从参苓白术散对宿主肠道菌群失衡及代谢产物表达的调节探讨健脾祛湿法抑制糖尿病发生的分子机制

Uncontrolled diabetes can lead to multiorgan dysfunction-initiated mortality and disability. Although the high prevalence and the severe complications have captured worldwide attention, no efficacious medicines were found to eradicate diabetes mellitus. In the opinion of traditional Chinese medicine theory, the occurrence of diabetes usually was due to the syndrome of spleen deficiency with dampness encumbrance. Clinically, diabetes mellitus can be prevented by fortifying the spleen and draining dampness, but the molecular mechanism failed to be clearly elucidated. In our preliminary study, Shen-Ling-Bai-Zhu-San (SLBZS, a representative formula on fortifying the spleen and draining dampness) attenuated gut microbial dysbiosis. Based on the above, we speculate that fortifying the spleen and draining dampness formula can restore the damaged intestinal microbiota structure, improve gut microbial metabolism, enhance the intestinal barrier function, block the body's inflammatory responses, and finally suppress the occurrence of diabetes mellitus. To prove this hypothesis, the following modern technologies will be applied in this project, including 16S rDNA microbial diversity analysis, HPLC-MS metabolomics assay, intestinal asepticization technology and related biological methods. Our project aims to establish the mouse model with glucose and lipid metabolism disorder induced by high-fat diet, the intestinal epithelial model with damaged barrier and the macrophagic inflammation model, to investigate the pharmacological mechanisms through which SLBZS prevents the formation of diabetes mellitus at molecular, cellular and histological level. It is expected that this project will find modern scientific elucidation of fortifying the spleen and draining dampness against diabetes in the perspective of micro-ecosystem. Also, our studies will provide theoretical support for the application of Shen-Ling-Bai-Zhu-San formula to the clinical prevention of diabetes mellitus.

糖尿病可导致多器官病变致残、致死，其高发病率和严重并发症已引起全球关注，但现今仍无彻底根治药物。中医认为糖尿病的形成阶段符合脾虚湿困证，且临床上运用健脾祛湿法防治糖尿病疗效确切，但其中蕴含的现代分子机制尚不完全清楚。我们前期研究表明，健脾祛湿代表方剂参苓白术散对糖脂代谢紊乱小鼠的肠道菌群失调具有显著改善作用。基于此，本申请推测健脾祛湿方药可通过调节肠道菌群结构、改善菌群代谢及增强肠道屏障功能，进而阻断炎症反应，最终抑制糖尿病的发生。为证明此假设，本申请拟采用16S rDNA微生物多样性分析、代谢组学分析、肠道无菌化技术和相关生物学手段，建立高脂饮食诱导的糖脂代谢紊乱小鼠模型、肠上皮细胞屏障模型及巨噬细胞炎症模型，从分子、细胞及组织水平探讨参苓白术散抑制糖尿病发生的药效机制。预期本申请将从肠道微生态角度阐释健脾祛湿法防治糖尿病的现代科学内涵，为参苓白术散在糖尿病临床预防中的应用提供理论支持。

糖尿病是全球重大慢性代谢疾病，中国糖尿病发病人数超过1.5亿，其病程漫长，病发后期存在缺血损伤、肾衰竭、眼部病变等多种风险。糖尿病目前尚无根治药物，在该病早期阶段阻滞糖尿病的发病进程具有重大意义，可以较小代价防范重大疾病风险。我国90%糖尿病患者为II型糖尿病，其并发早期存在肥胖与胰岛素抵抗症状，多数型符合脾虚湿困的中医证型。本项目以健脾渗湿方参苓白术散（SLBZS）防治高脂饮食诱导的糖脂代谢紊乱模型，探讨肠道菌群及其代谢过程规律，并从中阐释参苓白术散防治糖尿病的科学内涵。.项目主要完成了以下几方面工作：①首先制备并解析了参苓白术散的主要成分，并鉴定了其中关键活性组分含量，获得了该汤剂的质量控制基准。②本项研究建立了胆汁酸等代谢产物的分析方法。③建立了糖脂代谢紊乱动物模型，并基于16S rRNA高通量测序技术、非靶向代谢组学技术及常规细胞生物学方法，分析了糖脂代谢紊乱条件下肠道菌群及其胆汁酸类代谢产物的变化规律。④结合非靶向代谢组学与基因芯片数据库，通过多组学整合分析解析了糖脂代谢紊乱的关键靶标及代谢物。⑤发现了SLBZS可显著改善高脂饮食引起的体重过快增长、葡萄糖不耐受、脂肪细胞肥大等表现，对糖脂代谢紊乱有积极防治作用。⑥调控肠道菌群结构，提升菌群多样性，促进有益菌双歧杆菌属（Bifidobacterium）和乳酸杆菌属（Lactobacillus）相对丰度，降低布劳特氏菌（Blautia）、普拉梭菌（Faecalibaculum）以及肠杆菌（Enterorhabdus）水平。⑦通过细菌代谢与宿主信号互作研究，揭示了SLBZS对短链脂肪酸受体GPR43信号通路的调控作用，并最终促进胰岛素分泌，改善糖脂代谢稳态。.本项目确认了糖尿病早期的糖脂代谢紊乱时肠道菌群组成及代谢产物谱改变情况，探究了关键基因转录及代谢产物靶标，并证实了SLBZS经肠道菌群—短链脂肪酸受体—宿主代谢等改善脾虚湿困的科学内涵，为SLBZS临床早期干预糖尿病提供了理论支撑。