【作者】 闫华；

【导师】 李浩； 刘剑刚；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2011， 硕士

【摘要】 背景：老年人轻度认知功能障碍(mild cognitive impairment,MCI),现在已经被公认为是“痴呆临床前期状态”, MCI患者与早期AD患者存在着很多相同点,二者皆为以进行性认知功能减退为特征的中枢神经系统(Central nervous system,CNS)病变。流行病学调查显示,随着全球人口的老龄化提速,认知功能障碍和痴呆的发病率呈不断上升趋势,到2030年全球痴呆患病人数将达到6570万,到2050年将增至1亿1540万,迄今为止,认知功能障碍及痴呆发病机制尚不十分清楚,更缺乏有效的治疗药物及措施。祖国传统医学根据辨证论治的观点,充分发挥中药复方所特有的多环节、多途径、整体调节的作用特点,在防治MCI与早期AD方面具有一定的优势,基于中医“虚”、“瘀”、“浊”、“毒”病机理论组方的还脑益聪方,前期临床及基础研究现证实对MCI及AD有较好的防治作用,实验将还脑益聪方经现代工艺提取其组分,研究其对APP695V717I转基因痴呆小鼠模型的早期干预作用,并初步探讨其作用机制,为未来中医中药更为广泛地应用于临床MCI的预防及治疗提供依据。目的：基于胆碱能系统功能异常和自由基(Free radicals,FR)失衡理论导致认知功能减退的发病机制,以APP695V717I转基因小鼠为实验动物模型,应用以补肾益气、活血解毒功用为组方配伍依据的还脑益聪方对模型小鼠进行早期干预,观察动物的行为学改变,研究此中药复方对APP转基因小鼠脑组织Ap生成途径及胆碱能系统和氧化应激(Oxidative stress,OS)等相关指标的影响,探讨还脑益聪方提取物早期干预认知功能障碍的作用机制,为MCI患者与AD患者的中医药防治提供药理依据。方法：选用3月龄APP695V717I转基因小鼠,雌雄各半,随机分为模型组、阳性药物(盐酸多奈哌齐)对照组(按0.65mg/kg-d给药)、还脑益聪方提取物大剂量组(按2.8g/kg·d给药)及还脑益聪方提取物小剂量组(按1.4g/kg-d给药),并设立遗传背景相同的非转基因小鼠作为正常对照组(C57BL/6J小鼠),连续灌胃4个月(至7月龄)后进行相关指标检测。本实验共分为三个部分。实验一,采用Morris水迷宫观测APP转基因模型小鼠学习、记忆等认知相关能力,采用跳台实验(被动回避性条件反射)观测小鼠一次性被动回避反应记忆功能,研究还脑益聪方提取物对APP转基因模型小鼠行为学的影响；实验二,采用免疫组织化学方法检测APP、Aβ1-42在小鼠海马CA1区的表达,通过还脑益聪方提取物早期干预对APP695转基因小鼠脑组织Aβ生成途径的影响,探讨还脑益聪方防治的作用机制。实验三,采用酶联免疫吸附试验(ELISA)测定小鼠皮层及海马中乙酰胆碱(Acetylcholine,ACh)、乙酰胆碱酯酶(Acetylcholinesterase,AchE)、胆碱乙酰转移酶(Choline acetyltransferase,ChAT)的含量,研究还脑益聪方对APP转基因模型小鼠中枢胆碱能系统功能的影响；并采用比色法检测APP转基因小鼠血清超氧化物歧化酶(Superoxide Dismutase,SOD)的活性,硫代巴比妥酸法测定小鼠血清丙二醛(Malondialdehyde,MDA)的含量,观察还脑益聪方对APP转基因模型小鼠脑组织氧化应激损伤的影响。结果：实验一,还脑益聪方提取物早期干预对APP转基因模型小鼠行为学的影响。Morris水迷宫实验结果表明：与正常对照组相比,7月龄模型组小鼠穿越原平台位置的次数、第四象限游泳时间及路程均显著缩短和减少(P<0.01或P<0.05)；药物治疗后,小鼠在Morris水迷宫中穿越原平台位置次数、第四象限游泳时间及路程均较模型组显著提高(P<0.05或P<0.01)。跳台实验结果表明：与正常对照组相比,模型组错误次数显著增高(P<0.05),潜伏期显著缩短(P<0.05)；与模型组相比,还脑益聪方提取物大剂量组错误次数明显减少,有显著性差异(P<0.05),盐酸多奈哌齐组、还脑益聪方提取物小剂量组错误次数呈现减少趋势,但无显著性差异(P>0.05)；还脑益聪方提取物大剂量组潜伏期显著延长(P<0.01),盐酸多奈哌齐组、小剂量组潜伏期亦有延长趋势,但无显著性差异(P>0.05)。实验二,还脑益聪方提取物早期干预对APP转基因小鼠脑组织APP、Aβ蛋白表达的影响。APP转基因小鼠海马CA1区均可见棕黄色阳性表达,分布于神经元胞浆和胞膜。与正常组比较,模型组APP阳性细胞数目和面积比例均显著增多(P<0.01)。各给药组治疗后,APP阳性细胞数目较模型组均显著减少(P<0.01)；还脑益聪方提取物大、小剂量组APP阳性细胞面积比例均显著减少(P<0.05或P<0.01)；盐酸多奈哌齐组有减小趋势,但无统计学差异(P>0.05)。与正常组比较,模型组Aβ阳性细胞数目和面积比例均显著增多(P<0.01)。各给药组治疗后,Aβ阳性细胞数目和面积比例均较模型组显著减少(P<0.05或P<0.01)。实验三,还脑益聪方提取物早期干预对APP转基因模型小鼠中枢胆碱能系统功能及氧化应激损伤指标的影响。与正常对照组相比较,7月龄模型组小鼠海马区ACh及ChAT的含量均明显降低,AchE的含量明显增加,经统计学处理,有显著性差异(P<0.05)；与模型组相比较,盐酸多奈哌齐组、还脑益聪方提取物大、小剂量组均能提高小鼠海马区ACh的含量,经统计学处理,有显著性差异(P<0.05,P<0.01)；与模型组相比较,多奈哌齐组、还脑益聪方提取物大剂量组均能显著降低小鼠海马区AchE的含量(P<0.05)；与模型组比较,盐酸多奈哌齐、还脑益聪方提取物大、小剂量组均能显著提高小鼠海马区ChAT的含量,经统计学处理,有显著性差异(P<0.05,P<0.01)。与正常对照组比较,7月龄模型组小鼠脑皮层中ACh、ChAT的含量均明显降低,AchE含量明显增加,经统计学处理,差异有显著性(P<0.05)；与模型组相比较,还脑益聪方提取物小剂量组能明显提高模型组小鼠脑皮层中ACh的含量,经统计学处理,差异有显著性(P<0.05)；与模型组相比较,各组小鼠经给药治疗后测定脑皮层中AchE的含量,经统计学处理,无显著性差异(P<0.05)；与模型组相比较,还脑益聪方提取物小剂量组能提高模型组小鼠脑皮层中ChAT的含量,经统计学处理,差异有显著性(P<0.05),而盐酸多奈哌齐组及还脑益聪方提取物大剂量组ChAT的含量无明显变化(P<0.05)。与正常对照组比较,模型组小鼠血清SOD活性显著降低(P<0.01)；与模型组比较,还脑益聪方提取物大、小剂量组的血清SOD活性有显著升高(P<0.05或P<0.01)；与模型组比较,盐酸多奈哌齐组的血清SOD活性有显著升高(P<0.05)；与正常对照组比较,模型组小鼠血清MDA含量显著升高(P<0.01)；与模型组比较,各给药组血清MDA含量有减少的趋势,但无显著性差异(P>0.05)。结论：应用还脑益聪方提取物进行早期干预可以在一定程度上改善APP转基因模型小鼠空间学习、记忆等认知功能及一次性被动回避反应记忆功能,其机制可能与改善小鼠脑皮层及海马的中枢胆碱能系统功能及抑制脑组织的氧化应激损伤有关。更多还原

