四妙方通过增加肠道Akkermansia muciniphila改善胰岛素抵抗的作用机制研究

Mechanism study of Si Miao Formula on alleviating insulin resistance by increasing the abundance of Akkermansia muciniphila in mice

四妙方能够改善高糖高脂饮食诱导的糖代谢紊乱,并调控肠道菌群组成,特别是增加肠道Akkermansia muciniphila (A. muciniphila)的丰度。但是,肠道菌群和A. muciniphila在四妙方改善糖代谢中的确切作用,以及四妙方上调A. muciniphila的机制尚不清楚。本研究将通过体内外实验探讨四妙方改善胰岛素抵抗与A. muciniphila的相关性及四妙方升高A. muciniphila的作用机制。利用肠道菌群移植及抗生素干扰等方法,研究肠道菌群组成和A.muciniphila丰度变化对四妙方改善高糖高脂诱导的小鼠胰岛素抵抗的作用;通过四妙方拆方动物实验和药效评价,明确升高A. muciniphila的关键药味和组分;利用体外厌氧培养系统结合细胞动物实验,探索四妙方关键组分增加A. muciniphila含量的作用机制。动物福利和实验过程均遵循上海中医药大学动物伦理委员会的规定。结果显示,四妙方改变的肠道菌群可在受体小鼠中发挥改善高糖高脂饮食诱导的胰岛素抵抗的作用,且改善效果与A.muciniphila丰度呈正相关;君药黄柏是四妙方中调控肠道菌群组成并增加A. muciniphila的关键药物,而黄柏中主要活性成分小檗碱与黄柏在上调A. muciniphila丰度上作用相近。体外机制研究发现,小檗碱不能直接促进A.muciniphila的生长,但能通过激活肠细胞黏蛋白的表达,间接促进A. muciniphila的增殖。上述结果表明,四妙方改善胰岛素抵抗的作用很可能依赖于增加肠道A. muciniphila数量,且四妙方升高A. muciniphila的作用很可能与君药黄柏中小檗碱激活肠细胞黏蛋白的表达有关。

In our previous study,we found that Si Miao Formula(SMF) had the effect of improving the disorder of glucose metabolism caused by high fat and high sucrose diet,and significantly altered the composition of gut microbiota,especially increasing the level of Akkermansia muciniphila(A.muciniphila).However,it is unclear that the role of intestinal flora and A.muciniphila play in SMF improving blood glucose homeostasis,and the mechanism of how SMF increases the level of A.muciniphila.Therefore,this study will explore the correlation between SMF improving the insulin resistance and increasing the level of A.muciniphila,as well as the mechanism of SMF-induced growth of A.muciniphila using the in vitro and in vivo experiments.We explored the effect of intestinal flora and A.muciniphila on SMF-improved insulin resistance through fecal microbiota transplantation(FMT) and antibiotic intervention.In order to study the mechanisms underlying SMF on elevating A.muciniphila,we disassembled SMF to find the key component which can particularly elevate the number of A.muciniphila.Using the in vitro anaerobic culture system combined with cell and animal experiments,we explored the mechanism of the key component in elevating A.muciniphila.The research was approved by the Animal Ethical and Welfare Committee of Shanghai University of Traditional Chinese Medicine.Our results showed that the gut microbiota altered by SMF can improve high fat and sucrose diet induced insulin resistance in recipient mice,and the improvement was closely related to the abundance of A.muciniphila.Cortex Phellodendri played the most important role in regulating the composition of intestinal flora and increasing the number of A.muciniphila,of which,berberine was the key component of Cortex Phellodendri which up regulated A.muciniphila.We have found that berberine cannot directly promote the growth of A.muciniphila in vitro,but it can stimulate the expression of mucin,which,in turn,promote the growth of A.muciniphila.The above results show that the improved insulin sensitiviy by SMF depends on the increased level of A.muciniphila.The effect of SMF on elevating the amount of A.muciniphila might be correlated with the increased expression of mucin stimulated by berberine.