Ⅰ、KMBZ-009 改善心理应激所致认知障碍和抑郁样行为的研究 Ⅱ、银杏叶提取物及复方制剂改善老年鼠空间学习记忆的突触可塑性机理

1、KMBZ-009 改善高台应激所致认知障碍和应激相关的抑郁样行为及其相关机理 研究。 虽然适当的应激会提高动物的学习记忆功能，但过量的应激特别是无法逃避 的应激，往往导致依赖海马或前额叶的学习记忆功能受损，这与应激改变脑内应 激激素（皮质酮，皮质醇等）和神经递质的释放，影响突触传递和可塑性（包括 长时程增强和长时程抑制，LTP 和LTD）有关。一些疾病的发生、发展和恶化， 比如抑郁症（Depression）、创伤后应激障碍（PTSD），往往也和应激相关联，其 神经化学基础被证实与内分泌系统和单胺类（如五羟色，去甲肾上腺素，多巴胺） 神经递质系统的功能密切相关。遗憾的是，到目前为止还没有发现能治疗应激的 药物。本实验室过去的研究证实：KMBZ-009(申报新药时的名称为芬克罗酮，英 文名Phenchlobenpyrrone)——一种新的取代吡咯烷酮类化合物，通过调节细胞 内钙，改变脑内神经递质的释放，从而影响脑高级功能。KMBZ-009 对神经递质 释放影响是否能减轻应激导致的认知障碍及应激相关疾病的发生还没有进行研 究。本研究采用Morris 水迷宫、行为操作箱、绝望游泳、膜片钳和活体电生理 技术研究了KMBZ-009 对高台应激所致认知障碍和应激相关的抑郁样行为的影响 及其相关机理。 研究结果发现，高台应激或皮质酮注射造成大鼠空间记忆提取障碍，这与其 导致的海马CA1 区突触可塑性改变有关，而KMBZ-009 能成剂量依赖性地逆转应 激对空间记忆提取的损伤作用，这与它阻断应激或皮质酮异化的LTD 和恢复应激 或皮质酮损伤的LTP 密切相关。KMBZ-009 能部分地降低因应激而升高的血清皮 质酮含量，此外，KMBZ-009 对大鼠海马CA1 区锥体神经元的兴奋和抑制电流的 影响可能也参与了其对应激的调节作用。KMBZ-009 能显著增加海马CA1 区锥体 神经元上AMPA 受体介导的兴奋性突触后电流（EPSC）的幅度，但不影响其动力 学特性。NMDA 受体介导的EPSC 不受KMBZ-009 的影响；GABA 受体介导的抑制突 触后电流（IPSC）的幅度几乎不受KMBZ-009 的影响，而其受体动力学特性明显 被KMBZ-009 改变，表现为IPSC 恢复的时间显著延长。KMBZ-009 对CA1 区兴奋 抑制电流的调节作用，使大鼠海马细胞具有更强的维持细胞稳态的能力，从而避免应激导致神经元功能的损害。KMBZ-009 对抗应激对认知得损伤作用提示其可 能会减少动物的抑郁样行为，本实验结果发现，KMBZ-009 确实能明显减少小鼠 在强迫游泳（FST）中的不动时间，增加大鼠在72 秒低频差式强化（DRL-72s） 模型中的强化率，并降低其反应率。其机制是KMBZ-009 增加正常动物中枢神经 系统胞外NE 水平，激活alpha 和beta 肾上腺素受体，从而使得实验动物的抑郁 样行为明显减少。 2、KMBZ-009 减轻氧化应激对细胞活力、线粒体电位及海马LTP 的损伤作用。 前人的研究表明，氧自由基过多是导致老年痴呆患者和老年人神经细胞凋亡 与认知障碍的因素之一。KMBZ-009 和阿尼西坦是吡咯烷酮类化合物，研究显示 均具有促智作用。有报道指出阿尼西坦能减少神经胶质细胞在缺血缺氧时氧自由 基的生成，从而避免细胞受到氧应激损伤。本研究采用神经元原代培养和离体电 生理学方法，观察了KMBZ-009 和阿尼西坦对氧应激神经元的保护作用。结果发 现，KMBZ-009 和阿尼西坦均能保护氧应激神经元的线粒体的功能，对抗氧自由 基对神经元细胞活力的损伤，从而有效逆转了氧化应激对海马脑片CA1 区LTP 的 损伤作用。KMBZ-009 的作用效果比阿尼西坦的效果强10 倍。 3、银杏叶提取物及复方制剂改善老年大鼠空间学习记忆的突触可塑性机理。 有研究表明，银杏叶和三七叶提取物能调节神经系统的功能。本研究采用 Morris 水迷宫和活体电生理技术研究了银杏三七复方制剂及银杏叶提取物（以 标准银杏叶提取物——金纳多作为阳性对照药）改善老年大鼠空间学习记忆障碍 的突触可塑性机理研究。结果发现：老年大鼠空间学习记忆能力较差，高频诱导 不能在其海马CA1 区引发LTP，当长期服用金纳多或复方制剂一个月后，老年动 物的空间学习记忆功能得到明显改善，这可能与药物增强海马LTP 有密切关系。 复方制剂的作用效果与金纳多的效果相当。 4、悬尾应激损伤避暗作业学习行为的多巴胺D1 受体机制。 近年来的研究表明，DA 系统对应激非常敏感，应激改变PFC 内DA 的含量， 从而导致依赖于PFC 的工作记忆受损。但目前尚不知道应激对DA 系统的影响是 否涉及依赖杏仁核和海马的情绪学习记忆功能。因此，我们采用被动回避作业和 行为药理学的方法，初步探讨了此问题。结果发现：和对照组动物相比，随着悬 尾应激持续时间的增加（5min、10min、20min），动物在避暗作业作业重测试中的步入潜伏期明显缩短，当动物被悬尾应激后回到鼠笼中休息20min，其步入潜 伏期无明显变化；腹腔注射DA D1 受体拮抗剂SCH23390 呈剂量依赖性地缩短动 物的步入潜伏期，但SCH23390 腹腔注射和悬尾应激共同处理实验动物时，此种 D1 受体拮抗剂能有效逆转应激对步入潜伏期的影响；进一步的研究发现，应激 或D1 受体拮抗剂对痛觉感受的影响不是其改变动物步入潜伏期的主要因素。本 研究结果表明悬尾应激导致脑内多巴胺释放过度增加，杏仁核（可能还有海马及 相关神经回路）内的D1 受体被过度激活，从而导致小鼠在操作被动回避任务时 的记忆获得障碍。

何首乌炮制前后生物活性和化学成分的变化研究

