Premature Ovarian Failure Rats Research Articles

Repair effect of human umbilical cord mesenchymal stem cell-derived small extracellular vesicles on ovarian injury induced by cisplatin.

Small extracellular vesicles (sEVs) secreted by human umbilical cord have therapeutic effects on various degenerative diseases. However, the characteristics and potential functions of human umbilical cord mesenchymal stem cells (huMSCs)-derived sEVs, especially the role of premature ovarian failure (POF), are poorly understood. Here, we isolated and characterized huMSCs and their sEVs. huMSCs highly expressed CD73, CD90, and CD105. huMSC-sEVs showed typical exosomal features, highly expressing CD9, TSG101, and CD63. It was shown that huMSC-sEVs could be taken up by granulosa cells (GCs) and damaged ovarian tissue, which increased the levels of hormone secretion and reduced GCs apoptosis. We further confirmed that the levels of follicle-stimulating hormone in rat serum decreased dramatically, while the levels of estrogen (E2）and anti-mullerian hormone (AMH) increased significantly with the treatment of huMSC-sEVs. Meanwhile, huMSC-sEVs treatment greatly reduced cell apoptosis and autophagy, while increased the phosphorylation levels of p-PI3K and p-Akt. Therefore, treatment with huMSC-sEVs significantly inhibited GCs apoptosis, improved ovarian morphology, promoted follicular development, inhibited follicular over-atresia, and improved ovarian reserve capacity in POF rats. Our study verified that activation of PI3K/Akt signaling pathway and regulation of cellular autophagy, thus reducing GCs death, are the mechanisms by which huMSC-sEVs restore ovarian tissue function.

Exosomal YB-1 facilitates ovarian restoration by MALAT1/miR-211-5p/FOXO3 axis

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. Mesenchymal stem cells-derived small extracellular vesicles (MSCs-sEVs) are attractive candidates for ovarian function restoration and folliculogenesis for POF due to their safety and efficacy, however, the key mediator in MSCs-sEVs that modulates this response and underlying mechanisms remains elusive. Herein, we reported that YB-1 protein was markedly downregulated in vitro and in vivo models of POF induced with H2O2 and CTX respectively, accompanied by granulosa cells (GCs) senescence phenotype. Notably, BMSCs-sEVs transplantation upregulated YB-1, attenuated oxidative damage-induced cellular senescence in GCs, and significantly improved the ovarian function of POF rats, but that was reversed by YB-1 depletion. Moreover, YB-1 showed an obvious decline in serum and GCs in POF patients. Mechanistically, YB-1 as an RNA-binding protein (RBP) physically interacted with a long non-coding RNA, MALAT1, and increased its stability, further, MALAT1 acted as a competing endogenous RNA (ceRNA) to elevate FOXO3 levels by sequestering miR-211-5p to prevent its degradation, leading to repair of ovarian function. In summary, we demonstrated that BMSCs-sEVs improve ovarian function by releasing YB-1, which mediates MALAT1/miR-211-5p/FOXO3 axis regulation, providing a possible therapeutic target for patients with POF.

Inhibition of p38 MAPK/NF-κB p65 signaling pathway activity by rare ginsenosides ameliorates cyclophosphamide-induced premature ovarian failure and KGN cell injury

Ethnopharmacological relevancePanax ginseng C. A. Mey., one of the most used herbs in the world, shows effective treatment in reproductive injury. Recent studies have proven that the processed product, red ginseng, which is more active than ginseng itself. Therefore, it is speculated that its main functional component, rare ginsenosides (heat-transformed saponin, HTS), may be effective in treating premature ovarian failure (POF), but its efficacy has not yet been experimentally confirmed. Aim of the studyTo evaluate whether HTS could attenuate cyclophosphamide-induced inflammation and oxidative damage in POF model rats and the human granulosa-like KGN cell line and protect granulosa cell proliferation. Material and methodsHTS were isolated from ginsenosides and high performance liquid chromatography (HPLC) analysis was used to analyze the HTS components. Cyclophosphamide (CP) was used to establish a POF rat model and KGN cell injury model. Reactive oxygen species (ROS) and antioxidant enzyme production was determined using specific assays, while inflammatory cytokine secretion was measured by enzyme-linked immunosorbent assay (ELISA). The proliferative function of granulosa cells was assessed using high-content screening and immunohistochemistry to determine the Ki67 protein level. Protein expression in ovarian tissues and KGN cells was analyzed by Western blotting, quantitative real-time PCR (qRT-PCR) was used to determine the transcriptional changes in ovarian tissues and KGN cells. ResultsIn CP-treated POF model rats, HTS significantly decreased malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels, increased glutathione oxidase (GSH) levels, and upregulated Ki67 expression in ovarian granulosa cells. In addition, HTS significantly increased cell survival and Ki67 expression levels in CP-treated cells, and superoxide dismutase (SOD) levels were significantly increased. HTS significantly downregulated IL-6, TNF-α, and interleukin-1β (IL-1β) mRNA expression and significantly inhibited nuclear factor kappa-B p65 (NF-κB p65) and p38 mitogen activated protein kinase (p38 MAPK) phosphorylation in POF model rats and KGN cells. Moreover, NF-κB p65 and p38 MAPK levels were significantly increased in ovarian granulosa cells. p65 and p38 protein and gene expression was significantly downregulated. ConclusionHTS ameliorated CP-induced POF and human granulosa cell injury, possibly by inhibiting inflammation and oxidative damage mediated by the p38 MAPK/NF-κB p65 signaling pathway.

