on memory impairment(1)The memory obstruction model model and drug administrationAnimals were divided into 7 groups:normal, model, oxiracetam, edaravone, three doses of catalpol as treatment group. In all groups, except for the saline control, learning and memory dysfunction in mice was induced by SCOP (2 mg·kg-1) intraperitoneal injection 30 mins before behavioral testing after drugs were injected into the tail vein for three days..(2) Morris water maze testMorris water maze tesconsists of positioning navigation experiment and space exploration experiment.Part Three:Effect of catalpol on memory impairment of mechanisms(1) Following the Morris water maze test, blood and serum were collected via the fossa orbitalis vein. The hippocampus of the brains were removed after decapitation.(2 ELISA analysisThe activity of ChAT and AChE were measured by chromometry,the level ACh and BDNF were measured by ELISA.(3) Western blot analysisWestern blot analysis of the effect of Catapol on the expression of M 1、M 2、ChAT and AChE in hippocampus. Results:(1) Effect of catalpol on NeuroethologyCatapol and edaravone significantly reduced Zea Longa’s scored<0.05) compared with the model group. The results suggested that catapol can improve neurobehavioral outcome.(2) Effect of catalpol on cerebral blood flow ratiosCompared with the model group,catapol and edaravone significantly increased cerebral blood flow (P< 0.05).It showed that catapol can improve rCBF.(3) Effect of catalpol on cerebral infract areaThere was significant decreasment in catapol and edaravone groups (P< 0.01),compared with the model group. It showed that catapol can decrease the cerebral infract area.(4) Effect of catalpol on cytomorphology in hippocampusMouse in sham group did not show any histopathological abnormalities and those in ischemia group showed marked cell damages with pycnotic nucleus and eosinophilia in hippocampus. Oxiracetam also reduced neuronal loss and moderate morphologic changes. Edaravone and catalpol significantly decreased neuronal cell damage induced by pMCAO. Catapol and edaravone obviously improved the nerve cells in hippocampus compared with the model group.(5) Effect of catalpol on Morris water maze testCompared with the model group,catapol and oxiracetam significantly decreased escape latency time(P< 0.05) and increased the numbers of crossing platform area(P < 0.05). The results suggested that catapol can improve memory.(6) Effect of catalpol on ChAT and AChEThe AChE activity was no significant difference in the blood serum among all the groups. The hippocampal AChE activity was not significantly different between among the model and catalpol groups. ChAT activity in the hippocampus significantly increased in the oxiracetam group and catalpol (P< 0.05). The results suggested that catapol can increase ChAT activity. (7) Effect of catalpol on ACh and BDNFCompared with the model group, catapol and oxiracetam significantly increase the level of ACh and BDNF, the mechanism may be related with cholinergic system and higher BDNF expression.(8) Effect of catalpol on M 1、M2、ChAT and AChE in hippocampusCompared with the model group, catapol advanced the expression ofM 1、M2、ChAT protein,lowered the expression of AChE protein.Conclusion:Compared with memory improvement drug as oxiracetam, edaravone treated cerebral ischemic. Compared with a neuroprotectives as edaravone, catalpol and oxiracetam promoted memory. Catalpol has neurprotection effects and memory-enhancing.更多还原