何首乌为常用中药，由何首乌及含何首乌的中成药制剂所引起的不良反应也时见报道，科学阐明不良反应的物质基础并提出解决方案对何首乌的使用十分重要。本论文研究了何首乌炮制前后KM小鼠肝脏毒性基因表达谱、生物活性及化学成分的变化。所获结果支持何首乌炮制的目的是减毒、改性（改变药效），何首乌生、熟异治的观点。制首乌对抑郁症的效果显著优于生首乌，这与本草所记载的何首乌炮制后补肝肾、益精血，归肝、肾经一致。 主要结果如下： 1、 生、制首乌的毒理基因芯片研究结果 何首乌的不良反应主要表现在肝损害方面。本研究建立了生何首乌和制何首乌不同剂量的肝毒性作用模型，体重指标统计发现生何首乌各剂量组平均体重显著下降，中剂量组（10 g/kg.d）体重下降20 %，高剂量组（20 g/kg.d）体重下降42%，50%动物死亡，提示动物机体能量代谢障碍；基因芯片研究结果表明何首乌是CYP450的抑制剂，生何首乌相对于制何首乌CYP3A4、CYP4A5显著下调，导致毒性成分在体内的吸收增加，服用大剂量的生何首乌后产生明显的肝毒性；主要对以下六条Pathway产生影响：①PPAR signaling pathway，主要毒性靶基因有RXRB CYP7a1、Acadl、Apoa2、Cyp4a、 FABP2 、MAPKKK5等基因。②Calcium signaling pathway，主要毒性靶基因有CAMK2B、CACNA1F、S100A1、 F2R、Ryr1、Slc8a2、Camk4 ③Neuroactive ligand-receptor interaction，主要毒性靶基因有Chrm4、 Ntsr2 、 GABRR1、 GRIK3、F2R等基因。④Wnt signaling pathway，主要毒性靶基因有Daam2、Rac1 等基因。⑤Complement and coagulation cascades，主要毒性靶基因有F2R、Serpina1b、Cfi 、FGA等基因。⑥Oxidative hosphorylation，主要毒性靶基因有Atp5e、NDUFA1等基因。生何首乌毒性明显强于制首乌，且生何首乌水煎液的毒性大于生何乌首丙酮提取物的毒性，这一结果表明，何首乌主要的毒性成分很可能并不仅仅是传统所认为的以大黄素为代表的蒽醌类化合物，而是何首乌中大量存在的有效组分二苯乙烯苷与大黄素相互作用的结果，这一研究结果与前述的何首乌对肝药酶的影响是一致的。后续生、制首乌的化学成分差异研究表明，炮制后二苯乙烯苷含量明显降低：生首乌为5.512 %、清蒸制首乌为3.811 %、豆制首乌为3.538 %,大黄素的含量炮制后显著升高,生首乌为0.094 %、清蒸制首乌为0.119 %、豆制首乌为0.126 %。 2 生、制首乌药效差异研究结果 本文采用慢性中等强度不可预知应激刺激模型(chronic unpredictable mild stress, CUMS)和动物行为绝望实验法，研究生、制首乌抗抑郁活性的差异，制首乌（5 g/kg.d）与模型组相比有显著差异（P< 0.01），生首乌制首乌（5g/kg.d）与模型组相比无显著差异，这一结果表明制首乌抗抑郁活性显著优于生首乌。 本文比较了生、制首乌对四氧嘧啶糖尿病模型小鼠血糖的影响的差异，生首乌（5 g/kg.d）与模型组相比有显著差异（P< 0.01），制首乌（5 g/kg.d）与模型组相比无显著差异，这一结果表明生首乌降糖活性优于制首乌。这一结果与历代中医古书中生首乌治疗消渴症（糖尿病）的记载一致。 3生、制首乌化学成分差异的研究结果 本文选用HPLC-DAD指纹图谱技术结合药效成分含量测定来研究生、制首乌化学成分的差异。炮制后，何首乌中的主要化学成分并未消失，只是其含量发生了改变。炮制后二苯乙烯苷含量明显降低：生首乌为5.512 %、清蒸制首乌为3.811 %、豆制首乌为3.538 %,大黄素的含量炮制后显著升高,生首乌为0.094 %、清蒸制首乌为0.119 %、豆制首乌为0.126 %。 综上所述，炮制前后何首乌中二苯乙烯苷和大黄素含量比的变化可能是何首乌炮制减毒、改性的物质基础。 根据上述结果我们建立了生、制首乌的质量控制新模式。 In recent years, some adverse drug reactions (ADR) about some traditional Chinese medicine were reported at times. As a Chinese medicine most in use, the ADRs of Radix Polygoni multiflori (RPM) and the medicines containing the RPM were also mentioned. The resolution of the problems caused by the ADRs is very important for the use of the RPM as a medicine. The process (or preparation) is a significant feature for the clinical use of the Chinese medicine and an important technology for the safe use and good effect of the Chinese medicine. By processing, the toxicity of the Chinese medicine can be reduced, its properties can be changed and curative effect can be enhanced at the same time. The changes of the gene expression profiles for KM mice hepatotoxic effects, and the change of the biological activity and the chemical composition after being processed of the