【Abstract】 Background:The mild cognitive impairment of old people, has now been recognized as the "Early clinical dementia status",patients with MCI and early AD have many similarities,both of them are central nervous system lesions characterized by Progressive cognitive decline. Epidemiological survey shows,with the accelerated aging of the global population,mild cognitive impairment and the incidence of dementia showed a continually rising trend,the number of Global prevalence of dementia by 2030 will reach 65.7 million,the year of 2050 will be increased to 100 million and 15.4 million,but so far, cognitive impairment and dementia as a worldwide medical problem, the pathogenesis is not very clear,In particular, the lack of effective treatments and measures. The treatment of Traditional Chinese Medicine (TCM), based on syndrome differentiation, has superiority and prospect at the prevention and treatment of MCI and AD which is beyond the reach of western medicine, especially by the role of Chinese herbal compound, which has many curative pathways and targets to MCI and AD. Huannao Yicong Fang (HNYCF) is a Chinese herbal compound based on"Deficiency-Blood Stasis-Turbidity-Toxin",the pathogenesis of MCI and AD in TCM.It has been confirmed by Early experiments and clinical research that HNYCF has good preventive and therapeutic effects to MCI and early AD,In this experiment, we use modern technology to extract the fractions of HNYCF, In-depth study its mechanism and effective channel to APP695V717I transgenic mouse model,so as to provide basis to the TCM is more widely used in clinical treatment of MCI and early Alzheimer’s disease.Objective:Based on cognitive dysfunction in the pathogenesis of the Cholinergic system dysfunction and free radical imbalance theory, APP695V717I transgenic mice 3 month-old were used as models in this study,application of the extract of HNYCF (herbal extract) on the model of early intervention,observe the behavior of the model, generation of beta amyloid protein(Aβ),cholinergic system and oxidative stress-related index changes,discussion the mechanism of HNYCF in the treating of MCI and AD.Methods:APP695V717I transgenic mice 3 month-old were used as models in this study, which were randomly divided into model group, Donepezil group, the extract of HNYCF large dose group and the extract of HNYCF small dose group. Normal control adopted the same age and background C57BL/6J mice. The animals were administered intragastrically by the drug or water from 3 month-old to 7 month-old. This research was divided into three parts. PartⅠ: Morris water maze test was performed to measure the spatial learning、memory and other cognitive-related capacity.Step-down test (Passive avoidance conditioning) was performed to observe the