【作者】 张莲珠；

【导师】 周秋丽； 张扬；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 认知是指人的大脑接受外界信息，经过加工处理后，转换成内在的心理活动，从而获取知识或应用知识的过程。认知功能障碍泛指由各种原因导致的不同程度的认知功能损害，从轻度认知功能损害到痴呆。所有直接或间接导致大脑皮层结构和功能慢性损伤的因素均可通过不同机制引起认知功能障碍。乙酰胆碱、多巴胺和去甲肾上腺素等神经递质及其受体异常，脑组织蛋白质异常修饰、异常聚集等脑神经细胞退行性变性疾病，自由基损伤、能量供应障碍、酸中毒、兴奋性毒性等引起的慢性脑缺血性损伤，以及一些会造成脑部血液供应不足的疾病等都会引起认知功能障碍。认知功能障碍多见于60岁以上老年人，属于老年痴呆的初级阶段，如果不能很好的控制，将很快发展成为老年痴呆。因此轻度认知功能障碍阶段是预防、控制和治疗老年痴呆的最佳时期。近年来，随着国际上对认知功能障碍研究的进展，其危害和重要性逐渐被认识。目前治疗认知功能障碍的药物，基本上与抗阿尔茨海默病或老年痴呆的药物一致，主要针对相应的发病原因如胆碱能神经异常、β-淀粉样蛋白堆积、炎症、铝中毒、钙平衡失调、代谢紊乱、自由基损伤以及线粒体损伤等加以治疗，可以控制患者的病情或一定程度上缓解认知功能障碍的快速发展。其中有一种药物疗效显著且被广泛接受，即以银杏叶提取物为原料的金纳多。银杏叶提取物中主要为黄酮类成分，且大部分为双黄酮。研究表明银杏叶提取物能增加脑血管流量，降低脑血管阻力，改善脑部血液循环，保护脑细胞免受缺血损害，清除有害的氧化自由基，并具有提高免疫能力和抗衰老作用。由于银杏树分布范围很小，且银杏叶产量有限，由此我们想到，其他植物中含有的双黄酮如具有与银杏双黄酮类似的功效，则可以弥补银杏叶资源不足的问题。对多种中药黄酮及双黄酮类成分活性筛选的结果表明，垫状卷柏总黄酮（total flavonids of Selaginella pulvinata，TFSP）具有较好的抗氧化和改善模型动物认知功能障碍的作用。文献资料表明，卷柏总黄酮具有抗氧化、抗糖尿病、促进人脐静脉内皮细胞增殖及血管内皮细胞生长因子表达等活性。垫状卷柏双黄酮能清除二苯代苦味肼基自由基，显著抑制溶血性磷脂酰胆碱所致内皮细胞乳酸脱氢酶的泄漏，并能减轻溶血性磷脂酰胆碱所致人脐静脉内皮细胞ECV304的损伤，且对黄嘌呤氧化酶体外抑制作用较强。目前尚未见有关TFSP对认知功能障碍影响的报道。为此，本文首先从整体水平观察了TFSP对不同阶段学习记忆功能障碍模型小鼠的治疗作用，进而在细胞水平探讨TFSP和穗花杉双黄酮（amentoflavone,AF）对正常神经细胞的促增殖作用以及对多种损伤因子致神经细胞损伤的保护和修复作用。在此基础上，本文从内质网应激、线粒体功能障碍及氧化应激等方面初步探讨了TFSP的活性成分——穗花杉双黄酮（AF）改善小鼠学习记忆功能障碍可能的作用途径。整体试验结果表明，TFSP对东莨菪碱、亚硝酸钠及乙醇造成的小鼠记忆获得、记忆巩固及记忆再现等不同阶段、不同层面的记忆功能障碍具有较明显的治疗作用。TFSP能够抑制东莨菪碱所致小鼠大脑乙酰胆碱含量降低，调节乙酰胆碱酯酶和胆碱乙酰转移酶活性，通过对胆碱能系统酶活性的影响，拮抗东莨菪碱对M-胆碱能受体的阻断作用，对抗东莨菪碱所致小鼠记忆获得障碍。对亚硝酸钠诱导的记忆巩固障碍小鼠大脑组织的过氧化损伤，TFSP不但能激活脑组织抗氧化酶活性而且能有效遏阻过氧化产物在脑内堆积，通过抗氧化途径治疗记忆巩固障碍。D-半乳糖能够导致小鼠全身性衰老，包括免疫器官萎缩、体内过氧化物和自由基堆积及抗氧化酶活性下降，TFSP通过增强免疫和抗氧化遏制小鼠全身性衰老的进程。上述整体试验结果为TFSP在细胞水平的深入研究奠定了基础。离体试验以人神经母细胞瘤细胞SH-SY5Y为对象，首先观察了TFSP及AF对正常状态下SH-SY5Y细胞生长的影响，发现在一定浓度范围内TFSP和AF对SH-SY5Y细胞具有促增殖作用。当SH-SY5Y细胞与过氧化氢、冈田酸、Aβ25-35三种细胞毒性因子共孵育时，光镜下看到大量细胞死亡，细胞存活率明显下降。提示，过氧化氢、冈田酸、Aβ25-35造成神经细胞不同程度的损伤，可以作为神经细胞损伤研究的离体模型。从计算的IC50值分析，冈田酸的细胞毒性最强。我们的研究发现，AF对三种毒性因子造成的细胞损伤呈现出不同程度的保护和修复作用，其中对过氧化氢造成的细胞损伤保护作用最明显。推测， AF对细胞的保护和修复作用可能主要是通过抗氧化应激损伤（抗过氧化氢损伤）实现。鉴于内质网应激、线粒体功能障碍及氧化应激是三个引发认知功能障碍的可能作用途径，并结合细胞水平的研究结果，本研究首先选择了与内质网应激密切相关的3种蛋白caspase-12、葡萄糖调节蛋白、增强子结合蛋白同源蛋白，观察AF对冈田酸诱导的SH-SY5Y细胞中3种蛋白表达的影响。发现，AF对caspase-12具有一定的调节作用，而对葡萄糖调节蛋白、增强子结合蛋白同源蛋白的表达没有产生明显的影响，提示AF对神经细胞的保护作用可能主要不是通过内质网应激途径介导。与此同时在冈田酸诱导的SH-SY5Y细胞中，我们发现AF可以使已经降低的线粒体膜电位得到部分恢复，并能抑制SH-SY5Y细胞凋亡。提示我们，AF对SH-SY5Y细胞的保护作用可能与线粒体功能障碍途径有关。已知线粒体功能障碍导致的必然结果是氧化应激加剧，自由基和过氧化物在细胞内大量堆积，多种抗氧化酶活性降低。AF对处于氧化应激状态的SH-SY5Y细胞表现出较强的抗氧化水平。提示，抗氧化应激可能是AF保护SH-SY5Y细胞免受冈田酸损伤最重要的作用途径之一。本项研究首次将垫状卷柏总黄酮（TFSP）用于学习记忆功能障碍模型小鼠的治疗研究，并初步探讨了TFSP和穗花杉双黄酮（AF）抗认知功能障碍可能的作用途径。为TFSP和AF作用机制的深入研究提供了有价值的细胞生物学和分子生物学线索，为TFSP的开发利用奠定实验医学基础。更多还原

【Abstract】 Cognition is the process of human brain for acquiring and processing ofinformation, translating into psychological action, and finally gaining and applyingknowledge. Cognition is a group of mental processes that includes attention, memory,producing and understanding language, learning, reasoning, problem solving, anddecision making. Cognitive impairment (CI) refers to the dysfunction of the cognitioninduced by any reasons. It includes several steps, from mild cognitive impairment todementia. All factors that can directly or indirectly lead to chronic damnification tothe cortical structure and function of the brain will cause CI, for example, abnormitiesof neurotransmitters like dopamine, acetylcholine, noradrenaline and their receptors,abnormal aggregation and modification of protein in the brain, neural degenerativedisease, chronic ischemia injury to the cerebrum induced by disorder of