RPm were studied in the present dissertation. The RPm heatotoxicity mechanism and the toxicity target genes were explained on the gene level for the first time. With the antidepressant activity, and the hypoglycemic effect as the target, the differences on the pharmacodynamics between the processed RPm and unprocessed RPm, for the first time, were investigated. The results obtained show that the antidepressant activity of the processed RPM is far higher than the ones of unprocessed RPm. As we know, the results were reported for the first time. The quality control systems (QCS) for the processed and the unprocessed RPm were founded. The HPLC-DAD was used in the systems founded on the basis of the toxicology and the pharmacodynamics experiments. As we know, the OCSs were reported for the first time. The above-mentioned experimental results confirm that the unique process theory of the traditional Chinese medicine (TCM) used for the process of the Radix Polygoni multiflori (RPm) is correct, i.e after being processed the toxicity of the RPm decreases and its Pharmacodynamic effects change. It is known to author that there have been no similar reports in the literatures up to now. The main experimental results are summarized as follows: 1 The results on the mice toxicology gene chip for the unprocessed and processed RPm The KM mice hepatotoxic model caused by the RPm at the different dosages was established in the present study. The results obtained show that the mouse average body weight obviously decreased in the groups at the different dosages of the unprocessed RPm: the 10 g/kg.d .group decreased 20%; 20 g/kg.d. group decreased 42%, and 50% mice died at 20 g/kg.d. group. The main experimental results on the mice toxicology gene chip The RPm is the CYP450 inhibitor. As compared with the processd RPm, the CYP3A4, CYP4A5 of the unprocessed RPm demonstrate the marked downregulation, which leads to the increase of the poison absorbtion into the body with the result that the unprocessed RPm yields the marked hepatotoxication. The hepatotoxication was produced because the following 6 pathways were affected: ①PPAR signaling pathway, the chief toxicity target genes are RXRB, CYP7a1, Acadl, Apoa2, Cyp4a, FABP2 and MAPKKK5 etc. ②Calcium signaling pathway, the chief toxicity target genes are CAMK2B, CACNA1F, S100A1, F2R, Ryr1,Slc8a2 and Camk4 etc. ③Neuroactive ligand-receptor interaction, the chief toxicity target genes are Chrm4, Ntsr2, GABRR1, GRIK3 and F2R etc. ④Wnt signaling pathway, the chief