黄连解毒汤多维调控APP/PS1转基因小鼠脑内炎症微环境与胆固醇合成代谢紊乱交互影响的作用机制研究

The neurodegenerative process of Alzheimer's disease (AD) was accelerated via the mutual effects of inflammation microenvironment in the brain coupled with the biosynthetic and metabolism pathway disorder of cholesterol. In our previous study, Huang-Lian-Jie-Du decoction (HLJDD), which was effective in the treatment of both AD and hyperlipidemia in clinic, could lower the level of inflammatory cytokine in AD rats depended on its multi-constituents character by "chemistry-pharmacokinetics-pharmacodynamics" relationship study. Based on our former research, the present project aims to establish a qualitative and quantitative technique using ultra high performance liquid chromatography (UHPLC) with high resolution mass spectrometry and triple quadrupole mass spectrometry to quickly monitor the specialized pro-resolving mediators (SPMs) and metabolites of cholesterol steroid in APP/PS1 mice (normal group and hyperlipidemia group with high fat diet) after a long-term administration of clinical dose of HLJDD. Specifically, this study aims to interpret the endogenous metabolic fingerprint of cholesterol and polyunsaturated fatty acid such as ω-3 and ω-6 along with the development of AD rats with hyperlipidemia. In addition, the pro-inflammatory and anti-inflammatory cytokines were measured using a liquid chip technology and the improvement of NFT、SP together with the content of p-tau were determined. According to the cluster network analysis method combining the aforementioned indexes with the serum concentration of the ingredients in HLJDD, a visual characteristics network including the effects and advantages of the above indexes was built to clarify the action mechanism of HLJDD based on its anti-inflammation activity and regulation of cholesterol metabolism to treat AD.

脑内炎症微环境与胆固醇合成代谢紊乱交互影响，加速阿尔茨海默病（AD）神经变性。本项目以临床治疗AD及高脂血症均有疗效的黄连解毒汤为研究对象，在前期“化学-药动-药效”三维关联研究基础上，拟以APP/PS1双转基因小鼠为模型（正常组、高脂饲料组），在黄连解毒汤临床剂量长期干预下，通过液相-高分辨及三重四极杆质谱联用技术，实现对促炎症消退介质（SPMs）及胆固醇类甾体的快速监测，建立基于ω-3，ω-6多元不饱和脂肪酸、胆固醇体内生物合成代谢途径的、可反映AD病程发展的内源性特征代谢指纹图谱；同时测定促炎及抗炎因子，以NFT、SP、p-tau水平等AD病理特征改善为实验终点，结合黄连解毒汤主成分及主代谢产物血药浓度，借助集群网络分析方法，衡量SPMs、促炎及抗炎因子、胆固醇类甾体和病理学指标在网络中的位置和优势差异，明确黄连解毒汤针对中枢炎症及胆固醇合成代谢紊乱的多维调控AD进程的作用机制。

本研究以脑内促炎症消退介质、炎症因子及胆汁酸甾体类等内源性物质调控为切入点，结合AD主要病理特征，包括水迷宫实验、Aβ淀粉样蛋白沉积、星形胶质细胞及小胶质细胞活化程度为主要评价指标，同时结合中枢及外周炎症环境改善、脂质代谢、胆汁酸代谢、肠道菌群改善等环节，深入开展了黄连解毒汤干预AD 进程的作用机制研究。研究发现，黄连解毒汤可显著改善APP/PS1转基因小鼠认知功能障碍、减少脑内Aβ淀粉样蛋白沉积、调节中枢神经递质分泌、缓解星形胶质细胞活化程度、降低脑内神经炎症水平，其作用机制与中枢及外周脂质代谢调控、肠道菌群改善及胆汁酸分泌具密切相关性。.同时，研究发现APP/PS1转基因小鼠给予高脂饲料饲养后，出现了中枢炎症因子、脑内炎症及氧化应激状态降低的现象，提示适当的高脂饮食干预，对于阿尔茨海默患者存在潜在的临床使用价值。针对高脂叠加的APP/PS1转基因小鼠模型，在黄连解毒汤干预后，与正常饮食的APP/PS1转基因小鼠的效果趋势一致，可减轻脑内炎症反应及淀粉样蛋白沉积情况，有效降低部分炎症因子表达，改善脑内氧化应激状态，并降低外周胆固醇水平，其作用机制与脂质代谢调控、菌群调节及胆汁酸调节等多个途径相关。.在国家自然科学基金等项目支持下，基于黄连解毒汤开发的六类中药新药黄连解毒丸，已经获得我国第一个证候类中药新药临床批件，正在开展临床试验。通过项目实施，目前该项目已形成文章4篇，其中3篇已发表，1篇投稿中，申请发明专利1项，培养研究生3人。