Dingkun Pill replenishes diminished ovarian reserve through the PI3K/AKT/mTOR signaling pathway in TWP-induced mice.

Diminished ovarian reserve (DOR) can lead to poor fertility and shorten the reproductive lifespan of females. The Dingkun Pill (DKP), a traditional Chinese-patented medication, has been an integral part of traditional Chinese medicinal treatment for the management of gynecological diseases for centuries. Relevant clinical studies have shown that DKP is able to protect against DOR, however, its mechanism of action is not yet fully elucidated. This study was conducted with the aim of exploring the impact of tripterygium wilfordii polyglycosidium (TWP) on the PI3K/AKT/mTOR pathway in the context of the pathophysiology of DOR and the mechanism of action of DKP. Eighty female balb/c mice with regular estrous cycles were assigned to Blank, Model, DKP and hormone replacement therapy (HRT) groups in a random manner. With the exception of the Blank group, mice in the other groups were exposed to 40 mg/kg/d TWP suspension for 30 days to DOR induction. Following this, either DKP or hormones were orally administrated to determine their effect on disease progression. During the experiment, changes in body weight and the estrous cycles of the mice were observed. Post treatment, serum sample anti-mullerian hormone (AMH), estradiol (E2), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were quantified using enzyme-linked immunosorbent assay (ELISA). The mice were then sacrificed in order to harvest their ovaries for hematoxylin and eosin (HE) staining. This process allowed for the assessment of ovarian morphology and follicular quantification. Apoptotic ovarian cells of the ovary were assessed using TUNEL technique, while Caspase-3 and Cytochrome C (Cyt C) expressions of the ovary were examined through immunohistochemistry (IHC). Western blotting analysis was used to quantify levels of Bax, Bcl-2, Caspase-3, Cyt C, mTOR, P-mTOR, AKT, P-AKT, P-PI3K and PI3K proteins, while mRNA levels of Bax, Bcl-2, PI3K, AKT and mTOR were measured in ovarian tissue using RT-PCR. The findings revealed that DKP was able to improve levels of serum hormones and promote the recovery of the estrous cycle. DKP augmented the total amount of primordial follicles while reducing the number of follicles that were atretic follicles. The apoptosis index of growing follicles and Bax, Cyt C and Caspase-3 expressions decreased, while the Bcl-2: Bax ratio increased. DKP suppressed levels of phosphorylation and the mRNA expressions of mTOR, AKT and PI3K. It was demonstrated that DKP was able to increase ovarian reserves through inhibition of the PI3K/AKT/mTOR signaling pathway, which lead to the suppression of primordial follicle activity and a reduction in levels of apoptosis of early growing follicles. This highlights its potentially beneficial role for the treatment of DOR.

Ma K ，Chen Y ，Fan X ，Yuan Y ，Wang K ，Tian C ，Li M ... -  《-》

Zihuai recipe alleviates cyclophosphamide-induced diminished ovarian reserve via suppressing PI3K/AKT-mediated apoptosis.

Zihuai recipe (ZHR), a Chinese herbal prescription, is widely used for the clinical treatment of Diminished ovarian reserve (DOR) infertility. However, little is known regarding its underlying mechanisms of DOR treatment. This study aimed to investigate the beneficial effects of ZHR on the treatment of DOR and to reveal the underlying mechanisms. Sixty female 8-week-old Sprague-Dawley rats were randomly divided into the following six groups (n=10 per group): control, DOR, low-dose(2.7 g/kg/day) ZHR (L-ZHR), medium-dose(5.4 g/kg/day), ZHR (M-ZHR), high-dose(10.8 g/kg/day) ZHR (H-ZHR), and hormone replacement therapy (HRT) treatment groups. The DOR model was established in all the groups, except the control group, by a single intraperitoneal injection of 90 mg/kg cyclophosphamide. After the induction of the DOR model, rats were weighed and administered either the relevant dose of ZHR or an equal volume of saline solution (in the control and DOR groups). Rats in the HRT group received estradiol valerate tablets (0.16 mg/kg/day), and with medroxyprogesterone acetate tablets (0.86 mg/kg/day) added on day 4. After 32 days of treatment, the rats were euthanized and the ovaries were collected for sampling. Ovarian morphology was observed by hematoxylin and eosin staining and the number of follicles was counted under a microscope. The serum levels of anti-Müllerian hormone (AMH), gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were quantified by ELISA. A TUNEL assay was used to analyze the level of apoptosis of the ovarian cells. The protein expressions of p-PI3K, p-AKT, PI3K, AKT, cleaved caspase-3, BAX, and Bcl-2 were measured by western blotting and immunohistochemistry. Data analysis was performed with SPSS 20.0 software. ZHR administration increased the ovarian index and the serum levels of AMH, GnRH, and E2, while lowering those of FSH and LH. ZHR treatment also increased the number of primordial, primary, secondary, and antral follicles, as well as the number of corpora lutea, but decreased the number of atretic follicles. Furthermore, ZHR administration decreased the percentage of TUNEL-positive ovarian cells. After treatment with ZHR, the protein expression levels of p-PI3K/PI3K, p-AKT/AKT, cleaved caspase-3 and BAX were decreased, whereas the level of Bcl-2 was increased. ZHR improved the ovarian reserve in CTX-induced DOR rats. The mechanisms of ZHR on DOR may be mediated through the regulation of gonadal hormones of the hypothalamic-pituitary-ovarian axis (HPOA), and the inhibition of PI3K/AKT-mediated apoptosis in granulosa cells.

Liu W ，Chen Q ，Liu Z ，Weng Z ，Nguyen TN ，Feng J ，Zhou S ... -  《-》

Dingkun Pill modulate ovarian function in chemotherapy-induced premature ovarian insufficiency mice by regulating PTEN/PI3K/AKT/FOXO3a signaling pathway.

Su C ，Zhang R ，Zhang X ，Lv M ，Liu X ，Ao K ，Hao J ，Mu YL ... -  《-》

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 ... -  《-》

[Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on PI3K/AKT/FOXO3a pathway in mice with poor ovarian response].

Hao J ，Wang H ，Cao Y ，Ji L ... -  《-》