Ameliorative effect of salidroside on the cyclophosphamide-induced premature ovarian failure in a rat model

Background Oxidative stress injury is an important pathological factor of premature ovarian failure (POF). Salidroside, extracted from the Chinese herb-Rhodiola rosea, has advantages in antioxidant characteristics. However, their therapeutic efficacy and mechanisms in POF have not been explored. Purpose This study aims to assess the therapeutic effects of salidroside in chemotherapy-induced ovarian failure rats. Methods A POF rat model was established by injection of cyclophosphamide, followed by treatment with salidroside. The therapeutic effect of salidroside was evaluated based on hormone levels, follicle count, and reproductive ability. Oxidative stress injury was assessed by the detection of SOD enzyme activity and MDA levels. Differential gene expression of Keap1, Nrf2, HMOX1, NQO1, AMH, BMP15, and GDF9, were identified by qRT‑PCR. The protein expression of Keap1, Nrf2, P53, and Bcl-2 were detected by western blot. Results Salidroside treatment markedly restored FSH, E2, and AMH hormone secretion levels, reduced follicular atresia, and increased antral follicle numbers in POF rats. In addition, salidroside improves fertility in POF rats, activates the Nrf2 signaling pathway, and reduces the level of oxidative stress. The recovery function of high dose salidroside (50 mg/kg) in a reproductive assay was significantly improved than that of lower dose salidroside (25 mg/kg). Meanwhile, the safety evaluation of salidroside treatment in rats showed that salidroside was safe for POF rats at doses of 25–50 mg/kg. Conclusions Salidroside therapy improved premature ovarian failure significantly through antioxidant function and activating Nrf2 signaling.

Efficacy and safety of recombinant human granulocyte colony-stimulating factor in patients with poor ovarian response: protocol for a multicentre, randomised, controlled trial

IntroductionApproximately 17.5% of couples of reproductive age have experienced infertility. Women with a poor ovarian response (POR) must undergo in vitro fertilisation (IVF) and embryo transfer to achieve pregnancy. However,...

LIPUS combined with TFSC alleviates premature ovarian failure by promoting autophagy and inhibiting apoptosis

Objective Premature ovarian failure (POF), also known as primary ovarian insufficiency, is a major cause of infertility in female worldwide. Excessive apoptosis and impaired autophagy in ovarian granulosa cells are the main pathological mechanisms of POF. The total flavonoids from semen cuscutae (TFSC) are often used in the treatment of gynecological endocrine disorders. In addition, low intensity pulsed ultrasound (LIPUS) is report as an effective method to improve ovarian function. This study aims to investigate the protective effect of POF by the combined use of TFSC and LIPUS. Methods POF rats model and granulosa cell model were successfully induced by tripterygium glycosides and cyclophosphamide, respectively. After that, model rats and cells received TFSC plus LIPUS administration. Then ovarian histomorphology, senescence, estrus cycle, and serum sex hormone levels were detected in rats. Ovarian tissue and granulosa cells autophagy and apoptosis levels were also assessed. Results Disturbed sex hormone levels, atrophied and senescent ovaries, and abnormal estrous cycle were found in POF rats. Meanwhile, cell autophagy was inhibited and cell apoptosis was activated in POF ovarian tissue and granulosa cells. However, TFSC combined with LIPUS improved these changes, and this combination treatment exhibited synergistic effects. The abnormal expression of the cell apoptosis-, autophagy-, and PI3K/AKT/mTOR signaling pathway-related proteins were also improved by combination treatment. Conclusion The study found that the combination of TFSC and LIPUS can alleviate POF by modulating cell autophagy and apoptosis. The findings may provide a viable scientific basis for POF treatment.

Synergistic Effects of Capsaicin and Quercetin Improved Induced Premature Ovarian Failure in Rat.

