Premature Ovarian Insufficiency Mouse Research Articles

Human Amniotic Epithelial Stem Cells Alleviate Autoimmune Premature Ovarian Insufficiency in Mice by Targeting Granulosa Cells via AKT/ERK Pathways.

Autoimmune factors play an important role in premature ovarian insufficiency (POI). Human amniotic epithelial stem cells (hAESCs) have recently shown promising treatment effects on chemotherapy-induced POI. However, the therapeutic efficacy and underlying mechanisms of hAESCs in autoimmune POI remain to be investigated. In this study, we showed for the first time that intravenous transplantation of hAESCs could reside in the ovary of zona pellucida 3 peptide (pZP3) induced autoimmune POI mice model for at least 4weeks. hAESCs could improve ovarian function and fertility, alleviate inflammation and reduce apoptosis of granulosa cells (GCs) in autoimmune POI mice. The transcriptome analysis of mice ovaries and in vitro co-cultivation experiments suggest that activation of the AKT and ERK pathways may be the key mechanism in the therapeutic effect of hAESCs. Our work provides the theoretical and experimental foundation for optimizing the administration of hAESCs, as well as the clinical application of hAESCs in autoimmune POI patients.

Deletion of Fbxw7 in oocytes causes follicle loss and premature ovarian insufficiency in mice.

Premature ovarian insufficiency (POI) is one of the important causes of female infertility. Yet the aetiology for POI is still elusive. FBXW7 (F-box with 7 tandem WD) is one of the important components of the Skp1-Cullin1-F-box (SCF) E3 ubiquitin ligase. FBXW7 can regulate cell growth, survival and pluripotency through mediating ubiquitylation and degradation of target proteins via triggering the ubiquitin-proteasome system, and is associated with tumorigenesis, haematopoiesis and testis development. However, evidence establishing the function of FBXW7 in ovary is still lacking. Here, we showed that FBXW7 protein level was significantly decreased in the ovaries of the cisplatin-induced POI mouse model. We further showed that mice with oocyte-specific deletion of Fbxw7 demonstrated POI, characterized with folliculogenic defects, early depletion of follicle reserve, disordered hormonal secretion, ovarian dysfunction and female infertility. Impaired oocyte-GCs communication, manifested as down-regulation of connexin 37, may contribute to follicular development failure in the Fbxw7-mutant mice. Furthermore, single-cell RNA sequencing and insitu hybridization results indicated an accumulation of Clu and Ccl2 transcripts, which may alter follicle microenvironment deleterious to oocyte development and accelerate POI. Our results establish the important role of Fbxw7 in folliculogenesis and ovarian function, and might provide valuable information for understanding POI and female infertility.

Exploring the effect of Gouqi Nuzhen Liuhe decoction on the PI3K/mTOR signaling pathway for premature ovarian insufficiency based on system pharmacology

ObjectiveTo explore the effect of Gouqi Nuzhen Liuhe Decoction (GNLHD) on the PI3K/mTOR Signaling Pathway for Premature Ovarian Insufficiency (POI) based on system pharmacology. MethodsFirst, the system pharmacology approach was used to predict the mechanism of GNLHD. Then, mice were randomly divided into model group, positive group, GNLHD high-dose group, GNLHD medium-dose group, and GNLHD low-dose group. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of ovarian tissue under light microscope. The expression levels of estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were detected by enzyme-linked immunosorbent assay. The expressions of PI3K, AKT1 and mTOR proteins in ovarian tissue were detected by immunohistochemistry. ResultsThe results of system pharmacology showed that GNLHD may regulate biological processes and signaling pathways such as: reproductive structure development, reproductive system development, Oocyte meiosis and so on. Compared with the model group, the levels of E2 in the GNLHD group were increased, and the levels of FSH and LH were decreased (P < 0.05). Compared with the model group, the number of mature follicles in the GNLHD group was significantly increased, the number of atretic follicles was relatively decreased, and the expressions of PI3K, AKT1, and MTOR proteins in the GNLHD group were significantly increased (P < 0.05). ConclusionGNLHD may improve the ovarian function of POI mice by affecting the expression of PI3K, AKT1 and mTOR proteins, promote the growth and development of follicles, increase the E2 level, reduce FSH and LH level, and maintain the stability of the ovarian internal environment.