learning and memory ability of single passive avoidance response, This part was to study HNYCF on the behavior of APP transgenic mice. PartⅡ:The expression ofβ-amyloid precursor protein (APP),β-site APP cleaving enzyme (BACE) and Aβin hippocampus CA1 region was detected by immunohistochemistry with image analysis. PartⅢ:Determination the content of ACh, AchE, ChAT by ELISA and Bradford method,This part was to study the extract of HNYCF on central cholinergic function of APP transgenic mice. The activity of superoxide dismutase (SOD) in serum was detected with colorimetry. The content of malondialdehyde (MDA) in serum was detected with Thibabituric Acid (TBA). This part was to study effects of HNYCF on free radical-induced oxidative stress in the brain of APP transgenic mice.Results:PartⅠ:The Early intervention with extract of HNYCF to improve the disorder of cognitive function in APP transgenic mice of the experimental observation of behavior.Morris water maze test results show that:Compared with the control group, model mice through the security platform for the location of the original number, in the fourth quadrant swimming time and distance were significantly shortened and reduced (P<0.01 or P<0.05); after treatment, mice in the Morris water maze in the position of crossing the number of security platform, in the fourth quadrant swimming time and distance compared with model group was significantly increased (P<0.05 or P<0.01);Step down test results show that:Compared with the control group, the number of errors of model group was significantly higher (P<0.05), latency was significantly shorter (P<0.05); Compared with model group, the extract of HNYCF large dose group had significantly fewer errors, there was significant difference (P<0.05), Donepezil hydrochloride group, the extract of HNYCF small dose group group showed a decreasing trend in the number of errors, but no significant difference (P>0.05);Compared with model group,the extract of HNYCF large dose group was significantly longer latency (P<0.01),Donepezil hydrochloride group, low dose group also extended incubation period of trend, but no significant difference (P>0.05).PartⅡ:Effect of early intervention with extract of HNYCF on the pathway of APP transgenic mouse brain AβgenerationComparing to the model group, the levels of expression of APP in hippocampus CA1 region of mice in the HNYCF group decreased obviously (P<0.01), including the number and area proportion of positive cells;the levels of expression of APP in hippocampus CA1 region of mice in the Donepezil group had the trend to reduce, but there was no statistical significant difference (P>0.05); The expression of Aβin hippocampus CA1 region of mice in all the treated groups decreased obviously (P<0.05 or P<0.01).Part III:Effect of early intervention with extract of HNYCF fractions on the cholinergic system and related indicators of Free radical-induced oxidative stress of APP