toxicity target genes are Daam2, Rac1 etc. ⑤Complement and coagulation cascades, the chief toxicity target genes are F2R, Serpina1b, Cfi and FGA etc. ⑥Oxidative phosphorylation, the chief toxicity target genes are Atp5e, NDUFA1 etc. The above experimental results, for the first time , demonstrate on the gene level that the unprocessed Rpm toxicity is far stronger than the processed RPm one, and the toxicity of the water decoction of the unprocessed RPm is greater than the one of its acetone extracts, which shows that the chief toxicity components of the RPm are probably not only the anthraquinones, for example, the emodin, but the complex compounds produced by the interaction between the emondin and the stilbene glucoside which is the largest component of the unprocessed RPm. The result is accordance with the above effect of the RPm on the hepatic drugenzyme. Aftter being processed, in fact, the content of the stibene glucoside in the RPm markedly decreases. 2. The results on the pharmacodynamic differences between the unprocessed and processed RPm The results obtained show that the effects of processing on RPm pharmacodynamic behaviour received in the Chinese Material Medica are correct. It is known to author that this is the first experimental result in the research materials now available. The chief results are as follows: For the treatment of the antidepressant, the curative effect of the processed RPm is far better than the one of the unprocessed RPm. By contrast with the above results, the hypoblycemic effect of the unprocessed RPm is better than the one of the processed RPm. 3. The results on the Chemical Composition The results obtained by using HPLC-DAD fingerprint and by the determination of effective component content show that the main chemical components in the RPm after being processed do not disappear, but their contents change. The contents of the stilbene glucoside (SG) and emodin in the different samples were determined as follows: SG contents 5.512 % for the unprocessed RPm 3.811 % for the processed RPm (Steamed) 3.588 % for the processed RPm (black soybean) Emodin contents 0.094 % for the unprocessed RPm 0.119 % for the processed RPm (Steamed) 0.126 % for the processed RPm (black soybean) The combination of above experimental results on the toxicity, the pharmacodynamics and the chemical composition indicates that the changes of the content ratio of SG/emodin may be the substance base of the toxicity decrease and pharmacodynamic changes of the RPM by the processing.

