基于肠道菌-短链脂肪酸-肠PPARγ途径探讨补肾化痰方改善PCOS胰岛素抵抗的机制研究

Polycystic ovary syndrome (PCOS) is a hotspot and a difficult point in the field of reproduction. Professor Xia Guicheng, a master of Chinese medicine, thinks that the disease are caused by kidney deficiency with phlegm stagnation. He treats PCOS with Bushen Huatan formula. This formula which could resolve insulin resistance (IR) by activating PPARγ and AKT pathway. And we also find it can increase the synthesis of intestinal short-chain fatty acids (SCFAs). It is supposed that the effect is exerted by the intestinal flora. In this study, PCOS mice and in vitro gut models were used, combined with antibiotic interference experiments, the technique of 16sRNA, PCR, Westernblot, immunofluorescence, and Laser Confocal Microscopy, to clear the causal relationship between intestinal flora and Bushen Huayu formula to improve IR, and demonstrate that this side stimulates intestinal PPARγ signal, inhibits nitrate synthesis and restores bacterial homeostasis by up-regulating SCFAs. Maintain mucosal barrier function, reduce intestinal permeability, and exert anti-PCOS-IR effect. Look for the flora associated with the development of PCOS, clarify the pathogenesis from the perspective of intestinal microecology, provide new ideas for the etiology, provide new methods for clinical diagnosis and treatment, and provide a scientific basis for the prevention and treatment of Chinese medicine.

多囊卵巢综合征（PCOS）是生殖领域研究热点与难点，国医大师夏桂成教授认为本病肾虚为本，痰湿为标，创制补肾化痰方，临床疗效显著。该方通过激活PPARγ和AKT通路改善胰岛素抵抗（IR），并能提高肠道代谢产物短链脂肪酸（SCFAs）合成，后者作为肠道细菌关键性代谢产物参与宿主健康。推测该方通过肠道菌群发挥效应。本研究以PCOS小鼠和体外肠道模型为研究对象，结合抗生素干扰实验，运用16sRNA高通量测序、激光共聚焦显微镜、PCR、免疫荧光及免疫印迹技术，筛选PCOS特异菌群，明确肠道菌群在补肾化痰方改善IR中作用及分子机制，论证该方通过调整菌群结构，上调SCFAs合成，激活肠PPARγ信号，抑制硝酸盐合成，保护肠黏膜屏障功能，发挥抗PCOS-IR效应。该研究将PCOS发生相关菌群与补肾化痰方建立联系，从肠道微生态角度阐明发病机制及该方干预机理，为病因学说提供新思路，为中医药防治提供科学依据。

本研究从体内外多种途径证实，PCOS小鼠肠道门、属、种水平多种菌群均和丁酸具有相关性。证实补肾化痰方通过重塑PCOS肠道菌群结构，增加SCFAs水平，尤其是丁酸水平，从而增强其激活肠道PPARγ信号的作用，使肠上皮细胞iNOS表达下调，宿主肠道中硝酸盐含量降低，从而降低肠道通透性，改善肠屏障功能；并能提高卵巢内PPAR-γ和GLUT4表达，改善卵巢胰岛素抵抗，抑制卵巢NF-KB通路，并通过调控m6A甲基化修饰，减轻卵巢炎症，起到治疗PCOS的作用。本项目从中医整体观念、辨证论治的角度出发，基于SCFAs介导“肠道菌-宿主”对话模式，从肠道菌群、代谢产物、PPARγ信号、肠屏障功能、m6A表观遗传学等角度，阐述肠道菌群在PCOS胰岛素抵抗中的分子机制，从肠道菌、代谢、炎症角度，为PCOS新的病因学说提供依据。采用名老中医经验方作为干预物，进一步丰富、完善名老中医验方对PCOS胰岛素抵抗的作用及分子机制，为开发天然安全的、防治PCOS的药物提供科学依据；也为名老中医的学术经验的继承与研究提高新思路。