transgenic mice.Comparing to the model group, model mice hippocampus ACh and ChAT levels were significantly reduced, AchE were significantly increased, through statistical analysis, the difference of the data was significant (P<0.05); Compared with model group, group of donepezil hydrochloride, the extract of HNYCF large and small dose group can effectively improve the content of ACh in hippocampus of mice, through statistical treatment, there was significant difference (P<0.05, P<0.01);Compared with model group, donepezil group and the extract of HNYCF large dose group could decrease the AchE consent of the hippocampus in mice, the difference was significant (P<0.05);Compared with model group, donepezil hydrochloride, the extract of HNYCF large and small dose group could significantly increase the content of ChAT in hippocampus of mice, through the statistical treatment, there was significant difference (P<0.05, P<0.01).Compared with the control group, model group mice cortex ACh, ChAT levels were significantly reduced, AchE was significantly increased, the statistical difference was statistically significant (P<0.05); Compared with model group, the extract of HNYCF large and small dose group could significantly improve the model mice in the ACh content of cortex, the statistical difference was statistically significant (P<0.05); Compared with model group, the mice treated by the administration to determine the content of AchE cerebral cortex, the statistical treatment, no significant difference (P<0.05); Compared with model group, the extract of HNYCF small dose group can improve the model mice brain cortex ChAT levels, the statistical difference was statistically significant (P<0.05),the donepezil hydrochloride group and the extract of HNYCF large dose group have no significant change in ChAT levels (P<0.05).Compared with the control group,serum SOD activity in model group were significantly different (P<0.01); Compared to the Serum SOD activity of the model group,the extract of HNYCF large and small dose group were significantly higher (P<0.05 or P<0.01); Compared to the Serum SOD activity of the model group,Donepezil hydrochloride group significantly increased (P<0.05); Compared with the control group, the Serum MDA levels of the model group,were significantly different (P<0.01); Compared to the Serum MDA levels of the model group,each treatment group of serum MDA content showed a decreasing trend, but no significant difference (P>0.05).Conclusions:Apply HNYCF effective fractions to early intervention, to a certain extent, improve the APP transgenic mouse model of spatial learning, memory and other cognitive function as well as the learning and memory ability of single passive, The mechanism may be related to the protective effect of the extract of HNYCF on the Cerebral cortex and hippocampus of the central cholinergic system function, and to improve the free radical induced oxidative stress in brain.更多还原