Combination of bone marrow mesenchymal stem cells and moxibustion restores cyclophosphamide-induced premature ovarian insufficiency by improving mitochondrial function and regulating mitophagy.

BACKGROUND: Premature ovarian insufficiency (POI) is a major cause of infertility. In this study, we aimed to investigate the effects of the combination of bone marrow mesenchymal stem cells (BMSCs) and moxibustion (BMSCs-MOX) on POI and evaluate the underlying mechanisms. METHODS: A POI rat model was established by injecting different doses of cyclophosphamide (Cy). The modeling of POI and the effects of the treatments were assessed by evaluating estrous cycle, serum hormone levels, ovarian weight, ovarian index, and ovarian histopathological analysis. The effects of moxibustion on BMSCs migration were evaluated by tracking DiR-labeled BMSCs and analyzing the expression of chemokines stromal cell-derived factor 1 (Sdf1) and chemokine receptor type 4 (Cxcr4). Mitochondrial function and mitophagy were assessed by measuring the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, and the mitophagy markers (Drp1, Pink1, and Parkin). Furthermore, the mitophagy inhibitor Mdivi-1 and the mitophagy activator CCCP were used to confirm the role of mitophagy in Cy-induced ovarian injury and the underlying mechanism of combination therapy. RESULTS: A suitable rat model of POI was established using Cy injection. Compared to moxibustion or BMSCs transplantation alone, BMSCs-MOX showed improved outcomes, such as reduced estrous cycle disorders, improved ovarian weight and index, normalized serum hormone levels, increased ovarian reserve, and reduced follicle atresia. Moxibustion enhanced Sdf1 and Cxcr4 expression, promoting BMSCs migration. BMSCs-MOX reduced ROS levels; upregulated MMP and ATP levels in ovarian granulosa cells (GCs); and downregulated Drp1, Pink1, and Parkin expression in ovarian tissues. Mdivi-1 significantly mitigated mitochondrial dysfunction in ovarian GCs and improved ovarian function. CCCP inhibited the ability of BMSCs-MOX treatment to regulate mitophagy and ameliorate Cy-induced ovarian injury. CONCLUSIONS: Moxibustion enhanced the migration and homing of BMSCs following transplantation and improves their ability to repair ovarian damage. The combination of BMSCs and moxibustion effectively reduced the excessive activation of mitophagy, which helped prevent mitochondrial damage, ultimately improving ovarian function. These findings provide a novel approach for the treatment of pathological ovarian aging and offer new insights into enhancing the efficacy of stem cell therapy for POI patients.

BCAA insufficiency leads to premature ovarian insufficiency via ceramide-induced elevation of ROS.

Premature ovarian insufficiency (POI) is a disease featured by early menopause before 40 years of age, accompanied by an elevation of follicle-stimulating hormone. Though POI affects many aspects of women's health, its major causes remain unknown. Many clinical studies have shown that POI patients are generally underweight, indicating a potential correlation between POI and metabolic disorders. To understand the pathogenesis of POI, we performed metabolomics analysis on serum and identified branch-chain amino acid (BCAA) insufficiency-related metabolic disorders in two independent cohorts from two clinics. A low BCAA diet phenotypically reproduced the metabolic, endocrine, ovarian, and reproductive changes of POI in young C57BL/6J mice. A mechanism study revealed that the BCAA insufficiency-induced POI is associated with abnormal activation of the ceramide-reactive oxygen species (ROS) axis and consequent impairment of ovarian granulosa cell function. Significantly, the dietary supplement of BCAA prevented the development of ROS-induced POI in female mice. The results of this pathogenic study will lead to the development of specific therapies for POI.

Erxian decoction alleviates cisplatin-induced premature ovarian failure in rats by reducing oxidation levels in ovarian granulosa cells.