HUCMSC-derived extracellular vesicles relieve cisplatin-induced granulosa cell apoptosis in mice by transferring anti-apoptotic miRNAs.

Premature ovarian insufficiency (POI) caused by chemotherapy is a common complication in female cancer survivors of childbearing age. Traditional methods including mesenchymal stem cell (MSC) transplant and hormone replacement therapy have limited clinical application due to their drawbacks, and more methods need to be developed. In the current study, the potential effects and underlying mechanisms of human umbilical cord MSC-derived extracellular vesicles (hUCMSC-EVs) were investigated in a cisplatin (CDDP)-induced POI mouse model and a human granulosa cell (GC) line. The results showed that hUCMSC-EVs significantly attenuated body weight loss, ovarian weight loss, ovary atrophy, and follicle loss in moderate-dose (1.5 mg/kg) CDDP-induced POI mice, similar to the effects observed with hUCMSCs. We further discovered that the hUCMSC-EVs might inhibit CDDP-induced ovarian GC apoptosis by upregulating anti-apoptotic miRNA levels in GCs, thereby downregulating the mRNA levels of multiple pro-apoptotic genes. In general, our findings indicate that moderate-dose chemotherapy may be a better choice for clinical oncotherapy considering the effective rescue of oncotherapy-induced ovarian damage with hUCMSC-EVs. Additionally, multiple miRNAs in hUCMSC-EVs may potentially be used to inhibit chemotherapy-induced ovarian GC apoptosis, thereby restoring ovarian function and improving the life quality of female cancer patients.

Human umbilical cord mesenchymal stem cells alleviate chemotherapy-induced premature ovarian insufficiency mouse model by suppressing ferritinophagy-mediated ferroptosis in granulosa cells

Primary ovarian insufficiency (POI) in younger women (under 40) manifests as irregular periods, high follicle-stimulating hormone (FSH), and low estradiol (E2), often triggered by chemotherapy. Though mesenchymal stem cell (MSC) therapy shows promise in treating POI, its exact mechanism remains unclear. This study reveals that human umbilical cord-derived MSCs (hUC-MSCs) can protect ovarian granulosa cells (GCs) from cyclophosphamide (CTX)-induced ferroptosis, a form of cell death driven by iron accumulation. CTX, commonly used to induce POI animal model, triggered ferroptosis in GCs, while hUC-MSCs treatment mitigated this effect, both in vivo and in vitro. Further investigations using ferroptosis and autophagy inhibitors suggest that hUC-MSCs act by suppressing ferroptosis in GCs. Interestingly, hUC-MSCs activate a protective antioxidant pathway in GCs via NRF2, a stress-response regulator. Overall, our findings suggest that hUC-MSCs improve ovarian function in CTX-induced POI by reducing ferroptosis in GCs. This study not only clarifies the mechanism behind the benefits of hUC-MSCs but also strengthens the case for their clinical use in treating POI. Additionally, it opens up a new avenue for protecting ovaries from chemotherapy-induced damage by regulating ferroptosis.

Human mesenchymal stem cells derived exosomes improve ovarian function in chemotherapy-induced premature ovarian insufficiency mice by inhibiting ferroptosis through Nrf2/GPX4 pathway.

Chemotherapy exposure has become a main cause of premature ovarian insufficiency (POI). This study aimed to evaluate the role and molecular mechanism of human umbilical cord mesenchymal stem cell-derived exosomes (hUMSC-Exos) in ovarian function protection after chemotherapy. hUMSC-Exos were applied to cyclophosphamide-induced premature ovarian insufficiency mice and human ovarian granulosa tumor cells (KGN) to determine their effects on follicular development and granulosa cell apoptosis. Evaluation was done for iron ion and reactive oxygen species (ROS) production, lipid peroxidation levels, and changes in iron death-related molecules (nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Glutathione Peroxidase enzyme 4 (GPX4), and Solute carrier family 7 member 11 cystine glutamate transporter (SLC7A11; xCT)). Furthermore, rescue experiments using an Nrf2 inhibitor were performed to assess the therapeutic effects of hUMSC-Exos on granulosa cells. hUMSC-Exos promoted ovarian hormone levels and primary follicle development in POI mice and reduced granulosa cell apoptosis. After hUMSC-Exos treatment, the ROS production, free iron ions and lipid peroxidation levels of granulosa cells decreased, and the iron death marker proteins Nrf2, xCT and GPX4 also decreased. Furthermore, the Nrf2 inhibitor ML385 significantly attenuated the effects of hUMSC-Exos on granulosa cells. hUMSC-Exos inhibit ferroptosis and protect against CTX-induced ovarian damage and granulosa cell apoptosis through the Nrf2/GPX4 signaling pathway, revealing a novel mechanism of hUMSC-Exos in POI therapy.

