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 (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. 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. 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) metformin, and 7) medium-dose GFW plus LY294002. In all non-control groups, we induced PCOS through daily letrozole 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), 17β-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. 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, Atg5, 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. 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.

Liu M ，Zhu H ，Zhu Y ，Hu X ... -  《-》

Liuwei Dihuang Pills alleviate the polycystic ovary syndrome with improved insulin sensitivity through PI3K/Akt signaling pathway.

Polycystic ovary syndrome (PCOS) is a complex gynecological endocrine disease commonly occurred in women of childbearing age. The main hallmark of PCOS includes elevated androgen production and insulin resistance (IR). Liuwei Dihuang Pills (LWDH Pills), a commonly prescribed traditional Chinese medicine (TCM) is widely used as a tonic prescription to treat diabetes, female menopause syndrome and other symptoms with'Kidney-Yin' deficiency. It has been reported the effects LWDH pills on PI3K/Akt signaling pathway in T2DM treatment. Recent studies have also indicated that the treatment of menopausal syndrome may be associated with the ovarian sexual hormone levels regulated by LWDH pills to alleviate female infertility. However, its potential benefits on PCOS have not been fully elucidated. The primary aim of this study was to investigate the alterations of PI3K/Akt pathway in polycystic ovary syndrome-insulin resistance (PCOS-IR) progression induced by letrozole combined with high fat diet (HFD) and then to explore the detailed mechanism of LWDH Pills to alleviate PCOS. The female Sprague-Dawley rats were continuously treated with letrozole (p.o administration at 1 mg kg-1·day-1) and HFD for 21 days to establish the PCOS-IR model. Concurrently, metformin (200 mg kg-1·day-1) or LWDH Pills was orally administrated (1.2 or 3.6 g kg-1·day-1) to intervene disease progression. The ovarian pathology was evaluated by HE (hematoxylin-eosin) staining. The serum sexual hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, testosterone, progesterone and fasting insulin (FINS) were determined by radioimmunoassay. The protein expressions of IRS-1, PI3Kp85α, Akt and FoxO1a were analyzed by western blotting, while the mRNA levels of follicle-stimulating hormone receptor (FSHR) and Cyp19a1 in ovarian tissue were measured by qPCR. The upregulated phosphorylation of IRS-1 (S307), down-regulated phosphorylation of PI3Kp85α, Akt, and FoxO1a were significantly reversed by LWDH Pills (3.6 g kg-1·day-1) in PCOS-IR rats with up-regulated mRNA levels of FSHR and Cyp19a1 in ovary. Also, the index of insulin resistance was gradually adjusted to normal by LWDH Pills. The serum levels of FSH, estradiol, progesterone levels were significantly raised while LH, testosterone were reduced. The ovarian polycystic changes were alleviated while the atresia follicles were reduced. LWDH Pills therapy obviously improved the ovarian polycystic pathogenesis and regained the development of follicles via upregulating Cyp19a1, alleviated insulin resistance through acting on PI3K/Akt signaling pathway. These findings have provided scientific evidence for LWDH Pills to treat PCOS.

Qiu Z ，Dong J ，Xue C ，Li X ，Liu K ，Liu B ，Cheng J ，Huang F ... -  《-》

The ameliorating effects of Guizhi Fuling Wan combined with rosiglitazone in a rat ovarian model of polycystic ovary syndrome by the PI3K/AKT/NF-κB and Nrf2/HO-1 pathways.

Ye Y ，Zhou W ，Ren Y ，Lu J ，Chen A ，Jin R ，Xuan F ... -  《-》

Acupuncture regulates the autophagy of ovarian granulosa cells in polycystic ovarian syndrome ovulation disorder by inhibiting the PI3K/AKT/mTOR pathway through LncMEG3.

The main features of polycystic ovary syndrome (PCOS) are abnormal follicular development and ovulation dysfunction, which are caused by the excessive autophagy of ovarian granulosa cells. Acupuncture has been shown to improve ovulation dysfunction and abnormal follicular development in PCOS patients, but its mechanism is unclear. This study hypothesized that the beneficial effects of acupuncture are the result of LncMEG3-mediated effects on the PI3K/AKT/mTOR pathway. Acupuncture (CV-4, RN-3, CV-6, SP-6 and EX-CA 1) was used to treat a rat model of polycystic ovary syndrome. Hematoxylin-eosin staining was used to observe ovarian morphology and enzyme-linked immunosorbent assay, western blotting, immunohistochemistry and real-time PCR were used to detect LH, E2, FSH, T, AMH, LncMEG3, PI3K, AKT, mTOR, P62 and LC3II/I expression. The ovarian morphology of 90% of the rats in the acupuncture treatment group was significantly improved after 11 consecutive days of therapy. Acupuncture also resulted in a significant decrease in serum LH, FSH, T and AMH levels and a significant increase in E2 level (P<0.01). LncMEG3, PI3K, AKT, mTOR, P62 and LC3II/I expression was decreased in ovarian granulosa cells after acupuncture compared with PCOS and lentiviral Intervention Group (P<0.05), while the expression of follicle stimulating hormone receptor was increased (P<0.05). These results indicate that acupuncture can down-regulate the expression of LncMEG3 and thereby inhibit the PI3K/AKT/mTOR pathway, reducing granulosa cell autophagy and normalizing their proliferation. These factors ultimately remedy abnormal follicular development. These findings suggest that acupuncture has clinical potential as a safe treatment for PCOS ovulatory dysfunction.

Chen X ，Tang H ，Liang Y ，Wu P ，Xie L ，Ding Y ，Yang P ，Long B ，Lin J ... -  《-》

Bu-shen-zhu-yun decoction inhibits granulosa cell apoptosis in rat polycystic ovary syndrome through estrogen receptor α-mediated PI3K/AKT/mTOR pathway.

Bu-shen-zhu-yun decoction (BSZYD) is a traditional chinese herbal prescription is widely used in the treatment of infertility. We aimed to elucidate the impact of a traditional herbal prescription BSZYD on polycystic ovary syndrome (PCOS). The candidate active compounds in BSZYD and their putative targets were investigated by bioinformatics analysis. A deydroepiandrosterone (DHEA)-induced PCOS rat model was then constructed using female Sprague-Dawley (SD) rats. Serum hormone levels were measured by enzyme-linked immunosorbent assay (ELISA). Pathological changes in ovarian tissues were analyzed with hematoxylin and eosin (H&E) staining. The expressions of estrogen receptor α (ER α)-mediated PI3K/AKT/mTOR pathway were measured by immunofluorescence and western blotting. Bioinformatics analysis showed that the putative targets of active compound candidates in BSZYD were enriched in PI3K/AKT and estrogen signaling pathways related to regulating ovarian ovulation. Animal experiments showed that BSZYD significantly alleviated pathological changes in the ovary, altered hormone levels of serum and reduced apoptosis rate of granulosa cells. In addition, BSZYD treatment notably upregulated the expressions of proteins in ER α-mediated PI3K/AKT/mTOR pathway and downregulated apoptosis-related proteins in PCOS rats. BSZYD can restore ovary lesions and ameliorate apoptosis through ER α-mediated PI3K/AKT/mTOR pathway, which might partly contribute to the treatment of PCOS.

Jiang X ，Yuan Y ，Shi M ，Zhang S ，Sui M ，Zhou H ... -  《-》