ETHNOPHARMACOLOGICAL RELEVANT: Erxian Decoction (EXD) has been used empirically for more than 70 years to treat premature ovarian failure (POF), but more research is needed to understand how it works. AIM OF THE RESEARCH: The study aims to ascertain both in vivo and in vitro rewards of EXD. MATERIALS AND METHODS: EXD is composed of Curculiginis Rhizoma, Epimedii Folium, Morindae Officinalis, Angelicae Sinensis, Anemarrhenae Rhizoma, and Phellodendri Chinensis Cortex. UPLC/MS analysis was used to investigate the components of EXD. Using a POF model created by administering cisplatin to rats intraperitoneally, the pharmacodynamic effects of EXD were investigated. Three dose groups of EXD were garaged into rats: high (15.6 g/kg), medium (7.8 g/kg), and low (3.9 g/kg). By using a vaginal smear, the impact of EXD on the rat estrous cycle was evaluated. An ELISA test was used to measure the anti-Mullerian hormone (AMH), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels in the serum of rats. By using HE stains, pathological alterations in the ovaries may be seen. MDA and SOD levels in ovarian samples were used to measure the degree of ovarian oxidation. TUNEL labeling of ovarian sections was used to find apoptosis levels. By using ATP, energy production was evaluated. The relative expression of proteins connected to aging and the RAGE pathway was assessed using Western blot. Then, using H2O2, a model of senescent human ovarian granulosa cells (KGN) was created in vitro. The impact of EXD and H2O2 on cellular senescence was discovered using-galactosidase staining. Cell apoptosis levels were found using PI/Hoechest33342. By using DCFH-DA, intracellular ROS was examined. MDA and SOD concentrations were used to measure the degree of cellular oxidation. RAGE-related mRNA and protein expression were evaluated using RT-qPCR and western blotting. RESULTS: Using UPLC/MS analysis, 39 chemicals in EXD were found. Rats' estrous cycles were enhanced by EXD, which increased ovarian index and follicle count and reduced the proportion of atretic follicles in the rats. EXD reduced LH and FSH output while restoring AMH and E2 secretion. In ovarian tissues, EXD reduced the amount of apoptosis and MDA while raising SOD activity and ATP levels. The protein levels of p16, p21, p53, and Lamin A/C were among the senescence-related proteins that EXD lowered, along with the levels of RAGE, PI3K, BAX, and CASPASE 3. Anti-apoptotic protein BCL-2 was also raised in the RAGE pathway. Senescence, apoptosis, ROS, and MDA levels in the KGN cells were lowered in vitro by EXD. Additionally, EXD increased the anti-apoptotic potential by changing the expression of CAT, SOD2, and SIRT1. RAGE, BAX, BCL-2, CASPASE 3, and p38 expression levels were altered by EXD, enhancing its anti-apoptotic capability. CONCLUSION: EXD boosted the ovary's antioxidant and anti-apoptotic capabilities while enhancing the estrous cycle and hormone output. These findings strongly suggested that EXD may contribute to the alleviation of POF and ovarian granulosa cells senescence.

Protective Effects of Antioxidants on Cyclophosphamide-Induced Ovarian Toxicity.

The most common limitation of anticancer chemotherapy is the injury to normal cells. Cyclophosphamide, which is one of the most widely used alkylating agents, can cause premature ovarian insufficiency and infertility since the ovarian follicles are extremely sensitive to their effects. Although little information is available about the pathogenic mechanism of cyclophosphamide-induced ovarian damage, its toxicity is attributed to oxidative stress, inflammation, and apoptosis. The use of compounds with antioxidant and cytoprotective properties to protect ovarian function from deleterious effects during chemotherapy would be a significant advantage. Thus, this article reviews the mechanism by which cyclophosphamide exerts its toxic effects on the different cellular components of the ovary, and describes 24 cytoprotective compounds used to ameliorate cyclophosphamide-induced ovarian injury and their possible mechanisms of action. Understanding these mechanisms is essential for the development of efficient and targeted pharmacological complementary therapies that could protect and prolong female fertility.

Electro-Acupuncture Regulates Metabolic Disorders of the Liver and Kidney in Premature Ovarian Failure Mice.