The mechanisms of MicroRNA 21 in premature ovarian insufficiency mice with mesenchymal stem cells transplantation

Umbilical cord-derived mesenchymal stem cell (UCMSC) transplantation has been deeply explored for premature ovarian insufficiency (POI) disease. However, the associated mechanism remains to be researched. To explore whether and how the microRNA 21 (miR-21) functions in POI mice with UCMSCs transplantation, the autoimmune-induced POI mice model was built up, transplanted with or without UCMSCs transfect with the LV-hsa-miR-21-5p/LV-hsa-miR-21-5p-inhibition, with the transfection efficiency analyzed by QRT-PCR. Mice hormone secretion and the anti-Zona pellucida antibody (AZPAb) levels were analyzed, the ovarian morphological changes and folliculogenesis were observed, and the ovarian apoptosis cells were detected to evaluate ovarian function. The expression and localization of the PTEN/Akt/FOXO3a signal pathway-related cytokines were analyzed in mice ovaries.Additionally, the spleen levels of CD8 + CD28-T cells were tested and qualified with its significant secretory factor, interleukin 10 (IL-10). We found that with the LV-hsa-miR-21-5p-inhibition-UCMSCs transplantation, the mice ovarian function can be hardly recovered than mice with LV-NC-UCMSCs transplantation, and the PTEN/Akt/FOXO3a signal pathway was activated. The expression levels of the CD8 + CD28-T cells were decreased, with the decreased levels of the IL-10 expression. In contrast, in mice with the LV-hsa-miR-21-5p-UCMSCs transplantation, the injured ovarian function can be reversed, and the PTEN/AKT/FOXO3a signal pathway was detected activated, with the increased levels of the CD8 + CD28-T cells, and the increased serum levels of IL-10. In conclusion, miR-21 improves the ovarian function recovery of POI mice with UCMSCs transplantation, and the mechanisms may be through suppressing the PTEN/AKT/FOXO3a signal pathway and up-regulating the circulating of the CD8 + CD28-T cells.

TRIM28-Mediated Excessive Oxidative Stress Induces Cellular Senescence in Granulosa Cells and Contributes to Premature Ovarian Insufficiency In Vitro and In Vivo.

Premature ovarian insufficiency (POI) is a clinical syndrome of ovarian dysfunction characterized by the abnormal alteration of hormone levels such as FSH and E2. POI causes infertility, severe daily life disturbances, and long-term health risks. However, the underlying mechanism remains largely unknown. In this study, we found that POI is associated with the cellular senescence of ovarian granulosa cells, and TRIM28 mediates oxidative stress (OS)-induced cellular senescence in granulosa cells. Mechanistically, OS causes a decrease in TRIM28 protein levels in KGN cells. Subsequently, it triggers an increase in the levels of autophagy marker proteins ATG5 and LC3B-II, and the downregulation of P62. Abnormal autophagy induces an increase in the levels of cellular senescence markers γ-H2A.X, P16, and P21, provoking cellular senescence in vitro. The overexpression of ovarian TRIM28 through a microinjection of lentivirus attenuated autophagy, cellular senescence, and follicular atresia in the ovaries of POI mice and improved mouse fertility in vivo. Our study highlights the triggers for POI, where the reduction of TRIM28, which is regulated by reactive oxygen species, causes follicular atresia and POI via triggering autophagy and inducing granulosa cell senescence. Shedding light on TRIM28 may represent a potential intervention strategy for POI.

Ningxin-Tongyu-Zishen formula alleviates the senescence of granulosa cells on D-galactose-induced premature ovarian insufficiency mice.

