基于microRNA 调控胰岛素信号通路的多囊卵巢综合征痰证分子生物学基础研究

Polycystic ovary syndrome(PCOS) is an important disease that affects women's lifetime health. Our former research showed that phlegm syndrome were the main traditional Chinese medicine(TCM) pathogenesis of insulin resistance in PCOS.Based on the research and TCM theory "fat people are phlegmatic",we are to make animal model of PCOS and PCOS with phlegm syndrome by means of androstanolone and high fat diet.Firstly to investigate the relationship between phlegm syndrome and Endocrine and metabolic abnormalities of PCOS by assaying the level of follicle stimulating hormone,luteinizing hormone,prolactin,estradiol,testosterone,insulin,blood sugar and blood fat of different model rat.Secondly to explore the molecular biological mechanism of insulin resistance caused by phlegm syndrome of PCOS by determining the expression of microRNAs (miR-143、miR-29、let-7) and mRNA of INSR,IRS,PI3K,MAPK and ERK which are target gene of former microRNA in insulin signinaling pathway and analyzing the expression of protein and phosphorylation of INSR,IRS,PI3K,MAPK and ERK.Thirdly to provide evidence for identifying PCOS phlegm syndrome and clues for therapeutic mechanism research on phlegm syndrome from symptomatic treatment with QIGONG Soup.So the study sought to enrich the pathogenesis theory of phlegm syndrome,interpretate the scientific meaning of "fat people are phlegmatic",and to underlie the prophylaxis,diagnosis and individual treatment of PCOS.

多囊卵巢综合征(PCOS)是影响女性健康的重要疾病。本课题组前期基础表明，痰证是PCOS胰岛素抵抗的核心病机。本研究拟在此基础上，基于"肥人多痰"理论,采用双氢睾酮造模和高脂膳食诱导法建立PCOS、PCOS+痰证大鼠模型,①测定不同模型大鼠促卵泡激素、促黄体生成素、催乳素、雌二醇、睾酮、胰岛素、血糖、血脂，分析痰证与PCOS内分泌代谢系统异常的关系。②检测大鼠卵巢、肝脏、骨骼肌组织、脂肪组织microRNA(miR-143、miR-29、let-7)及其调控的胰岛素信号通路中靶基因INSR、IRS、PI3K、AKT、MAPK、ERK的mRNA表达水平、蛋白表达水平及磷酸化水平，研究PCOS痰证引起胰岛素抵抗的分子生物学机制。③采用启宫汤干预，从方证对应角度为PCOS痰证辨识和治疗机制研究提供依据。进一步丰富痰证病机理论，诠释"肥人多痰"科学内涵，为PCOS预防、诊断和个性化治疗提供依据。

多囊卵巢综合征(PCOS)是影响女性健康的重要疾病。胰岛素抵抗是PCOS的核心病理机制，中医证素痰从病理机制上与西医胰岛素抵抗密切相关。本课题采用来曲唑造模和高脂膳食诱导法建立PCOS、PCOS+痰证大鼠模型,①测定不同模型大鼠促卵泡激素、促黄体生成素、催乳素、雌二醇、睾酮、胰岛素、血糖、血脂；②检测大鼠卵巢、肝脏、骨骼肌组织、脂肪组织microRNA(miR-125b、miR-598、miR-143、miR-29)及其调控的胰岛素信号通路中靶基因INSR、IRS、PI3K、AKT、ERK1、ERK2的mRNA表达水平、蛋白表达水平及磷酸化水平；③采用具有健脾化痰功效的启宫汤、痰脂消汤、穴位埋线干预，从方证对应角度为PCOS痰证辨识和治疗机制研究提供依据。本课题成功建立了一种新的PCOS胰岛素抵抗模型及适宜针刺研究的PCOS痰证动物模型，模型大鼠具有典型的PCOS 生殖内分泌改变，如阴道涂片无明显的动情周期，卵巢见多个呈囊性扩张的卵泡，颗粒细胞层减少，未见黄体，血清T、LH 均显著增高。体质量增长显著，FINS、HOMA-IR、TC、LDL 均显著增高。大鼠卵巢INSR，IRS，PI3K，Akt mRNA 表达水平均降低； ERK1、ERK2mRNA 表达水平均升高。INSR，p-INSR，IRS，p-IRS，PI3K，p-PI3K，Akt，p-Akt 蛋白表达水平均降低；ERK1、p-ERK1、ERK2、 p-ERK2蛋白表达水平均升高；INSR，IRS，PI3K，Akt 磷酸化比例均显著降低；ERK1、ERK2磷酸化比例均显著升高。miR-125b的表达量与ERK1、ERK2mRNA表达量成负相关。进一步阐明了PCOS痰证与胰岛素抵抗有关，其分子生物学机制可能与miRNA及其调控的胰岛素信号通路异常有关，验证了“肥人多痰”的中医理论。经二甲双胍、中药、穴位埋线等治疗后，大鼠FINS，HOMA-IR以及INSR、IRS、PI3K、AKT、ERK1、ERK2的mRNA表达水平、蛋白表达水平及磷酸化水平等胰岛素抵抗指标均得到显著改善，初步阐释了中医健脾化痰疗法的治疗机理。本研究结果有助于丰富痰证的病机理论，有助于阐释“肥人多痰”理论在特定疾病的科学内涵，为PCOS预防、诊断和个性化治疗提供依据。