As per the theory of traditional Chinese medicine (TCM), the liver and kidney dysfunction are important pathogenies for premature ovarian failure (POF). POF is a common gynecological disease that reduced the pregnancy rate. Electro-acupuncture (EA) is a useful non-pharmaceutical therapy that supposedly regulates the function of the liver and kidney in the treatment of POF with TCM. However, the underlying mechanism of EA in the treatment of POF has not been adequately studied through metabonomics with reference to the theory of TCM. Accordingly, we investigated the effect of EA on the liver and kidney metabolites in POF mice through metabolomics. POF mice were established via intraperitoneal injection of cisplatin. Both Sanyinjiao (SP6) and Guanyuan (CV4) were stimulated by EA for 3 weeks. The biological samples (including the serum and the ovary, liver, and kidney tissues) were evaluated by histopathology, molecular biology, and hydrogen-1 nuclear magnetic resonance (1HNMR)-based metabolomics to assess the efficacy of EA. 1HNMR data were analyzed by the orthogonal partial least squares discriminant analysis (OPLS-DA). The results revealed that EA was beneficial to ovarian function and the menstrual cycle of POF. Both the energy metabolism and neurotransmitter metabolism in the liver and kidney were regulated by EA. Notably, EA played an important role in regulating energy-related metabolism in the kidney, and the better effect of neurotransmitter-related metabolism in the liver was regulated by EA. These findings indicated that the ovarian functions could be improved and the metabolic disorder of the liver and kidney caused by POF could be regulated by EA. Our study results thus suggested that the EA therapy, based on the results for the liver and kidney, were related to POF in TCM, as preliminarily confirmed through metabolomics.

The effect of Kuntai capsule on ovarian function in cisplatin-induced premature ovarian insufficiency rats.

Objective: This study aims to evaluate the effect of Kuntai capsule on ovarian function in cisplatin-induced premature ovarian insufficiency rats and to explore the mechanism of Kuntai capsule on the ovarian function of rats. Methods: Seventy-four female Sprague-Dawley rats were used for this study. Eight of the rats were randomly assigned to the Control group. The remaining sixty-six rats were utilized to establish the POI model via Cisplatin and then randomly divided into four groups: the model Control group, the Estradiol group, and groups treated with low and high doses of Kuntai capsule. For the 28-day administration, the Control and model Control groups were intragastrically administered with 2.0 mL of 0.9% sodium chloride daily, the Estradiol group with 2.0 mL of Estradiol suspension (0.2mg/kg/d), and the low dose Kuntai capsule group and the high dose Kuntai capsule group with 2.0 mL of Kuntai capsule suspension (0.6g/kg/d, 1.8g/kg/d, respectively). Sex hormone levels, estrous cycle, and ovarian coefficient of the five groups were compared, histological sections analyzed follicle counts, and the protein expressions of growth differentiation factor 9, light chain 3 A-II, and Beclin 1 in the ovarian tissue were detected by Western blotting. Results: After the 28-day administration, the serum Estradiol and Follicle-Stimulating Hormone levels of the group treated with low dose of Kuntai capsule were not significantly different from the Control group, the serum anti-Müllerian Hormone level of the group treated with high dose of Kuntai capsule was significantly higher than the Estradiol group. The estrous cycle of the group treated with low dose of Kuntai capsule was significantly lower than the model Control group. Regarding ovarian coefficient, resting and growing follicles, growth differentiation factor 9, light chain 3 A-II, and Beclin 1 expression, both Kuntai capsule groups outperformed the model Control group with the statistical difference (P<0.05). Conclusion: Kuntai capsule can improve the estrous cycle and ovarian coefficient of rats with premature ovarian insufficiency, maintain the number of resting and growing follicles, and up-regulate the protein expression of growth differentiation factor 9, light chain 3 A-II, and Beclin 1 of rats' ovaries.

Network pharmacology and experimental validation to explore the molecular mechanisms of Bushen Huoxue for the treatment of premature ovarian insufficiency.