Ningxin-Tongyu-Zishen formula (NTZF) is a clinical experience formula for the treatment of premature ovarian insufficiency (POI) in traditional Chinese medicine (TCM), and the potential mechanism is unknown. For in vivo experiments, POI mouse models (C57BL/6 mice), were constructed by subcutaneous injection of D-galactose (D-gal, 200 mg/kg). After treatment of NTZF (10.14, 20.27, 40.54 g/kg;) or estradiol valerate (0.15 mg/kg), ovarian function, oxidative stress (OS) and protein expression of Sirt1/p53 were evaluated. For in vitro experiments, H2O2 (200 μM) was used to treat KGN to construct ovarian granulosa cells (OGCs) cell senescence model. Pretreatment with NTZF (1.06 mg/mL) or p53 inhibitor (Pifithrin-α, 1 μM) was performed before induction of senescence, and further evaluated the cell senescence, OS, mRNA and protein expression of Sirt1/p53. In vivo, NTZF improved ovarian function, alleviated OS and Sirt1/p53 signaling abnormalities in POI mice. In vitro experiments showed that NTZF reduced the level of OS and alleviated the senescence of H2O2-induced KGN. In addition, NTZF activated the protein expression of Sirt1, inhibited the mRNA transcription and protein expression of p53 and p21. Alleviating OGCs senescence and protecting ovarian function through Sirt1/p53 is one of the potential mechanisms of NTZF in the treatment of POI.

Plasma-derived from human umbilical cord blood restores ovarian function and improves serum reproductive hormones levels in mice with premature ovarian insufficiency (POI) through cytokines and growth factors.

Premature ovarian insufficiency (POI) patients experience a decline in ovarian function and a reduction in serum reproductive hormones, leading to a significant impact on the outcomes of assisted reproductive technology. Despite the absence of an effective clinical treatment to restore fertility in POI patients, recent research has indicated that cord blood plasma (CBP) derived from human umbilical cord blood (hUCB) may offer therapeutic benefits for various degenerative diseases. The primary aim of this study is to explore approaches for enhancing ovarian function and serum reproductive hormones through the administration of CBP in a murine model. Initially, hUCB was utilized to obtain CBP (CBP), which was subsequently analyzed for cytokine and growth factor profiles in comparison to adult blood plasma (ABP) by use of flow cytometry. Subsequently, POI mouse models were established through the induction of 4-vinylcyclohexene diepoxide, followed by the injection of CBP into the tail. At 7, 14, and 21 days posttreatment, mouse ovaries and blood were collected, and their estrus cycle, body weight, and ovarian weights were evaluated using precise electronic balance. Finally, ovarian morphology and follicle number were assessed through HE staining, while serum levels of anti-Müllerian hormone (AMH), estradiol (E2) and follicle-stimulating hormone (FSH) were determined by ELISA. Our study revealed that individuals with CBP exhibited significantly lower concentrations of proinflammatory cytokines, including IL-β (p < 0.01) and IL-2 (p < 0.05), while displaying elevated levels of anti-inflammatory cytokines and chemokines, such as IL-2, IL-4, IL-6, IL-8, IL-12P70, IL-17A, IP-10, interferon-γ, and tumor necrosis factor-α (p < 0.01). Furthermore, CBP demonstrated remarkably higher levels of growth factors, including transforming growth factor-β1, vascular endothelial growth factor, and insulin-like growth factor-1 (p < 0.01) than ABP. Notably, our investigation also revealed that CBP restored the content of serum reproductive hormones, such as AMH, E2, and FSH (p < 0.05), and increased the number of primordial and primary follicles (p < 0.01) and decreased the number of luteal and atretic follicles (p < 0.01) in vivo. Our findings suggested that CBP-secreted cytokines and growth factors could be restored POI ovarian function, enhanced serum reproductive hormones and rescued follicular development in vivo. These findings further support the potential of CBP as a promising strategy in clinical applications for POI related infertility.

