基于肠道菌群驱动LPS/TLR4通路研究电针、小檗碱对PCOS模型大鼠的治疗机理

Polycystic ovary syndrome(PCOS)is considered to be a multifactorial disorder resulting from the interaction of several predisposing and protective genetic variants, with strong environmental influences, mostly dependent on diet and life-style habits. The human gut harbours a myriad of different microbes known together as the intestinal flora．Accumulating evidence indicates that the gut microbiota is closely linked with the development of obesity, insulin resistance and other metabolic diseases. The gut microbiota may become a new target for studying of PCOS. The toll-like receptors (TLRs) are a family of innate immune receptors that recognize evolutionarily conserved pathogen-associated molecular patterns. TLR4 may play a role in obesity-related insulin resistance and metabolic dysfunction. Lipopolysaccharides (LPS) is the major component of the outer membrane of Gram-negative bacteria, contributing greatly to the structural integrity of the bacteria, and protecting the membrane from certain kinds of chemical attack. LPS also increases the negative charge of the cell membrane and helps stabilize the overall membrane structure. Bruce Beutler was awarded a portion of the 2011 Nobel Prize in Physiology or Medicine for his work demonstrating that TLR4 is the LPS receptor. LPS can activate TLR4 signaling pathway, up-regulate the downstream inflammatory response pathway and promote of pro-inflammatory cytokine,weaken the intestinal local barrier function and injured the local mucosal. There is a correlation between plasma LPS and insulin resistance .As part of the cellular stress response, superoxide is one of the major ROS species induced by LPS in various TLR expressing cell types. We speculate that balancing intestinal microecology may be able to improve metabolic function, balance oxidative stress and improve chronic inflammation in treating PCOS. Electroacupuncture and berberine will be used as the main intervention medium for this study, and probiotics will be added as secondary intervention media to support the regulation of intestinal microflora in the treatment.

越来越多的证据表明肠道菌群与肥胖、胰岛素抵抗等代谢性疾病的发生密切相关。近年来微生态学与中医学关系引起了广泛关注, 肠道菌群已成为研究PCOS的新靶点。我们推测肠道微生态失衡，令脂多糖/Toll样4受体(LPS/TLR4)途径引起下游炎症反应通路的激活，触发促炎症细胞因子的释放，肠道局部屏障功能被削弱，造成肠粘膜局部损伤，使肠粘膜抵御外界有害因素的能力下降，进而造成肠道内LPS入血，从而引起体内慢性低度炎症反应，造成肥胖及IR，导致慢性炎症、氧化应激失衡，而促进PCOS的发展。通过平衡肠道微生态可能改善代谢功能、抑制氧化应激、慢性炎症，而治疗PCOS。本课题根据喂养方式，采取三种造模方式，分析各组的病理特征，肠道菌群微生态与PCOS的发病相关性。并基于“脾胃学说”以电针和小檗碱作为此次研究的主要干预媒介，并加入益生菌作为次要干预媒介以佐证调节肠道菌群对治疗PCOS的潜在作用。

PCOS临床表现多样，可引起闭经、无排卵性不孕及代谢性疾病等一系列并发症，包括肥胖症、糖耐量受损等。肠道菌群参与了多种代谢性疾病的发生和发展，已成为研究PCOS的新靶点。我们假设PCOS 大鼠造模过程对大鼠肠道菌群结构的改变具有影响。饮食对肠道菌群具有显著影响，高脂饮食对多囊卵巢综合征患者的内分泌和代谢功能具有重要影响。分别使用来曲唑和双氢睾酮进行造模，对比观察普通和高脂饲料喂养下，大鼠在内分泌、代谢和肠道菌群结构和多样性方面的影响。最终以来曲唑合并高脂饲料作为造模的主要手段，以电针和小檗碱作为此次研究的主要干预媒介，加入槲皮素作为次要干预媒介，并加入二甲双胍作为对照干预媒介。结果：①外源性雄激素5α-DHT刺激，降低肠道菌群的丰富度，降低β多样性，但不影响菌群Alpha 多样性和均匀性。影响肠道菌群的结构，导致Bacteroidetes菌门丰度明显下降，而Firmicutes菌门丰度明显升高。影响肠道菌群代谢功能，引起参与多条代谢通路的菌群富集。但单纯使用来曲唑对肠道菌群影响并不明显。②来曲唑合并高脂饲料喂养的PCOS大鼠模型，具备肥胖、胰岛素抵抗、卵巢多囊样改变的特点，肠道菌群丰富度明显下降，降低β多样性。导致Bacteroidetes菌门丰度明显下降，而Firmicutes菌门丰度明显升高。影响肠道菌群代谢功能，引起参与多条代谢通路的菌群富集。③四组干预方法，都有助于恢复动情周期和排卵，提高菌群丰富度，但对改变β多样性没有明显作用。电针、小檗碱、二甲双胍和槲皮素都可升高Bacteroidetes菌门丰度，而降低Firmicutes菌门丰度。但小檗碱组Bacteroidetes菌门丰度仍低于对照组。四组干预方式，对参与代谢通路的菌落的作用并不相同。槲皮素对参与多种代谢通路的菌群影响最少，而电针可 抑制参与脂代谢、萜类化合物和聚酮类化合物的代谢与碳水化合物代谢通路菌群丰度，但只有参与脂代谢的菌群丰度降到了与对照组无明显差异的水平。小檗碱可以影响的代谢通路最多，包括了参与 碳水化合物代谢、氨基酸代谢、萜类化合物和聚酮类化合物的代谢、能量代谢和脂代谢通路菌群 。与电针组一样， 参与脂代谢的菌群丰度降到了与对照组无明显差异的水平 。二甲双胍对参与脂代谢和氨基酸代谢通路的菌群影响不明显，但只在一定程度上抑制参与碳水化合物代谢、氨基酸代谢、萜类化合物和聚酮化合物的代谢。