Objectives: To explore the effect and mechanism of Chinese medicine Bushen Huatan formula in treatment of polycystic ovary syndrome (PCOS).
Methods: Twenty-four SPF female C57BL/6J mice were randomly divided into 3 groups with 8 animals in each group. Control group was given drinking water ad libitum; PCOS was induced by giving letrozole gavage and high-fat diet in model group and treatment group; treatment group received Bushen Huatan formula suspension for 35 d. The sex hormone levels of mice were detected by enzyme-linked immunosorbent assay. Ovary morphology was observed under light microscope after hematoxylin and eosin staining. The feces in the colon of mice were collected, and the gut microbiota was detected by 16S rRNA sequencing. The short chain fatty acids were detected by gas chromatography-mas spectrometry. The expression of peroxisome proliferator activated receptor (PPARγ) was detected by immunohistochemistry. The mRNA expression of mucin-2, occludin-1, tight junction protein zonula occludens 1 (ZO-1) and PPARγ in intestinal epithelium were detected by realtime RT-PCR. The expression of inducible nitric oxide synthase (iNOS) and PPARγ was detected by Western blotting.
Results: Compared with the control group, the body weight, serum levels of follicle stimulating hormone, luteinizing hormone and testosterone in the model group were increased, and serum levels of estradiol were decreased (all P<0.01); the ovarian structure under light microscope was consistent with the characteristics of PCOS. Compared with the model group, the serum levels of sex hormone and ovarian structure in treatment group were improved. The overall structure of gut microbiota in PCOS model mice changed. Compared with control group, there were significantly reduced abundance of Firmicutes, and increased abundance of Verrucomicrobia, Proteobacteria and Actinobacteria inthe model group at phylum level (all P<0.05); there were significantly reduced abundance of Lactobacillus, and increased abundance of Akkermansia, Lachnoclostridium, Lactococcus and Eubacterium_coprostanoligenes at genus level (all P<0.05). The disordered condition of gut microbiota was significantly improved in treatment group. Compared with control group, the contents of acetic acid, propionic acid and butyric acid in feces of model group were significantly decreased (all P<0.05); while the contents of propionic acid and butyric acid in treatment group were significantly increased compared with model control group (both P<0.05). Compared with control group, the mRNA expression of ZO-1 and protein expression of iNOS in model group were significantly increased, and the protein expression of PPARγ and the mRNA expressions of mucin-2 and occludin-1 were significantly decreased (all P<0.05). Compared with model group, the mRNA expression of ZO-1 and protein expression of iNOS in treatment group were decreased, and the protein expression of PPARγ and the mRNA expressions of mucin-2 and occludin-1 were increased.
Conclusions: PCOS induced by letrozole high-fat diet induces microflora imbalance in mice. Chinese medicine Bushen Huatan formula may increase the level of short chain fatty acid by regulating gut microbiota, thereby activating the intestinal PPARγ pathway and improving intestinal barrier function to act as a cure for PCOS.
目的: 探究补肾化痰方对多囊卵巢综合征（PCOS）模型小鼠肠道菌群多样性的调节作用及其治疗PCOS的相关机制。方法: 24只无特定病原体级成年雌性C57BL/6J小鼠随机分为三组：空白对照组、模型对照组（使用来曲唑联合高脂饲料诱导）、中药治疗组（造模结束后连续灌服补肾化痰方混悬液35 d）。采用酶联免疫吸附测定法检测小鼠性激素水平；苏木精-伊红染色后光镜下观察卵巢形态；收集各组小鼠结肠内粪便，采用16S rRNA测序进行肠道菌群检测；气相色谱-质谱法检测短链脂肪酸含量；免疫组织化学法检测结肠过氧化物酶体增殖物激活受体（PPARγ）表达；实时逆转录聚合酶链反应法检测肠道上皮黏蛋白2、闭合蛋白1和紧密连接蛋白1（ZO-1）、PPARγ的mRNA表达；蛋白质印迹法检测诱导型一氧化氮合酶（iNOS）、PPARγ蛋白表达。结果: 与空白对照组比较，模型对照组体重、血清卵泡刺激素、黄体生成素和睾酮水平均升高，血清雌二醇水平降低（均P<0.01），光镜下卵巢结构符合PCOS病理特征；中药治疗组血清性激素水平和卵巢结构较模型对照组改善。模型对照组肠道菌群多样性较空白对照组发生变化。与空白对照组比较，在门水平，模型对照组厚壁菌丰度下降，疣微菌、变形菌、放线菌丰度升高（均P<0.05）；在属水平，模型对照组乳酸菌丰度显著下降，阿克曼菌、乳梭菌、乳球菌、产粪甾醇真细菌丰度升高（均P<0.05）。补肾化痰方治疗后小鼠肠道菌群紊乱状态得到一定程度的改善。与空白对照组比较，模型对照组粪便中乙酸、丙酸、丁酸含量均下降（均P<0.05），而中药治疗组乙酸、丙酸、丁酸含量较模型对照组均增加，其中丙酸和丁酸含量与模型对照组差异有统计学意义（均P<0.05）。与空白对照组比较，模型对照组肠道PPARγ表达降低，ZO-1 mRNA表达增加，黏蛋白2、闭合蛋白1 mRNA表达减少，iNOS蛋白表达增加（均P<0.05）；与模型对照组比较，中药治疗组PPARγ 蛋白表达增加，闭合蛋白1、黏蛋白2 mRNA表达显著增加（均P<0.05），ZO-1 mRNA表达减少，iNOS蛋白表达减少。结论: 来曲唑联合高脂饲料诱导的PCOS小鼠存在菌群失调状态，补肾化痰方通过调节肠道菌群种属分布可能有助于提高短链脂肪酸水平从而激活肠PPARγ通路，改善肠屏障功能，达到治疗PCOS的目的。.
Keywords: Bushen Huatan formula; Gut microbiota; Intestinal barrier function; Mice; Peroxisome proliferator activated receptor; Polycystic ovary syndrome; Short chain fatty acids.