Bushen Huoxue (BSHX) has been applied in clinical traditional Chinese medicine treatment, and has definitive clinical efficacy in the treatment of Premature Ovarian Insufficiency (POI) in China. However, little is known of the underlying mechanism of BSHX. The purpose of this paper is to study the pharmacological mechanisms of BSHX acting on POI based on a pharmacology and experimental validation. The pharmacological database of chinese medicine system and analysis platform (TCMSP) were used to search the effective active ingredients and potential action targets of BSHX. Drugbank, Online Mendelian Inheritance in Man (OMIM), Genecards, and Disgenet databases were used to obtain relevant targets of POI. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and the visual network of protein-protein interaction network were constructed by FunRich3.1. Pymol software, and Auto Dock tools 1.5.6 were used for molecular docking. Murine model of POI was used to further investigate the mechanism of BSHX against on POI. Finally, 127 active compounds were collected from TCMSP database, and 215 active targets were identified. There were 1366 targets related to POI and 99 targets of BSHX for the treatment of POI. Quercetin, kaempferol, and stigmasterol were recognized as the most effective compounds corresponding to targets. The top three genes according to degree value are TP53, Akt1, and VEGFA. Further, the results of GO and KEGG enrichment analysis revealed that those core targets were mainly enriched on TRAIL and TGF-ß receptor signaling. The results of molecular docking showed that stigmasterol had good binding ability to Akt1. Moreover, experimental validation suggests that BSHX significantly Increased the expression of TGF-ß1 and Smad2/3, regulating the release of serum sex hormones, which include Follicular stimulating hormone (FSH), Estradiol (E2), and Antimullerin hormone (AMH).

HIGHLIGHTSBSHX treats POI by regulating TGF-ß1 and Smad2/3 signaling pathways; Quercetin, kaempferol, and stigmasterol were the most effective compounds in BSHX.

Thymopentin alleviates premature ovarian failure in mice by activating YY2/Lin28A and inhibiting the expression of let-7 family microRNAs.

OBJECTIVE: Thymopentin (5TP) significantly improved typical murine premature ovarian failure (POF) symptoms induced by a high-fat and high-sugar (HFHS) diet. However, its effect and mechanism remain unclear. MATERIALS AND METHODS: RNA-Seq was used to detect the differentially expressed genes among each group. HFHS-induced POF mouse model was generated and injected with siRNA using Poly (lactic-co-glycolic acid) (PLGA) as a carrier. RESULTS: RNA-Seq suggested that 5TP promoted the expression of Yin Yang 2 (YY2) in mouse ovarian granulosa cell (mOGC) of HFHS-POF mice. Luciferase reporter assay indicated that 5TP promoted the binding of YY2 to the specific sequence C(C/T)AT(G/C)(G/T) on the Lin28A promoter and promoted Lin28A transcription and expression. We continuously injected PLGA-cross-linked siRNA nanoparticles targeting YY2 into HFHS-POF mice (siYY2@PLGA), which significantly reduced the therapeutic effect of 5TP. siYY2@PLGA injection also significantly attenuated the upregulation of Lin28a expression in mOGCs induced by 5TP and enhanced the expression of let-7 family microRNAs, thereby inhibiting the proliferation and division of mOGCs. qPCR results showed that there was a significant difference in the expression levels of exosome-derived Yy2 mRNAs between POF patients and normal women, and that there was a specific correlation between the expression level of exosome-derived Yy2 and the peripheral concentrations of the blood hormones pregnenolone, progesterone and oestradiol. CONCLUSIONS: Thymopentin promotes the transcriptional activation of Lin28A via stimulating transcription factor YY2 expression, inhibits the activity of let-7 family microRNAs and alleviates the ageing of ovarian granulosa cells, ultimately achieving a therapeutic effect on POF in mice.

Diosmin Mitigates Cyclophosphamide Induced Premature Ovarian Insufficiency in Rat Model.

The current study was designed to investigate the protective role of diosmin against cyclophosphamide-induced premature ovarian insufficiency (POI). Female Swiss albino rats received a single intraperitoneal dose of cyclophosphamide (200 mg/kg) followed by 8 mg/kg/day for the next 15 consecutive days either alone or in combination with oral diosmin at 50 or 100 mg/kg. Histopathological examination of ovarian tissues, hormonal assays for follicle stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH), assessment of the oxidative stress status, as well as measurement of the relative expression of miRNA-145 and its target genes [vascular endothelial growth factor B (VEGF-B) and regulator of cell cycle (RGC32)] were performed. Diosmin treatment ameliorated the levels of E2, AMH, and oxidative stress markers. Additionally, both low and high diosmin doses significantly reduced the histopathological alterations and nearly preserved the normal ovarian reserve. MiRNA-145 expression was upregulated after treatment with diosmin high dose. miRNA-145 target genes were over-expressed after both low and high diosmin administration. Based on our findings, diosmin has a dose-dependent protective effect against cyclophosphamide-induced ovarian toxicity in rats.