Adenovirus-mediated Sirt1 and Tgfbr2 gene therapy improves fertility in natural ovarian aging and doxorubicin-induced premature ovarian insufficiency mice

Premature ovarian insufficiency (POI) may lead to early menopause, fertility loss and birth defects without effective treatment. Here, we explored the efficacy and safety of gene therapy to rescue ovarian function in both natural ovarian aging and doxorubicin (Dox) induced POI mice. Sirt1 and Tgfbr2 were screened out and identified as key genes in both murine and human ovarian tissues. Then, adenovirus (AdV) was selected as a suited carrier for ovarian infection after comparison of multiple viral vectors. In both two models, murine fertility was significantly improved after AdV-Sirt1 and AdV-Tgfbr2 invention individually or in combination, without obvious side effects to themselves and their offspring. Compared with the control group, the successful pregnancy rate in the 9-month-old-AdV-Sirt1 group increased by 60 % (67 % vs 42 %). Meanwhile, the pregnancy rate in the AdV-Tgfbr2 + Dox group increased by 85 % (55.6 % vs 20 %). The biological process of ovarian follicle development and fibrosis was rescued. Our work demonstrated that AdV-Sirt1 and AdV-Tgfbr2 therapy alleviates natural ovarian aging and Dox-associated POI, which may be potentially applicable for female fertility protection.

Exosomes derived from mesenchymal stem cells attenuate NLRP3-related pyroptosis in autoimmune premature ovarian insufficiency via the NF-κB pathway

Research questionWhat is the effect of exosomes derived from bone marrow mesenchymal stem cells (MSC-Exos) on the pyroptosis and recovery of granulosa cells in autoimmune premature ovarian insufficiency (POI)? DesignIn vitro, KGN cells were exposed to interferon-gamma to simulate immune injury. Samples were collected after a 48 h incubation with MSC-Exos (30 μg/ml). The cell viability, secretion of oestrogen and expression of key molecules in pyroptosis and the nuclear factor kappa B (NF-κB) pathway were tested. In vivo, the BALB/c mouse model of autoimmune POI model induced by zona pellucida glycoprotein 3 was used. Fertility testing and sample collection were applied 4 weeks after the ovarian subcapsular injection of MSC-Exos (150 μg for each ovary). Hormone concentration measurements, follicle counting and pyroptotic pathway analyses were conducted for each group. ResultsIn vitro, MSC-Exos significantly promoted the proliferation rate and secretion of oestrogen, while at the same time suppressing apoptosis and pyroptosis. In vivo, exosomal treatment normalized the irregular oestrous cycles, rescued the follicular loss and increased the pregnancy rate and number of offspring in POI mice. Elevated serum concentrations of oestrogen and anti-Müllerian hormone, as well as decreased concentrations of FSH and interleukin-1β, were shown. Furthermore, MSC-Exos down-regulated the expression of the NLRP3/Casp1/GSDMD pathway and inhibited activation of the NF-κB pathway. ConclusionsThese findings demonstrate for the first time that MSC-Exos exert a significant effect on restoring ovarian function in autoimmune POI in vivo and in vitro by suppressing the NLRP3/Casp1/GSDMD pathway and pyroptosis. The NF-κB pathway may contribute to the regulation of NLRP3-related pyroptosis.

Transcriptome analysis during 4-vinylcyclohexene diepoxide exposure-induced premature ovarian insufficiency in mice.

The occupational chemical 4-Vinylcyclohexene diepoxide (VCD) is a reproductively toxic environmental pollutant that causes follicular failure, leading to premature ovarian insufficiency (POI), which significantly impacts a woman's physical health and fertility. Investigating VCD's pathogenic mechanisms can offer insights for the prevention of ovarian impairment and the treatment of POI. This study established a mouse model of POI through intraperitoneal injection of VCD into female C57BL/6 mice for 15 days. The results were then compared with those of the control group, including a comparison of phenotypic characteristics and transcriptome differences, at two time points: day 15 and day 30. Through a comprehensive analysis of differentially expressed genes (DEGs), key genes were identified and validated some using RT-PCR. The results revealed significant impacts on sex hormone levels, follicle number, and the estrous cycle in VCD-induced POI mice on both day 15 and day 30. The DEGs and enrichment results obtained on day 15 were not as significant as those obtained on day 30. The results of this study provide a preliminary indication that steroid hormone synthesis, DNA damage repair, and impaired oocyte mitosis are pivotal in VCD-mediated ovarian dysfunction. This dysfunction may have been caused by VCD damage to the primordial follicular pool, impairing follicular development and aggravating ovarian damage over time, making it gradually difficult for the ovaries to perform their normal functions.

Human Bone Marrow Derived-Mesenchymal Stem Cells Treatment for Autoimmune Premature Ovarian Insufficiency.