无指盘臭蛙皮肤抗菌肽Odorgrin A和Odorgrin C基因表达载体的构建及其在毕赤酵母中的表达

研究背景与目的：近二十年来，抗生素的广泛使用以及一些不当应用导致临床上出现大量的耐药性病原菌，所以不易产生耐药性的抗菌肽就成为目前研究的热点。本课题组此前的研究表明无指盘臭蛙（Odorrana grahami）皮肤抗菌肽具有广谱抗菌活性，但对真核细胞没有毒性，因此有成为新型药物的潜力。本研究采用毕赤酵母真核表达系统来生物合成抗菌肽Odorgrin A和Odorgrin C，为大量获取抗菌肽资源提供技术支撑。 方法：依照Odorgrin A和C的氨基酸序列、采用酵母偏爱密码子分别设计并化学合成了相应的目的基因序列。目的片段从合成质粒上用Xho Ι和EcoR Ι双酶切下后，与经同样限制酶完全酶切pPIC9K载体所获得的两个大片段直接连接，并转化至大肠杆菌DH5α。用PCR扩增、酶切及测序检测，鉴定正确的重组质粒。提取大量表达载体pPIC9K - Odo A和C并使之线性化后经电击法分别转化毕赤酵母（Pichia pastoris）GS115宿主菌，用营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序检测，鉴定并筛选出对G418具高抗性的Odorgrin A和C重组酵母菌。用甲醇对之进行诱导表达，SDS - PAGE电泳及反相层析检测表达产物，并做抑菌活性检测。 成果：PCR扩增、酶切及测序等结果表明表达载体pPIC9K - Odo A和C构建成功。营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序等证实pPIC9K - Odo A和C已整合入酵母基因组中。SDS - PAGE电泳及反相层析结果表明抗菌肽Odorgrin A和C成功地获得了分泌表达。而抑菌活性实验则检测到部分阳性克隆菌诱导分泌表达的抗菌肽Odorgrin A和C都对测试菌的生长具有较高（>94%）的抑制率。 结论：无指盘臭蛙皮肤抗菌肽Odorgrin A和Odorgrin C基因的表达载体都构建成功，并且都在毕赤酵母系统中获得了成功表达。 Background ＆ Objective: In the recent twenty years, a lot of pathogenic bacteria have come forth in clinic with durable trait derived from making use of and abusing the traditional antibiotics. Therefore, studying antimicrobial peptides, not be easy to be invalidated by durable bacteria, are becomimg popular and important. The skin antimicrobial peptides of Odorrana grahami with broad spectrum antibacterial activity and no toxicity to eukaryotic cell, discovered by previous research work of our workgroup, are looked forward to being potential medication. Pichia pastoris expressional system was used for biosynthesis antimicrobial peptides Odorgrin A and Odorgrin C in this study, for producing abundant antimicrobial peptides. Methods: The foreign fragments which included Odorgrin A or Odorgrin C gene according to their amino acid sequence respectively were synthesized based on the biased codon usage of yeast. The DNA fragments, obtained from the plasmids containing them by digested with Xho Ι and EcoR Ι, were directly ligated with the two bigger fragments obtained from the vector pPIC9K by digested with the same restriction enzymes. And then they were transformed into Escherichia coli DH5α to be selected and amplified positive colonies. The recombinants were testified by using PCR amplification, enzymes digestion and sequencing of the foreign fragment. After the expressional vector pPIC9K - Odo A and pPIC9K - Odo C were linearized, they were transformed into Pichia pastoris GS115 strain by the electroporation. Then the positive colonies which were of the highest geneticin resistant were selected through auxotrophic screening, genetic resistant screening, PCR amplification and sequencing of the inserted fragment. Methanol was used to induce the recombinant yeasts to express the foreign gene. SDS-PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment were used to testify the expressional products. Results: The evidences of PCR, enzymes digestion and sequence analysis confirmed that the expressional vector pPIC9K - Odo A and pPIC9K - Odo C have been constructed correctly. The results of auxotrophic screening, of genetic resistant screening, of PCR and sequencing of the foreign fragment showed that Odorgrin A and Odorgrin C gene have been homologous integrated with the Pichia pastoris genome. And it was also testified that antimicrobial peptides Odorgrin A and Odorgrin C have been expressed successfully by using SDS - PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment. Conclusion: The expressional vector of the skin antimicrobial peptides Odorgrin A and Odorgrin C gene of Odorrana grahami have been constructed correctly and both of the genes have been expressed successfully in Pichia pastoris system in this study.

