中药知母降糖的肠道菌群机制及活性成分研究

The clinical efficacy of traditional Chinese medicines (TCM) is clear, but the oral bioavailability of many Chinese herbs and their components are poor. It is a scientific problem how to play the efficacy of the Chinese herbs with low oral bioavailability. Anemarrhena Rhizoma is one of the most common hypoglycemic in TCM. Pharmacological studies have shown that it has anti-diabetic activity, and it seems that steroidal saponins, flavonoids and polysaccharides from Anemarrhena asphodefoides all have this kinds of activities. We present the hypothesis that " Anemarrhena Rhizoma can play an anti-diabetic effect through gut microflora". In this study, we observe the anti-diabetic activity of A. asphodefoides through the diabetic rats. At the same time, we investigate the change of the composition and diversity of gut microbiota which was affected by A. asphodefoides. We isolate and culture bacterial strains which are in connection with diabetic to confirm the facilitation and inhibition of the bacterial strain by the main components or metabolites of A. asphodefoides. And to explore the molecular mechanism of regulation, so as to elucidate the anti-diabetic mechanism and active ingredients of A. asphodefoides based on gut microflora regulation. It is a new perspective to understand the effectiveness of low bioavailability of traditional Chinese medicine, and lay the foundation of the new type of anti-diabetic drugs research as the targets on gut microflora.

中药临床疗效明确，但许多中药成分口服生物利用度低，吸收很少甚至不吸收的中药如何发挥作用，一直是困扰人们的一个科学问题。知母为中医临床最常用的降糖药味之一，药理研究证明其具降糖活性，而且似乎其皂苷、黄酮、多糖均有活性。我们提出“知母通过肠道菌群调节发挥降糖作用”的假说。本课题利用糖尿病模型大鼠观察知母降糖药理活性，同时研究知母对肠道菌群组成及多样性的影响，分离和培养糖尿病相关菌株，证实知母主要成分及其代谢产物对相关菌株的促进或抑制作用，并探索调节菌株生长的分子机理，从而阐明知母降糖的肠道菌群调节机制及有效成分，为理解低生物利用度中药的有效性提供一个新的视角，也为以肠道菌群为靶点的新型糖尿病药物的研发奠定基础。

中药临床疗效明确，但许多中药成分口服生物利用度低，吸收很少甚至不吸收的中药如何发挥作用，一直是困扰人们的一个科学问题。知母为中医临床最常用的降糖药味之一，前期药理研究证明其具降糖活性，而且似乎其皂苷、黄酮、多糖均有活性。本课题采用高脂高糖饮食与链脲佐菌素造模的糖尿病模型大鼠观察到知母提取物显著降低了糖尿病大鼠餐后2小时血糖和胰岛素敏感指数等，具有降糖降脂活性。同时，LC-MS定量分析显示知母中一系列甾体皂苷和黄酮的原型及代谢产物在肠道中的暴露比血中高2-3个数量级。因此我们观察了知母对肠道菌群的影响。通过16S rRNA测序及生物信息学分析，观察到知母提高肠道菌群多样性，促进Blautia等产短链脂肪酸的有益菌属并抑制了潜在的有害菌属，首次发现了知母降糖的肠道菌群调节机制。进一步通过分离和培养得到归于4门24个属的133株糖尿病相关菌株，并分离得到知母中的主要皂苷和黄酮类成分共21个。通过体外共培养实验，观察到知母提取物及其代谢产物可以促进有益菌Blautia的生长并呈现出剂量依赖性，同时对潜在有害菌Oligella具有生长抑制作用。单菌生长曲线实验显示，知母多糖是促进潜在有益菌B. glucerasea增殖的主要活性成分，知母皂苷AIII为抑制潜在有害菌O. ureolytica的主要成分。此外，为更全面地了解了知母降糖的机制，通过定量蛋白质组学研究，定量了胰腺组织和肝脏组织中的蛋白表达。其中知母促进胰腺组织中的Peroxiredoxin 4表达上调，进而促进了胰腺细胞再生及胰岛细胞功能的恢复；知母缓解糖尿病模型大鼠肝脏脂肪酸代谢异常可能与关键蛋白Ndufa6和Prkar2b的表达水平回调有关。本研究从肠道菌群的调节和胰腺蛋白靶点两方面阐明了知母的降糖作用机制，为理解低生物利用度中药的有效性提供一个新的视角，也为以肠道菌群为靶点的新型糖尿病药物的研发奠定基础。