Premature ovarian insufficiency (POI) is a relatively common gynecologic endocrine disorder, which is hypogonadism associated with amenorrhea, increased levels of gonadotropins, and hypoestrogenism. POI resulting from ovarian autoimmunity is a poorly understood clinical condition lacking effective treatments. This study is aimed to investigate the therapeutic effect of mesenchymal stem cells (MSCs) on autoimmune premature ovarian insufficiency. In this study, in vivo and in vitro experiments were conducted to clarify the therapeutic effects and possible mechanisms of human bone marrow-derived MSCs (hBMSCs) on autoimmune POI, and to provide an experimental evidence for the treatment of autoimmune POI by hBMSCs. Noteworthy, in this study, we used interferon-gamma (IFN-γ) to induce autoimmune inflammation in human granulosa cell line KGN, simulating the pathophysiological changes of granulosa cells in autoimmune POI, and therefore sought to establish an in vitro cell model of autoimmune POI, which is still lacking in experimental methodology. And we found that, in vitro, co-culture of hBMSCs could promote granulosa cell proliferation, inhibit apoptosis, improve hormone synthesis capacity, and reduce the occurrence of pyroptosis; and in vivo, hBMSCs resulted in improved estrous cycle disorders in autoimmune POI mice, increased serum estradiol, decreased follicle-stimulating hormone, improved ovarian morphology, increased number of primordial and primary follicles, decreased number of atretic follicles, and decreased ovarian granulosa cell apoptosis. hBMSCs have therapeutic effects on autoimmune POI both in vitro and in vivo.

Effect of Ningxin-Yishen Formula on D-galactose-induced Premature Ovarian Insufficiency Mice by Inhibiting p53

: The prevalence of premature ovarian insufficiency (POI) is gradually increasing, safer and more effective treatments are urgently needed. The aim of this study was to evaluate the effects of Ningxin-Yishen formula (NXYSF) on D -galactose-induced POI mice as well as to shed a light on its potential mechanisms. : Six to eight weeks old C57BL/6 female mice were used in this study and randomly divided into six groups: control group; model group; estradiol valerate (EV) treatment group and NXYSF treatment group with graded doses (9.5, 19, and 38 g∙kg −1 /day). Both EV and NXYSF treatments were initiated at the 15th day of modeling and lasted for 28 days. Afterwards, the ovarian function was evaluated in each group by analyzing the proportion of primordial follicles as well as the serum sex hormone levels. Furthermore, network pharmacology approach was performed to elucidate the potential targets of NXYSF, which were verified through western blotting experiments finally. : NXYSF could significantly reverse the inefficiency of weight gain caused by POI, and promote the development of primordial follicles. In addition, it could restore the abnormal serum anti-Müllerian hormone (AMH), estradiol (E 2 ), luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Moreover, some crucial key gene targets including TP53 were proposed to be related with the effect of NXYSF through network pharmacology analysis. Last but importantly, western blotting experiments confirmed that NXYSF could inhibit the expression of p53 protein in mouse ovary. : Our findings proposed that NXYSF might be an effective alternative treatment for POI.

PDCD4 deficiency improved 4-vinylcyclohexene dioxide-induced mouse premature ovarian insufficiency

Research questionWhat role does programmed cell death 4 (PDCD4) play in premature ovarian insufficiency (POI)? DesignA PDCD4 gene knockout (PDCD4–/–) mouse model was constructed, a POI mouse model was established similar to human POI with 4-vinylcyclohexene dioxide (VCD), a PDCD4-overexpressed adenovirus was designed and the regulatory role in POI in vitro and in vivo was investigated. ResultsPDCD4 expression was significantly increased in the ovarian granulosa cells of patients with POI (P ≤ 0.002 protein and mRNA) and mice with VCD-induced POI (P < 0.001 protein expression in both mouse ovaries and granulosa cells). In POI-induced mice model, PDCD4 knockouts significantly increased anti-Müllerian hormone, oestrodiol and numbers of developing follicles, and the PI3K-AKT-Bcl2/Bax signalling pathway is involved in it. ConclusionThe expression and regulation of PDCD4 significantly affects the POI pathology in a mouse model. This effect is closely related to the regulation of Bcl2/Bax and the activation of the PI3K-AKT signalling pathway.

