基于“脑-肠-肠道菌群”轴的银杏酮酯和多奈哌齐治疗阿尔茨海默病联用增效的作用机理研究

Alzheimer's disease (AD) is a serious threat to the health of the elderly. Donepezil is a first-line therapeutic drug which is often used in combination with the extract of Ginkgo biloba leaves. The study found that the levels of various brain intestinal peptides in AD patients tend to normal after oral administration of the two drugs, and the therapeutic effect was significantly enhanced. However, the mechanism of synergistic effect between the two drugs is not clear. Recent studies have shown that brain-gut-microbiome axis (BGMA) is involved in the occurrence and development of AD. Our previous study found that combined use of the two drugs can regulate the level of brain intestinal peptide which is the signal molecule of BGMA, which suggests that the synergistic effect of the two drugs may be played through BGMA. In this study, a multi-index comprehensive evaluation system of anti-AD effect will be established to verify the synergistic effect of the extract of Ginkgo biloba leaves and donepezil in treatment of AD. Then based on BGMA, the distribution and metabolism of drugs in brain and intestinal tissue, as well as plasma pharmacokinetics, will be investigated to analyze the in vivo process after combined use of the two drugs. By the methods of immunohistochemistry, 16s rRNA-RT-PCR, high-throughput sequencing and bioinformatics, the effects of the two drugs on the expression of brain intestinal peptides and related genes, as well as the composition spectrum and functional spectrum of intestinal flora will be compared to reveal the mechanism of synergistic effect. This study will provide scientific basis for clinical application of related drugs.

阿尔茨海默病（AD）严重威胁老年人健康。多奈哌齐是一线治疗药物，临床常与银杏酮酯联合应用。研究发现，两药联用使AD患者体内多种脑肠肽水平趋于正常，药效显著增强，但两药联用增效作用机理尚不明确。近来研究提示“脑-肠-肠道菌群”轴（BGMA）参与了AD的发生发展过程，我们前期研究发现两药联用可调节BGMA的脑肠肽信号分子水平，由此设想两药联用增效作用可能经BGMA发挥。本项目拟建立AD多指标综合评价体系，对银杏酮酯和多奈哌齐治疗AD联用增效进行药效学验证。在此基础上，基于BGMA对药物在脑和肠组织分布和代谢进行研究，结合血浆药代动力学研究分析两药联用的体内过程。通过免疫组化、16s rRNA、RT-PCR、高通量测序、生物信息学等方法，比较两药联用对脑和肠中脑肠肽及相关基因表达的影响，对肠道菌群组成谱和功能谱的影响，揭示两药联用增效的作用机理，为相关药物临床应用提供科学依据。

本项目基于临床应用分析，选择银杏酮酯和多奈哌齐作为研究对象，采用适宜的方法与技术研究二者联用前后抗痴呆的生物效应与物质基础的变化规律，为阐明两药联用增效的基本规律提供思路与方法，为中西药联用现代研究提供参考，为指导创新中成药二次开发及中西医临床应用做贡献。.（1）对不同量比的银杏酮酯和多奈哌齐联用抗痴呆效应强弱进行研究，发现银杏酮酯和多奈哌齐的最佳配比为1:1、最佳剂量为临床等效量时，发挥抗痴呆的效果最佳；.（2）银杏酮酯-多奈哌齐联用后部分药源性成分可以增强透过血脑屏障的能力，有些药源性成分以原型发挥作用，有些药源性成分以其代谢产物的形式发挥药效；体外肠道菌代谢发现，药源性成分在正常和病理状态下的肠道微生物中的代谢反应时间、类型和产物等均存在差异；.（3）基于“脑-肠-肠道菌群”轴理论，从肠道菌组成谱、脑肠肽分子、神经递质和神经营养因子4个方面对银杏酮酯-多奈哌齐联用抗痴呆效应增强的机制进行研究，结果显示两药联用可提高厚壁菌门、乳酸杆菌属和埃希氏菌属的丰度，降低拟杆菌门、变形菌门和链球菌属的丰度；两药联用可使模型动物的脑、肠中单胺氧化酶b和血管活性肠肽的蛋白表达和mRNA表达出现显著回调作用，其余的脑肠肽信号分子表现出相应回调的趋势；两药联用可使模型动物的脑、肠中的L-谷氨酸释放减少，而抑制性氨基酸和单胺类神经递质模型动物的脑、肠中释放增加；两药联用后可显著提高模型动物脑中的神经营养因子BDNF和TrkB蛋白表达量。基于此，本项目提出银杏酮酯和多奈哌齐可能通过改善肠道菌群结构、提高益生菌比例、回调脑、肠中脑肠肽信号分子的异常水平、调节体内神经递质的释放及提高神经营养因子的水平来改善痴呆症状，进而发挥协同增效作用。.通过项目实施在国内外学术刊物已发表论文6篇，其中SCI收录5篇；培养研究生2名。