Exploring the mechanism of (-)-Epicatechin on premature ovarian insufficiency based on network pharmacology and experimental evaluation.

METHODS: Relevant potential targets for EC were obtained based on Traditional Chinese Medicine System Pharmacology Database (TCMSP), a bioinformatics analysis tool for molecular mechanism of Traditional Chinese Medicine (BATMAN-TCM) and STITCH databases. The Online Mendelian Inheritance in Man (OMIM) and GeneCards databases were utilized to screen the known POI-related targets, while Cytoscape software was used for network construction and visualization. Then, the Gene Ontology (GO) and pathway enrichment analysis were carried out by the Database for Annotation, Visualization and Integrated Discovery (DAVID) database. Furthermore, KGN cells were performed to validate the predicted results in oxidative stress (OS) model, and antioxidant effect was examined. RESULTS: A total of 70 potential common targets for EC in the treatment of POI were obtained through network pharmacology. Metabolic process, response to stimulus and antioxidant activity occupied a leading position of Gene Ontology (GO) enrichment. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that PI3K/protein kinase B (AKT), TNF, estrogen, VEGF and MAPK signaling pathways were significantly enriched. In addition, cell experiments showed that EC exhibited antioxidant effects in an H2O2-mediated OS model in ovarian granulosa cells by regulating the expression of PI3K/AKT/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and multiple downstream antioxidant enzymes. CONCLUSION: EC could regulate multiple signaling pathways and several biological processes (BPs). EC had the ability to down-regulate elevated OS level through the PI3K/AKT/Nrf2 signaling pathway and represented a potential novel treatment for POI.

Samul-tang ameliorates oocyte damage due to cyclophosphamide-induced chronic ovarian dysfunction in mice.

Samul-tang (SM), a traditional herbal medicine, has been used to treat menstrual irregularities and infertility in women. However, the cellular and molecular mechanisms underlying the effects of SM remain elusive. We investigated the potential protective effect of SM against chronic ovarian dysfunction and used bioinformatics analysis to identify its underlying mechanism in a mouse model of cyclophosphamide (CP)-induced diminished ovarian reserve. Female C57BL/6 mice were intraperitoneally injected with CP three times a week, followed by oral administration of distilled water (CP group) or SM (CP + SM group) for 4 weeks. Four weeks later, the effect of SM was assessed by ovarian tissue histological analysis, steroid hormone measurement, oocyte quality, and mRNA and microRNA microarray analysis in the ovaries. Although SM administration did not prevent CP-induced follicle loss in mice, the quality of oocytes was better in CP + SM mice than in CP mice. Gene expression analysis revealed that the expression of fertilisation- and ovarian follicle development-related genes was altered by CP treatment but normalized after SM administration. Further bioinformatics analysis showed possible interactions between differentially expressed mRNAs and microRNAs. Therefore, we demonstrated the protective effects of SM on ovarian function and oocyte maturation against CP-induced damage via multiple epigenetic mechanisms.

Acupuncture Reduces Apoptosis of Granulosa Cells in Rats with Premature Ovarian Failure Via Restoring the PI3K/Akt Signaling Pathway.

Acupuncture is widely recognized as an effective therapy for premature ovarian failure (POF) in clinical, but information about its potential mechanisms is rarely explored. To investigate the mechanism, fifty SD female rats were randomly divided into normal group, POF group, POF+estradiol-valerate group (abbreviated as estradiol group), and POF+acupuncture group (abbreviated as acupuncture group). The estrous cycle of the rats was tracked by vaginal smears. Their ovaries morphology was observed by hematoxylin-eosin staining. The apoptotic level of granulosa cells was detected by in situ TUNEL fluorescence staining assay. Serum follicle-stimulating hormone (FSH) and estrogen (E2) levels were measured by enzyme-linked-immunosorbent-assay (ELISA). Protein and gene expression of PI3K, Akt, bcl-2, and bax were detected by Western blotting and qPCR. In the acupuncture and estradiol groups, compared with the POF group as controls, the apoptosis number of granulosa cells was significantly decreased (p < 0.05). FSH levels were decreased, while E2 levels were increased (p > 0.05). The gene and protein expression levels of PI3K, Akt, and bcl-2 were increased, while the expression levels of bax were decreased (p < 0.05), and the protein expression level of p-Akt increased. There was no significant difference between the acupuncture group and the estradiol group (p > 0.05). Acupuncture was able to regulate hormone levels in POF rats, up-regulate PI3K/Akt signaling pathway, and reduce the apoptosis of granulosa cells. This may be one of the mechanisms of acupuncture treating premature ovarian failure.