energy supply,oxidosis, free radical injury, excitatory toxicity, and some diseases that will causeinsufficiency of cerebral blood supply. CI is in fact the primary stage of seniledementia, it is common in the people over60years old. CI will soon develop intodementia (or Alzheimer’s disease) if it is not treated properly. Therefore, CI is the beststage for preventing, intervening and curing senile dementia. In recent years, the harmand importance of CI have been recognized, attributed to the research development ofcognitive impairment.At present, CI is mostly treated in the same way as Alzheimer’s disease or seniledementia in clinic. The possible reseasons are considered, such as cholinergic nerve,β-amyloid protein, inflammation, aluminium poisoning, calcium imbalance, metabolicdisturbance, free radical insult and mitochondrial dysfunction, and the related drugsare prescribed. The CI condition of the patients can be controlled or relieved to someexetent after the drugs being administered. There is a medicine that is effective andwidely accepted, named Ginaton. It is made of leaf extract of Ginkgo Biloba. Themain constituents in leaf extract of Ginkgo Biloba are flavonoids, and mostly are bioflavonoids. It is proved that leaf extract of Ginkgo Biloba has many good effects,such as improving blood volume of cerebral vascular, reducing the resistance ofcerebral vascular, ameliorating blood circulation, protecting the brain cells fromischemia, clearing oxyradical, improving immunity and against aging. The efficacy ofleaf extract of Ginkgo Biloba enlightened us that the bioflavonoids in other plants mayhave analogous effects.We screened flavonoids extracted from different plants. Total flavonoids ofSelaginella Pulvinata (TFSP) showed good effects on anti-oxidant and improving CIin animal model. The results in the literaures showed that total flavonoids ofSelaginella tamariscina had the ability of anti oxidation, anti diabetes, improvinghuman umbilical vein endothelia cells proliferation and VEGF expression. Flavonoidsof Selaginella moellendorfii Hieron inhibit the expression of COX-2and proliferationof HT-29. Biflavones of Selaginella Pulvinata have the effects of clearing diphenylpicryl hydrazinyl radical, inhibiting lactate dehydrogenase release and damages ofendothelial cells induced by lysophosphatidylcholine, and decreasing the activity ofxanthinoxidase in vitro. There is not a report about the effect of TFSP on CI so far. Wedetected the effects of TFSP on different step of learning and memory in mice model.TFSP and AF promote the proliferation of SH-SY5Y, protect the cell insulted byhydrogen peroxide, okadaic acid and Aβ25-35, and they repair the damaged cell tosome extent. On the basis of the above research, the possible mechanism of AF onameliorating CI was explored from endoplasmic reticulum stress (ERS),mitochondrial dysfunction and oxidative stress.The animal experiment results showed, TFSP had significant therapeutic actionon the impairment of memory acquisition, memory consolidation and memoryevocation induced by scopolamine hydrobromide, sodium nitrite and45%alcoholrespectively. TFSP inhibited the acetylcholine reduction in the brain induced byscopolamine hydrobromide, increased the activity of acetylcholinesterase and cholineacetyltransterase. It suggested that TFSP resisted the memory acquisition impairmentinduced by scopolamine hydrobromide through its influence on enzyme