Guizhi Fuling Wan reduces autophagy of granulosa cell in rats with polycystic ovary syndrome via restoring the PI3K/AKT/mTOR signaling pathway
Guizhi Fuling Wan reduces autophagy of granulosa cell in rats with polycystic ovary syndrome via restoring the PI3K/AKT/mTOR signaling pathway
复制标题
桂枝茯苓丸通过恢复PI3K/AKT/mTOR信号通路减少多囊卵巢综合征大鼠颗粒细胞自噬
DOI：
10.1016/j.jep.2021.113821
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发表时间：
2021-02-01
影响因子：
5.4
通讯作者：
Hu, Xiaodan
中科院分区：
文献类型：
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作者：
Liu, Min;Zhu, Hongqiu;Hu, Xiaodan
人种药理学相关性：桂枝茯苓丸是东汉张仲景发明的治疗妇科疾病的化瘀化痰中药。近年来，GFW已被广泛用于治疗多囊卵巢综合征（PCOS）患者。临床和动物研究表明，它对PCOS的治疗有效，但其机制尚不清楚。研究目的：探讨GFW对多囊卵巢综合征（PCOS）胰岛素抵抗（IR）大鼠的治疗作用及其机制，为进一步了解GFW的临床疗效提供理论依据，为中医药的研究提供理论依据。材料与方法：84只雌性SD大鼠随机分为7组（每组n = 12）：1）对照，2）PCOS模型，3）低剂量GFW，4）中剂量GFW，5）高剂量GFW，6）甲氨蝶呤，和7）中剂量GFW加LY 294002。在所有非对照组中，我们通过每日给予来甲唑联合高脂乳剂灌胃21天来诱导PCOS。治疗后处死大鼠，采用酶联免疫吸附法（ELISA）测定血清促卵泡激素（FSH）、睾酮（T）、孕酮、黄体生成素（LH）、17 β-雌二醇、空腹胰岛素（FINS）和空腹血糖水平。计算LH/FSH比值和HOMA-IR值。苏木精-伊红染色观察卵巢形态，显微镜下计数所有卵泡。MDC阳性囊泡作为标记物检测自噬，并通过免疫染色检测p62、Beclin 1和LC 3-II的表达水平。结果：与PCOS模型组相比，GFW治疗组大鼠闭锁卵泡和囊性卵泡减少，成熟卵泡和黄体增多。与PCOS模型组相比，GFW治疗组大鼠血清T、LH和FINS水平降低，LH/FSH比值和HOMA-IR值降低。GFW处理导致切割的半胱天冬酶-3、切割的半胱天冬酶-9、BAX、Beclin 1、AtgS和LC 3-II的水平显著降低。与PCOS模型组相比，GFW治疗组大鼠PI 3 K、AKT和mTOR的磷酸化水平显著升高。PI 3 K，AKT和mTOR的磷酸化与使用PI 3 K拮抗剂降低。结论：我们的研究结果表明，GFW抑制颗粒细胞自噬，促进卵泡发育，以减轻PCOS-IR大鼠排卵障碍。这与PI 3 K/AKT/mTOR信号通路的激活有关。
Ethnopharmacological relevance: Guizhi Fuling Wan (GFW) is a traditional Chinese medicine used to remove blood stasis and dissipate phlegm for treating gynecological diseases that was invented by Zhang Zhongjing in the Eastern Han dynasty. In recent years, GFW has been widely used to treat patients with polycystic ovary syndrome (PCOS). Clinical and animal studies have shown that it is effective in the treatment of PCOS, but its mechanism is unknown. Generally, it works by regulating autophagy via the PI3K/AKT/mTOR signaling pathway.Aim of the study: This study investigated the effects and mechanism of GFW in PCOS rats with insulin resistance (IR) in order to provide better understanding of its observed clinical effects and a theoretical basis for the study of traditional Chinese medicine.Materials and methods: Eighty-four female Sprague-Dawley rats were randomly divided into seven groups (n = 12 per group): 1) control, 2) PCOS model, 3) low-dose GFW, 4) medium-dose GFW, 5) high-dose GFW, 6) met-formin, and 7) medium-dose GFW plus LY294002. In all non-control groups, we induced PCOS through daily letmzole combined with intragastric high-fat emulsion for 21 days. After treatment, rats were sacrificed and serum follicle-stimulating hormone (FSH), testosterone (T), progesterone, luteinizing hormone (LH), 17 beta-estradiol, fasting insulin (FINS), and fasting plasma glucose levels were measured by enzyme-linked immunosorbent assay (ELISA). The LH/FSH ratios and HOMA-IR values were calculated. Ovarian morphology was observed by hematoxylin and eosin staining, and all follicles were counted under a microscope. MDC-positive vesicles were used as markers to detect autophagy, and the expression levels of p62, Beclin1, and LC3-II were examined by immunostaining. Western blotting was used to measure PI3K/AKT/mTOR pathway activation, granulosa cell apoptosis, and autophagy.Results: Compared with the PCOS model group, GFW-treated rats had less atretic and cystic follicles, and more mature follicles and corpus lutea. The GFW-treated rats had lower serum T, LH, and FINS levels than the PCOS model group, as well as lower LH/FSH ratios and HOMA-IR values. GFW treatment resulted in significantly reduced levels of cleaved-Caspase-3, cleaved-Caspase-9, BAX, Beclin1, AtgS, and LC3-II. Phosphorylation of PI3K, AKT, and mTOR was significantly higher in GFW-treated rats compared with the PCOS model group. The phosphorylation of PI3K, AKT, and mTOR was decreased with the use of a PI3K antagonist.Conclusions: Our results indicate that GFW inhibited granulosa cell autophagy and promoted follicular development to attenuate ovulation disorder in PCOS-IR rats. This was associated with activation of the PI3K/AKT/mTOR signaling pathway.