Zigui-Yichong-Fang protects against cyclophosphamide-induced premature ovarian insufficiency via the SIRT1/Foxo3a pathway.

Zigui-Yichong-Fang (ZGYCF) is a traditional Chinese medicine prescription for the treatment of infertility and premature ovarian insufficiency (POI). It is clinically used to regulate hormone levels, improve ovarian reserve and increase pregnancy rate. However, the exact mechanism of action is not yet clear. This study aimed to explore the potential impact and mechanism of ZGYCF on POI, and provide a scientific basis for its clinical application. UHPLC‒MS/MS was used to identify the main compounds of ZGYCF. Female 8-week-old C57BL/6N mice were randomized into four group containing the vehicle control (Veh) group, the cyclophosphamide (CTX) model group, the low-dose ZGYCF (CTX-ZG-L) group and the high-dose ZGYCF (CTX-ZG-H) group. A mouse POI model was induced with a single intraperitoneal injection of CTX, and the therapeutic effects of different doses of ZGYCF on POI were evaluated according to the ovarian weight coefficient, serum AMH, serum E2, ovarian histomorphology and follicle counts. After the dose screening experiment, the CTX-ZG-L group was renamed the CTX-ZG group and subjected to follow-up experiments. RNA-seq was used to explore the mechanism of POI and the therapeutic mechanism of ZGYCF on POI in Veh group, CTX group and CTX-ZG group. The mechanism of action of ZGYCF on POI were determined by measuring serum hormone level, histomorphology, follicle counts, protein expression and acetylation modification in groups of Veh, CTX, CTX-ZG and CTX-ZG-Nam (SIRT1 inhibitor). A total of 37 compounds in ZGYCF were identified. ZGYCF attenuated the morphological changes in ovarian tissue in POI model mice, increased serum AMH and E2 levels, reduced the damage to primordial follicles and other follicles at all stages, and protected ovarian reserve. RNA-seq results suggested that the genes expression of the PI3K signaling and apoptosis signaling pathways was increased in POI mice, while ZGYCF upregulated SIRT1 gene and the expression of estradiol, apoptosis inhibition and other signaling pathway genes. Immunohistochemical staining, TUNEL staining, Western blot analysis and immunoprecipitation results showed that in CTX group, SIRT1 expression and Foxo3a nuclei localization were decreased, while Ac-Foxo3a, p-AKT, p-Foxo3a and apoptotic markers were upregulated. After administration of ZGYCF, these conditions were reversed, however, after treatment with the SIRT1 inhibitor, the results were opposite to those of ZGYCF. Acetylated Foxo3a plays an important role in the occurrence of POI. ZGYCF improves the ovarian reserve of CTX-induced POI mice by activating SIRT1-mediated deacetylation of Foxo3a, and played a role in the treatment of POI. SIRT1 may be a novel target for ZGYCF to ameliorate POI.

Xiu Z ，Tang S ，Kong P ，Yan M ，Tong X ，Liu X ，Liang X ，Li R ，Duan Y ... -  《-》

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 ... -  《-》

Jinfeng pills ameliorate premature ovarian insufficiency induced by cyclophosphamide in rats and correlate to modulating IL-17A/IL-6 axis and MEK/ERK signals.