细胞外信号调节蛋白激酶-cAMP反应元件结合蛋白信号通路在应激所致抑郁行为障碍中的作用

Stress is the most important factor in the vulnerability to depression and other behavioral disorders, but the mechanisms that stress signals are transferred into depression are far from understanding. To date, the neurotransmitters, neurotrophins and signal pathway have been concerned in the topic focusing on the pathophysiology of depression, but there are still many puzzles. Increasing evidence has indicated that the alteration in neuronal plasticity is the “trace” of stress-induced damages. The extracellular signal-regulated protein kinase(ERK)-cyclic-AMP-responsive element（CRE）-binding protein(CREB)signal pathway is a powerful intracellular signal transduction pathway participating in neuronal plasticity which is involved in higher brain cognitive functions such as learning and memory. However, so far, little is known about the role of the ERK-CREB signal pathway in response to stress and emotional modulations. Thus the aim of the study was to systematically investigate the role of the ERK-CEB signal pathway in depressive-like behaviors induced by stress. Depression animal models, antidepressant agent treatment and disruption of signal pathway in specific brain regions were applied. In the present study, three experiment sessions were designed to make sure whether the ERK-CREB signal pathway was indeed one of pathophysiological mechanisms of depressive-like behaviors induced by stress. In experiment one, two different stress animal models were applied, chronic forced swim stress and chronic empty water bottle stress. After stress, all animals were tested behaviorally using open-field, elevated-plus maze and saccharine preference test, and brain samples were processed for determination of ERK, P-ERK, CREB and P-CREB using western blot. The relationships between the proteins of ERK, P-ERK, CREB and P-CREB in the brain and the behavioral variables were also analyzed. In experiment two, rats were treated with antidepressant agent fluoxetine once a day for 21 consecutive days, then the brain levels of ERK, P-ERK, CREB and P-CREB was determined, the depressive-like behaviors were also examined. In experiment three, mitogen activated extracellular-signal-regulated kinase kinase (MEK) inhibitor U0126 was administrated to inhabit the activation of ERK in the hippocampus and prefrontal cortex respectively, then behavioral measurements and protein detection were conducted. The main results of the study were as the following: (1) Chronic forced swim stress induced animals to suffer depression and disrupted the ERK-CREB signal pathway in hippocampus and prefrontal cortex. There were significant correlations between P-ERK2, P-CREB and multiple variables of depressive-like behaviors. (2) Chronic empty water bottle stress did not induce depressive-like behaviors. Such stress decreased the brain level of P-ERK2 in hippocampus and prefrontal cortex, but the level of P-CREB in the hippocampus was increased. (3) The antidepressant agent fluoxetine relieved depressive-like behaviors and increased the activities of the ERK-CREB signal pathway in stressed animals. (4) Animals treated with U0126 injection into hippocampus showed decreased activities of the ERK-CREB signal pathway in the hippocampus, and suffered depression comorbid with anxiety. (5) Animals treated with U0126 injection into prefrontal cortex showed decreased activities of the ERK-CREB signal pathway in the prefrontal cortex, and exhibited depressive-like behaviors. In conclusion, The ERK-CREB signal pathway in the hippocampus and prefrontal cortex was involved in stress responses and significantly correlated with depressive-like behaviors; The ERK-CREB signal pathway in the hippocampus and prefrontal cortex participated in the mechanism that fluoxetine reversed stress-induced behavioral disorders, and might be the target pathway of the therapeutic action of antidepressants; The disruption of the ERK-CREB signal pathway in the hippocampus or prefrontal cortex led to depressive-like behaviors in animals, suggesting that disruption of ERK-CREB pathway in the hippocampus or prefrontal cortex was involved in the pathophysiology of depression, and might be at least one of the mechanisms of depression induced by stress.