Dehydroepiandrosterone supplementation attenuates ovarian ageing in a galactose-induced primary ovarian insufficiency rat model.

PURPOSE: Almost every female classic galactosemia patient develops primary ovarian insufficiency (POI). The unique pathophysiology of classic galactosemia, with a severely reduced follicle pool at an early age, requires a new therapeutic approach. This study evaluated the effect of dehydroepiandrosterone (DHEA) on ovarian tissue in a galactose-induced POI rat model. METHODS: Pregnant rats were fed with either a normal or a 35% galactose-containing diet from day 3 of conception continuing through weaning of the litters. Galactose-exposed female offspring were further divided into 5 groups on PND21. The first group received no application. Treatment groups were fed orally by gavage once daily with sesame oil (group 2), or DHEA at doses of 0.1 mg/kg (group 3), 1 mg/kg (group 4) or 10 mg/kg (group 5) until PND70. Fertility rates of mothers with galactosemia, body weights (BWs), and ovarian weights of the litters from PND21 to PND70 were recorded. Ovarian follicle count, immunohistochemistry for proliferation and apoptosis marker expressions and TUNEL for cell death assessment were performed in offspring ovaries. RESULTS: Decreased fertility, ovarian/body weights were observed under galactosemic conditions, together with decreased follicle number and increased atresia. Improved postnatal development, primordial follicle recruitment and follicular growth were observed after DHEA treatment. After DHEA treatment, the expression of Ki67 protein was found to be increased; elevated expression of cleaved-caspase-3 under galactosemia was found to be reduced. CONCLUSIONS: Our data suggests that DHEA treatment may be a potentially useful clinical therapy to improve ovarian ageing in women with POI-induced by galactosemia.

Icariin exerts a protective effect against d-galactose induced premature ovarian failure via promoting DNA damage repair.

Icariin is one of the most common active ingredients in traditional Chinese medicine, while its function against Premature ovarian failure (POF) has not been explored. POF animal model was induced by d-galactose, and icariin at different doses was administered. Ovarian structure and follicle counting were observed via hematoxylin and eosin staining. The levels of serum hormones were measured by ELISA. Primary ovarian granulosa cells were cultured to compare the protective effects of icariin on cell aging, and DNA damage markers including γH2AX and 53BP1 were assessed by Western Blot. Administration of icariin promoted ovary/body weight, follicles numbers and fertility outcomes. In addition, icariin downregulated the levels of follicle stimulating hormone and luteinizing hormone, and upregulated the levels of estradiol and anti-Müllerian hormone. Icariin protected ovarian granulosa cells from d-galactose induced aging, with increased cell viability and lower endogenous ß-galactosidase activity. The alterations of expression level of γH2AX and 53BP1 by icariin indicated that the protection is via promoting DNA damage repair. In this study we tested the biological function of icariin against the d-galactose induced POF. Our results demonstrated that icariin effectively attenuated ovarian injury via promoting DNA damage repair, suggesting that icariin can be developed as a protective agent against POF.

Zuogui Pills inhibit mitochondria-dependent apoptosis of follicles in a rat model of premature ovarian failure.