Jinfeng Pill (JFP) is a classical Chinese medicine formula and composed of 9 herbs, including Epimedium brevicornu Maxim (Yinyanghuo), Cervus elaphus Linnaeus (Lurong), Panax ginseng C.A.Mey. (Renshen), Equus asinus (EJiao), Ligustrum lucidum W.T.Aiton (Nvzhenzi), Reynoutria multiflora (Thunb.) Moldenke (Heshouwu), Curculigo orchioides Gaertn (Xianmao), Neolitsea cassia (L.) Kosterm. (Rougui) and Leonurus japonicus Houtt. (Yimucao). The formula is clinically used to regulate menstrual cycle and alleviate polycystic ovarian syndrome due to its capabilities of ovulation induction. It is therefore presumed that JFP could be used for the therapy of premature ovarian insufficiency (POI) but the assumed efficacy has not been fully substantiated in experiment. To evaluate the effectiveness of JFP on cyclophosphamide (CTX)-induced POI and preliminarily explore its potential mechanisms of action. An experimental rat model of POI was established by using CTX induction to assess the efficacy of JFP. The potential targets of action for JFP alleviating POI were predicted by the combination of network pharmacology and transcriptomics and finally validating by RT-qPCR and Western blot. JFP alleviated the damages of ovarian tissue induced by CTX in the rat model of POI via significantly decreasing serum levels of FSH and LH and the ratio of FSH/LH and increasing the levels of E2 and AMH, accompanied with promoting ovarian folliculogenesis and follicle maturity and reversing the depletion of follicle pool. With the analysis of network pharmacology, pathways in cancer, proteoglycans in cancer, PI3K-AKT, TNF and FoxO signaling pathways were predicted to be influenced by JFP. The results of RNA-seq further revealed that IL-17 signaling pathway was the most important pathway regulated by both CTX and JFP, following by transcriptional misregulation in cancer and proteoglycans in cancer. Combining the two analytical methods, JFP likely targeted genes associated with immune regulation, including COX-2, HSP90AA1, FOS, MMP3 and MAPK11 and pathways, including IL-17,Th17 cell differentiation and TNF signaling pathway. Finally, JFP was validated to regulate the mRNA expression of FOS, FOSB, FOSL1, MMP3, MMP13 and COX-2 and decrease the release of IL-17A and the protein expression of IL-6 and suppress the phosphorylation of MEK1/2 and ERK1/2 in CTX induced POI rats. Jinfeng Pill is effective to ameliorate the symptoms of POI induced by CTX in the model of rats and its action is likely associated with suppressing IL-17A/IL-6 axis and the activity of MEK1/2-ERK1/2 signaling.

Hu YY ，Zhong RH ，Guo XJ ，Li GT ，Zhou JY ，Yang WJ ，Ren BT ，Zhu Y ... -  《-》

Quercetin alleviates cyclophosphamide-induced premature ovarian insufficiency in mice by reducing mitochondrial oxidative stress and pyroptosis in granulosa cells.

Exposure to cyclophosphamide (CTX) induces premature ovarian insufficiency (POI). Quercetin is a natural flavonoid that exhibits anti-inflammatory and antioxidant properties, and its antioxidant activity is correlated with POI. However, the mechanism underlying its protective role in CTX-induced ovarian dysfunction is unclear. This study aimed to explore whether quercetin can protect ovarian reserves by activating mitochondrial biogenesis and inhibiting pyroptosis. Thirty-six female C57BL/6 mice were randomly subdivided into six groups. Except for the control group, all groups were injected with 90 mg/kg CTX to establish a POI model and further treated with coenzyme 10 or various doses of quercetin. The mice were sacrificed 48 h after 10 IU pregnant mare serum gonadotropin was injected four weeks after treatments. We used enzyme-linked immunosorbent assays to detect serum hormone expression and light and transmission electron microscopy to assess ovarian tissue morphology and mitochondria. Additionally, we tested oxidant and antioxidant levels in ovarian tissues and mitochondrial function in granulosa cells (GCs). The expression of mitochondrial biogenesis and pyroptosis-related proteins and mRNA was analyzed using western blotting and RT-qPCR. Quercetin elevated serum anti-Müllerian hormone, estradiol, and progesterone levels, decreased serum follicle-stimulating hormone and luteinizing hormone levels, and alleviated ovarian pathology. It reduced the mitochondrial DNA content and mitochondrial membrane potential. Furthermore, it upregulated ATP levels and the mRNA and protein expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), mitochondrial transcription factor A, and superoxide dismutase 2. In addition, it suppressed NOD-like receptor pyrin domain containing 3, caspase-1, interleukin-1β, and gasdermin D levels in the GCs of POI mice. Quercetin protected the ovarian reserve from CTX-induced ovarian damage by reversing mitochondrial dysfunction and activating mitochondrial biogenesis via the PGC1-α pathway. Moreover, quercetin may improve ovarian functions by downregulating pyroptosis in the CTX-induced POI model. Thus, quercetin can be considered a potential agent for treating POI.

Chen Y ，Zhao Y ，Miao C ，Yang L ，Wang R ，Chen B ，Zhang Q ... -  《Journal of Ovarian Research》

Danggui Shaoyao San protects cyclophosphamide-induced premature ovarian failure by inhibiting apoptosis and oxidative stress through the regulation of the SIRT1/p53 signaling pathway.

Chen H ，Zhang G ，Peng Y ，Wu Y ，Han X ，Xie L ，Xu H ，Chen G ，Liu B ，Xu T ，Pang M ，Hu C ，Fan H ，Bi Y ，Hua Y ，Zhou Y ，Luo S ... -  《-》