activities of cholinergic system and antagonizing scopolamine’s blockage on M-cholinergicreceptor. Sodium nitrite caused peroxidation injury to the mice cerebral tissue andbrought about the memory consolidation impairment. TFSP activated theantioxidative enzyme and suppressed the accumulation of hyperoxide in the brain.TFSP might improve the memory consolidation through anti-oxidation. D-galactosecaused the systemic aging of the mice, including the withering of the immune organs,the accumulation of hyperoxide and free radical in the body and the activitydepression of antioxidative enzyme. TFSP suppressed the systemic aging of the micethrough antioxidation and improving the immunity.On the basis of animal experiments, we studied the efficacy of TFSP and AF oncell model in vitro. Within the concentration range, TFSP and AF moderatelypromoted the proliferation of SH-SY5Y. Hydrogen peroxide, okadaic acid and Aβ25-35brought the cell to death when they were incubated with SH-SY5Y cell together. Thatmeans hydrogen peroxide, okadaic acid and Aβ25-35could be the damage factors in thestudy. We learned from the IC50of the three factors that okadaic acid was the mostpoisonous factor to the cell. Our results indicating, AF showed protection andrepairment to the cell damage caused by hydrogen peroxide, okadaic acid and Aβ25-35,especially to the insult induced by hydrogen peroxide. It is inferred that AF mightexert the action of improving memory consolidation through resistance to oxidativestress injury (resisting hydrogen peroxide injury).Since ERS, mitochondrial dysfunction and oxidative stress were three mainpossible paths that could cause CI, we determined the expression of three proteinclosely related with ERS (cysteinylaspartate specific proteinase-12, glucose regulatedprotein78and CCAAT/enhancer binding protein homologous protein; caspase-12,Grp78and Chop) induced by okadaic acid in SH-SY5Y treated with AF. AF regulatedcaspase-12to some extent that increased by okadaic acid in SH-SY5Y. Grp78andChop had no significant change in SH-SY5Y after treated with okadaic acid and AF,suggesting that the protective effect of AF on the cell may not attribute to ERS. Wefound that AF partially recovered the mitochondrial transmembrance potential and inhibited the cell apoptosis induced by okadaic acid. That indicated AF might exertprotective effect by regulating the mitochondrial dysfunction. Hyperphosphorylationof tau was detected induced by okadaic acid, while AF had the effect ofdephosphorylation on phosphorelated tau, suggesting that AF might protect the nervecell through resisting phosphorylation of protein and maintaining the structure andfunction of the microtube. The inevitable result of mitochondrial dysfunction is theincrease of oxidative stress, the accumulation of free radical and hyperoxide in celland the activity depression of antioxidative enzyme. AF displayed strong antioxidativeactivity on SH-SY5Y in oxidative stress. It suggested that anti oxidative stress mightbe the main important mechanism for AF protecting SH-SY5Y cell insulted byokadaic acid.We studied the effect of TFSP on the memory impairment mice model for thefirst time, and investigated the possible mechanism of TFSP and AF on improving CI.That supplied a valuable clue for further research of TFSP and AF in cell biology andmolecular biology, and provided experimental medicine foundation for theexploitation and utilization of TFSP.更多还原