ETHNOPHARMACOLOGICAL RELEVANCE: Zuogui Pills (ZGP), which is a classical prescription of Traditional Chinese Medicine (TCM), has been reported to be widely used in the treatment of premature ovarian failure (POF). AIM OF THE STUDY: To investigate the therapeutic effects of ZGP on the treatment of POF induced by chemotherapy, and elucidate the potential molecular mechanism. MATERIALS AND METHODS: Female 8-week-old Sprague-Dawley rats (N = 54) were randomized to six groups, containing the Control group, Model group, three ZGP groups and Triptorelin group which was served as a positive control. The Triptorelin group received triptorelin injection ten days before model establishment by cyclophosphamide. The three ZGP groups (high dose group, medium dose group and low dose group) were given a daily intragastric administration of ZGP at doses of 3.2, 1.6 and 0.8 g/kg for sixty days. We observed the general growth of rats and examed the estrous cycle and the rate of pregnancy, ovarian ultrastructures, follicles and corpora lutea numbers. The serum hormone concentrations were measured by Enzyme-linked immunosorbent assay (ELISA). To explore the molecular mechanism of the effect, gene and protein expression levels of Bax, Bcl-2 and Cyt-c related to apoptosis were determined by quantitative PCR (qPCR), Western Blot and Immunohistochemistry analysis, respectively. RESULTS: After treating with ZGP, though the rate of pregnancy showed no significant difference, the estrous cycle, ovarian ultrastructures, numbers of follicles and corpora lutea were improved significantly. And ZGP led to a significant lower concentration of follicle stimulating hormone (FSH) in the serum, and the concentration of oestradiol (E2) was increased. Furthermore, a significant downregulation of Bax, cytochrome c (Cyt-c), and upregulation of B cell lymphoma/leukemia-2 (Bcl-2) both on gene and protein levels were observed after the administration with ZGP. And effects showed a positive correlation with the dosages. CONCLUSIONS: Our study suggested that ZGP exerted significant effect on POF, which was meditated by inhibiting mitochondria-dependent apoptosis in the follicles.

Effects of the Modified Bazhen Decoction in the Treatment of Premature Ovarian Failure in Rats.

OBJECTIVE: This study aims to investigate the role of X-linked inhibitor of apoptosis protein (XIAP) in the pathogenesis of premature ovarian failure (POF) and the effects of the Modified Bazhen Decoction (MBD) in the treatment of POF. MATERIALS AND METHODS: Twenty-four eight-week-old Sprague Dawley (SD) rats were randomly divided into four groups: control group, POF group, MBD treatment group, and Fufang Ejiao Syrup (FES) treatment group. After adaptive feeding for one week, 18 SD rats in the POF, MBD and FES groups were subcutaneously injected with D-galactose (dissolved in saline) at the back of neck for eight weeks (150 mg/kg/day) to establish the POF model. Six SD rats in the control group received equal volumes of subcutaneous injection of saline. Tail blood was collected, and the concentration of follicle stimulating hormones (FSHs) and estradiol (E2) was measured, in order to evaluate the success of the POF model. SD rats in the MBD and FES treatment groups were intragastrically administered with MBD (10 ml/kg/day) and FES (10 ml/kg/day), respectively. Rats in the control and POF groups were intragastrically administered with saline (10 ml/kg/day). After four weeks of intragastrical administration with different medicines and saline, ovarian tissues were collected; and the expression level of XIAP, miR-23a and miR-27a were measured and compared among different groups. RESULTS: Compared with the control group, XIAP expression was significantly lower, and miR-23a and miR-27a expression significantly higher in the POF group. Furthermore, XIAP expression was significantly higher, and miR-23a and miR-27a expression was significantly lower in the MBD group. CONCLUSION: XIAP is involved in the regulation of oocyte and granulosa cells via the cysteinyl aspartate specific proteinase (caspase) pathway, and plays an important role in POF. MBD can dramatically activate XIAP, but inhibit the expression of miR-23a and miR-27a; preventing the apoptosis of oocyte and granulosa cells. Our study suggests that MBD may be a useful traditional Chinese medicine for the treatment of POF.

Research on the establishment of chronic stress-induced premature ovarian failure the rat model and effects of Chinese medicine Muniziqi treatment.

The purposes of this study were to establish and to explore the biological basis of the chronic stress-induced premature ovarian failure (POF) model and to explore the therapeutic effects of the traditional Chinese medicine Muniziqi. Sexually matured female Sprague-Dawley rats were fed with spinach and cilantro in cold and wet conditions for about 20 weeks until a chronic stress (CS) model was established. The CS rats were divided into a POF stress model group and a stress model group according to weekly biological characteristics and hormone level detection ( luteinizing hormone [LH], follicle stimulating hormone [FSH], and estrogen [E2]). To investigate the therapeutic effect of Muniziqi, the POF disease stress model group was divided into the high-, medium-, and low-drug intervention groups. The results showed that chronic stresses (special food, cold, damp) can lead to POF disease. The traditional Chinese medicine Muniziqi could not only improve the reproductive hormone level disorder, but also improve the function of the hypothalamus-pituitary-ovarian axis. The underlying mechanism may be a change in the E2, LH, and FSH hormone levels in serum and lower expression of ovarian premature aging-related protein PFN-1.

