基于肠道菌群研究古方千金黄连丸的配伍内涵和治消渴（糖尿病）机理

Qianjin Huanglian pill is derived from the ancient anti-diabetic prescription that documented in the medical book “Beiji Qianjin Yao Fang” written by Simiao Sun in Tang Dynasty, which is made up of Huanliang and fresh Shengdi, and exerts effective and definite anti-diabetic action in the modern clinical use, but the relative mechanisms are unclear. Berberine is a bioactive intergradient in Huanglian possessing significant hypoglycemic action, but simultaneously inducing gastrointestinal tract side effect for poor selectivity of bactericidal action. Stachyose is an important carbohydrate component in fresh Shengdi and evidently repairs disturbance of microbiota. The complementary effects of berberine and stachyose suggest that microbiota may be an important mechanism of Qianjin Huanglian pill in drug compatibility and action, which has not been reported up to now. Thus, this study aims to clarify the mechanisms of Qianjin Huanglian pill through studying the compatibility of berberine and stachyose based on microbiota. Firstly, the “baseline geometrically increasing and decreasing method” is used to explore the best dose ratio of berberine and stachyose, which will be applied in the following compatibility studies. Then high-throughput sequencing is utilized to study the influences of compatibility on microbiota in diabetic mice and screen the differential microbiotas compared to mice only treated with berberine or stachyose. The differential microbiotas will be cultured in vitro and treated with berberine, stachyose and compatibility to find out the target microbiotas that dominate the beneficial effects of compatibility. Meanwhile, the chip detection, library screening and bacteria transforming are used to screen and clarify the feces miRNAs that specifically bonded to the target microbiotas, thus revealing the molecular bases of target microbiotas. Ultimately, the target microbiotas will be transplanted to the intestine of germ-free mice, and the relative feces miRNAs will be knocked-out or -in through transfecting the miRNA mimics or inhibitors in diabetic mice, such as to clarify the mechanisms of compatible use of berberine and stachyose, and supply ample experimental bases for revealing the mechanisms of Qianjin Huangliang pill and developing novel compound preparations.

千金黄连丸源自古医药学家治消渴的名方，由黄连和鲜生地组成，疗效确切，但内涵和机理不清。黄连的活性成分小檗碱降糖作用显著，但因杀菌选择性差而具有胃肠道副反应；鲜生地的糖类成分水苏糖可以改善肠道菌群稳态。提示肠道菌群可能是阐明此方配伍内涵和治消渴机理的关键所在，但未见相关报道。本研究将基于肠道菌群，通过研究药味化学成分小檗碱与水苏糖的伍用来阐明千金黄连丸的内涵和作用机理。首先，运用基线等比增减法，研究小檗碱伍用水苏糖的最佳剂量比；然后采用高通量测序研究伍用对糖尿病小鼠肠道菌群的影响，筛选差异菌群并体外培养，揭示与伍用相关的靶标菌群；并采用芯片检测、文库筛选及转化菌群的方法，筛选并揭示与靶标菌群特异结合的粪便miRNA，阐明伍用影响菌群的分子机理；最后采用差异菌群移植无菌小鼠和miRNA转化糖尿病小鼠的方法，阐明二者伍用治消渴的机理，为揭示古方配伍内涵和机理、开发基于化学成分的复方制剂提供依据。

千金黄连丸是我国古代治疗消渴病的名方，由黄连和鲜生地组成，疗效确切，但内涵和机理不清。小檗碱是黄连的主要降糖活性成分，水苏糖是鲜生地中含量较高的糖类成分。本研究主要基于肠道菌群，通过研究小檗碱与水苏糖的伍用来阐明千金黄连丸方的内涵和作用机理。为此，首先采用四氧嘧啶小鼠研究并确定小檗碱与水苏糖以100 mg/k和200 mg/kg伍用的降糖效果相对最佳；接着将二者以此剂量配伍，分别给予2型糖尿病db/db小鼠、KKAy小鼠和ZDF大鼠，设置二者单用组，考察它们对动物糖代谢、肠道菌群多样性、小分子代谢物、肠道通透性和炎症等的影响，以及ZDF大鼠结肠miRNA和基因表达谱变化。结果显示，在db/db小鼠中，小檗碱单用和伍用水苏糖均可显著控制其血糖波动，且二者伍用还可显著改善其口服葡萄糖耐量异常和胰岛素抵抗；与小檗碱单用相比，二者伍用还可显著增加菌门Verrucomicrobia和菌属Akkermansia，尤其是菌种Akkmansia muciniphila的丰度，同时明显降低全反式七肾素二磷酸和升高富马酸含量，并分别与Akkermansia菌属呈现显著负相关和正相关。在KKAy小鼠和ZDF大鼠中，小檗碱单用和伍用水苏糖均可显著改善其糖代谢；但与小檗碱单用相比，二者伍用可并显著增加KKAy小鼠肠道Akkermansiaceae丰度和降低粪便短链脂肪酸含量，显著降低ZDF大鼠Desulfovibrionaceae丰度和增加其结肠miR-10a-5p表达和降低结肠egr1和hbegf表达。总之，随着给药时间的延长，小檗碱伍用水苏糖可以更好地改善糖代谢，而这与增加肠道Akkermansiaceae和降低Desulfovibrionaceae丰度，及上调结肠miR-10a-5p和降低egr1和hbegf表达密切相关，不仅揭示了千金黄连丸的配伍内涵，还为研发有效成分的复方制剂奠定了基础。