Qirong Tablets inhibits apoptosis of ovarian granulosa cells via PI3K/Akt/ HIF-1 signaling pathway

This study aims to observe the effect and explore the mechanism of Qirong Tablets in the treatment of premature ovarian insufficiency(POI) in mice via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/hypoxia inducible factor 1(HIF-1) signaling pathway. Sixty SPF female BALB/c mice were randomly divided into normal group, model group, positive control group, Qirong Tablets low-, medium-and high-dose group. The normal group was intraperitoneally injected with the same amount of normal saline, and the other groups were intraperitoneally injected with cyclophosphamide 120 mg·kg~(-1)·d~(-1) once to establish a POI animal model. After the model was successfully established, the low-, medium-and high-dose groups of Qirong Tablets were administered orally with 0.6, 1.2, 2.4 mg·kg~(-1)·d~(-1) respectively. The positive control group was given 0.22 mg·kg~(-1)·d~(-1) Clementine Tablets by intragastric administration, and the normal group and model group were given intragastric administration with the same amount of normal saline, and the treatment was 28 d as a course of treatment. After drug intervention, enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of estradiol(E_2), follicle-stimulating hormone(FSH), luteinizing hormone(LH), and anti-mullerian hormone(AMH) in peripheral blood, and hematoxylin-eosin(HE) staining to observe the ovarian tissue. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay was used to detect the apoptosis of granulosa cells, and Western blot to determine the expression levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, PI3K, Akt, and HIF-1. Compared with the normal group, the modeling of POI caused loose or destroyed ovarian tissue with vacuolar structures, edema and fibrosis in the ovarian interstitium, disordered or loose arrangement of granulosa cells, and reduced normal follicles. Compared with the model group, drug interventions restored the ovarian tissue and follicles at all the development stages and reduced atretic follicles. Compared with the normal group, the modeling of POI lowered the serum level of E_2 and AMH(P&lt;0.01), and elevated the level of FSH and LH(P&lt;0.01). Compared with the model group, high-dose Qirong Tablets elevated the levels of E_2 and AMH(P&lt;0.05), and lowered the levels of FSH and LH(P&lt;0.05). Compared with the normal group, the modeling of POI up-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 and down-regulated the protein level of Bcl-2 in the ovarian tissue(P&lt;0.01). Compared with the model group, low-, medium-, and high-dose Qirong Tablets down-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 proteins and up-regulated the protein level of Bcl-2 in the ovarian tissue(P&lt;0.05). In conclusion, Qirong Tablets can up-regulate the expression Bcl-2, down-regulate the expression of Bax and caspase-3 in POI mice. Qirong Tablets may inhibit the apoptosis of follicular granulosa cells in mice, thereby delaying ovarian aging, improving reproductive axis function, and strengthening ovarian reserve capacity, which may be associated with the inhibition of PI3K/Akt/HIF-1 pathway.

Network analysis and experimental approach to investigate the potential therapeutic mechanism of zishen yutai pills on premature ovarian insufficiency