Electro-acupuncture attenuates the mice premature ovarian failure via mediating PI3K/AKT/mTOR pathway.

AIMS: Electro-acupuncture (EA) is frequently recommended as a complementary therapy for premature ovarian failure (POF) in the clinical. However, little information exists about its potential treatment mechanisms. The study was designed to observe the effect of EA to ovarian function and fertility in POF mice model, and investigated its potential mechanisms on PI3K/AKT/mTOR signaling pathway. MATERIALS AND METHODS: Forty-five female C57/BL6 mice were divided into the Control, the Model and the EA group. The ovaries morphology of mice was observed by hematoxylin and eosin (HE) staining, and all follicles were counted under microscope. The protein expression of PI3K, phospho-PI3K, AKT, phospho-AKT, mTOR, phospho-mTOR, S6, phospho-S6, 4E-BP1 and phospho-4E-BP1 were detected by western blotting. The data was presented as the ratio of phosphorylation protein to total protein. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and anti-Mullerian hormone (AMH) levels were measured by enzyme-linked immunosorbent assay (ELISA). The fertility was observed by giving treated mice 8 weeks for breeding. KEY FINDINGS: We found that primordial follicle counts were increased in EA group compared to Model group. The phosphorylation of PI3K, AKT, mTOR, 4E-BP1 and S6K in EA group significantly reduced compared to Model group. Serum FSH and LH levels in EA group were decreased compared to Model group, while, serum E2 and AMH levels in EA group were increased compared with Model group. The litter size in EA group was improved compared to Model group. SIGNIFICANCE: The effects of EA on the PI3K/AKT/mTOR signaling pathway may represent one of the mechanisms involved in attenuating the mice POF.

Effect and mechanism of Bushen Huoxue recipe on ovarian reserve in mice with premature ovarian failure.

The aim of the present study was to explore the effect and mechanism of Bushen Huoxue recipe (BHR) on ovarian reserve in mice with premature ovarian failure (POF). Mice were divided into 3 groups: normal group, model group and BHR group. Intraperitoneal injection of cyclophosphamide was performed to create the POF model. Primordial follicular (PDF) number, ovarian wet weight, ovarian index, and estrous cycle were analyzed to evaluate the effect of BHR on POF. Meanwhile, the mRNA and protein level of Mouse Vasa Homologue (MVH) in the bone marrow, peripheral blood and ovary were detected, to explore the underlying mechanism of the treatment efficacy of BHR on ovarian reserve. By the time of BHR treatment for 28 days, BHR increased the PDF number and shortened the estrous cycle of POF mice. BHR also decreased the mRNA level of MVH in the bone marrow, and increased mRNA and protein level of MVH in the ovary of POF mice. Our results demonstrated a treatment efficacy of BHR on POF mice, and revealed that BHR might repair the dysfunction of germline stem cells in the bone marrow, and thus to improve the ovarian reserve and enhance the ovarian function of POF mice through neo-oogenesis.

The potential of follicle-stimulating hormone peptide-modified triptolide-loaded nanoparticles to induce a mouse model of premature ovarian insufficiency.

The use of triptolide (TP) is limited by its poor water solubility and severe toxicity. In this study, we developed an active drug delivery system (TP-loaded nanoparticles) that could help improve the water solubility of TP and decrease its toxicity. Then, we investigated whether TP-loaded nanoparticles could be used to establish a novel premature ovarian insufficiency mouse model. The mice treated with TP-loaded nanoparticles for 35 days displayed normal growth, decreased serum antimullerian hormone, prominent ovarian fibrosis and vacuolar changes, fewer follicles and corpus lutea, increased collapsed oocytes and follicle apoptosis, and sterility. In conclusion, this model appears to show the reproductive characteristics associated with premature ovarian insufficiency in women and will allow us to study the mechanism of the effects of traditional Chinese medicine on gonadal toxicity.