BackgroundAs society continues to develop, women are more at risk of gonadotoxic substance exposure. Consequently, the incidence of premature ovarian insufficiency (POI) has increased significantly in the past decades. Hormone replacement therapy (HRT) is recommended as the standard treatment to relieve hypoestrogenic symptoms; however, its potential side effects and contraindications have drawn widespread controversy and concern. As such, the Chinese medicine Zishen Yutai Pill (ZSYTP) commonly used for treating miscarriage and menoxenia, is a highly promising alternative drug candidate against POI, however its therapeutic mechanism has not been completely elucidated. ObjectiveTo systematically analyze the potential therapeutic targets of ZSYTP on POI, we combined network pharmacology analysis and molecular docking to predict critical target genes, with experimental validation on POI murine models. MethodsThe active compounds of ZSYTP were collected from three online databases, and the candidate targets were predicted based on the chemical structure. The POI-related targets were obtained from four databases. A PPI network was constructed to find the key target genes between ZSYTP and POI, while GO and KEGG enrichment analyses were employed to study the mechanism of ZSYTP against POI. The binding capability of the key co-targets with active components was examined by molecular docking. We used a cyclophosphamide (CTX)-inducible POI mouse model to verify our predictions by histopathological observation, immunohistochemical staining (caspase-3, TUNEL assay), hormone determination (FSH, AMH) and ribonucleic acid sequencing (RNA Seq). Progynova was also used to study the difference between ZSYTP and HRT. ResultWe identified 21 target genes as the hub between ZSYTP and POI. The GO and KEGG analyses revealed that the molecular mechanism of ZSYTP against POI were mainly based on the regulation of gene and protein expression. A variety of signaling pathways may be involved in the treatment of ZSYTP against POI, especially PI3K-AKT, HIF-1 and the AGE-RAGE cascades. Docking simulation showed that G1, C1, SR5, and F1 had relatively lower binding energy. In vivo, ZSYTP significantly reversed CTX-induced ovarian damage in follicle number, hormone level and apoptosis, with an overall improved therapeutic effect compared to Progynova. Results from RNA-Seq revealed that the PI3K-AKT, Hippo, AGE-RAGE, and Rap1 signaling pathways and regulation of inflammation, immune response, and lipid metabolism may mediate the protective effects of ZSYTP against POI, which is different than Progynova's mechanism of action. ConclusionsCollectively, this study indicates that ZSYTP could be a highly promising alternative as a non-HRT-based therapy for POI. Its mechanism involves multiple signaling pathways, alleviating ovarian apoptosis and recovering AMH and FSH level. However, the discrepancy between different research techniques highlight the necessity of further experimental verification from other aspects such as translation and posttranslational modification.

Transcription factor c-fos induces the development of premature ovarian insufficiency by regulating MALAT1/miR-22-3p/STAT1 network

BackgroundThe current study attempted to investigate the role of transcription factor c-fos in the development of premature ovarian insufficiency (POI) as well as the underlying mechanism involving the MALAT1/miR-22-3p/STAT1 ceRNA network.MethodsBioinformatics analysis was performed to extract POI-related microarray dataset for identifying the target genes. Interaction among c-fos, MALAT1, miR-22-3p, and STAT1 was analyzed. An in vivo POI mouse model was prepared followed by injection of sh-c-fos and sh-STAT1 lentiviruses. Besides, an in vitro POI cell model was constructed to study the regulatory roles of c-fos, MALAT1, miR-22-3p, and STAT1.Resultsc-fos, MALAT1, and STAT1 were highly expressed in ovarian tissues from POI mice and CTX-induced KGN cells, while miR-22-3p was poorly expressed. c-fos targeted MALAT1 and promoted MALAT1 transcription. MALAT1 competitively bound to miR-22-3p and miR-22-3p could suppress STAT1 expression. Mechanically, c-fos aggravated ovarian function impairment in POI mice and inhibited KGN cell proliferation through regulation of the MALAT1/miR-22-3p/STAT1 regulatory network.ConclusionOur findings highlighted inducing role of the transcription factor c-fos in POI through modulation of the MALAT1/miR-22-3p/STAT1 ceRNA network.

Berberine protects cyclophosphamide and busulfan-induced premature ovarian insufficiency in mouse model

Premature ovarian insufficiency (POI) is a clinical syndrome that declines ovarian function in women. Berberine (BBR) is a compound with anti-inflammatory, antioxidant, and anti-apoptotic activities. However, the role of BBR on POI is still unknown. In this study, we investigated the role of BBR on ovarian function decline by establishing a POI mouse model using cyclophosphamide (CTX) and busulfan (BU). Our results showed that POI was attenuated by BBR, which was evidenced by enhanced body weight and ovarian weight, improved morphology of ovary, increased the number of healthy follicles, decreased the production of atretic follicles and restored serum hormone levels, including estradiol, anti-Müllerian hormone and follicle-stimulating hormone. In addition, we showed that germ cell function markers, mouse vasa homologue (MVH) and octamer-binding transcription factor 4 (OCT4) were enhanced by BBR, at both protein and mRNA levels. Furthermore, our results revealed that BBR inhibited inflammation and oxidative stress by reducing nuclear factor kappa B (NF-κB) and enhancing nuclear factor erythroid 2-related factor 2 (Nrf2) pathways. Taken together, we demonstrate that BBR can effectively improve ovarian function in POI mice, which is mainly mediated by reducing oxidative stress and inflammatory response. Our study also provides new strategy for POI treatment.