Premature ovarian failure (POF) is a heterogeneous disorder. POF is defined as hypergonadotropic hypoestrogenism in women under 40 years. There is no effective treatment to cure POF patients. Antioxidants prevent ovarian damage by reducing the lipid peroxidation cascades affecting folliculogenesis, meiosis and ovulation. Hence; the aim of present study was to investigate the effects of Capsaicin (CAP) and Quercetin (QUR) on cyclophosphamide (CYC)-induced POF in rat model. In this experimental study, POF was induced by intraperitoneal injection of 200 mg/kg CYC on first day and then 8 mg/kg/day for the following 3 days. After 4 days of CYC administration, rats were randomly divided into five groups (n=6/group) as follows: POF, dimethyl sulfoxide (DMSO), CAP (0.5 mg/kg/day), QUR (100 mg/kg/day) and CAP+QUR. Biochemical, hormonal, gene expression, and histological evaluations were performed on blood serum and tissue samples after 14 days of treatment with the CAP and QUR. CAP, QUR and CAP+QUR groups showed signs of restored ovarian function in the form of a significant increase in serum total antioxidant capacity (TAC), estrogen, progesterone and anti-mullerian hormone (AMH) levels versus POF and DMSO groups and a significant improvement in histological parameters and follicle numbers in treatment groups compared to POF and DMSO groups. Polymerase chain reaction (PCR) analysis demonstrated that CAP and QUR upregulate the expression of BAX gene and decreased the expression of apoptosis inducing genes (BCL-2 and P53). CAP and QUR treatment of CYC-induced POF rats showed a positive effect on reducing ovarian damage by improving TAC levels, expression of apoptotic genes, levels of ovarian reserve markers, and histological parameters. Our results suggest that treatment with CAP or QUR may be a conservative treatment approach for CYC -induced POF.

Hydrogen-rich water treatment targets RT1-Db1 and RT1-Bb to alleviate premature ovarian failure in rats.

Premature ovarian failure (POF) is defined as the cessation of ovarian function before the age of 40 years, imposing a significant health burden on patients. However, effective etiological therapy for POF is scarce. Thus, we aimed to explore the protective role and targets of hydrogen-rich water (HRW) in POF. Based on cyclophosphamide (CTX)-induced POF rat models, the protective role of HRW treatment was mainly determined through serum 17-β-estradiol (E2), follicle-stimulating hormone (FSH), anti-mullerian hormone (AMH) levels, ovarian histomorphological analysis, and TUNEL assay. Tandem mass tag (TMT)-based quantitative proteomic analysis was then conducted on ovarian tissues, and the targets of HRW in POF were identified integrating differential expression analysis, functional enrichment analysis, and interaction analysis. In HRW treatment of POF rats, the serum AMH and E2 levels significantly increased, and FSH level significantly reduced, indicating the protective role of HRW. After TMT quantitative proteomic analysis, a total of 16 candidate differentially expressed proteins (DEPs) were identified after the cross analysis of DEPs from POF vs. control and POF+HRW vs. POF groups, which were found to be significantly enriched in 296 GO terms and 36 KEGG pathways. The crucial targets, RT1-Db1 and RT1-Bb, were finally identified based on both protein-protein interaction network and GeneMANIA network. The HRW treatment could significantly alleviate the ovarian injury of POF rats; RT1-Db1 and RT1-Bb are identified as two crucial targets of HRW treatment in POF rats.

LCZ696 (sacubitril/valsartan) mitigates cyclophosphamide-induced premature ovarian failure in rats; the role of TLR4/NF-κB/NLRP3/Caspase-1 signaling pathway

AimCyclophosphamide (CP) is used to treat a variety of cancers and autoimmune illnesses. CP has been found to frequently cause premature ovarian failure (POF). The study's objective was to assess LCZ696's potential for protection against CP-induced POF in a rat model. Main methodsRats were randomly assigned into seven groups as follows: control, valsartan (VAL), LCZ696, CP, CP + VAL, CP + LCZ696, and CP + triptorelin (TRI). Ovarian malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), interleukin-18 (IL-18), IL-1β, and tumor necrosis factor-alpha (TNF-α) were assessed using ELISA. Serum anti-mullerian hormone (AMH), estrogen, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were also measured using ELISA. The expression of NLRP3/Caspase-1/GSDMD C-NT and TLR4/MYD88/NF-B P65 proteins was estimated using western blot assay. The histopathology of the ovaries was also investigated. The estrous cycle, body, and ovarian weights were also monitored. Key findingsCP treatment significantly elevated levels of MDA, IL-18, IL-1β, TNF-α, FSH, LH, and up-regulated TLR4/NF-κB/NLRP3/Caspase-1 proteins, as compared to the control group, however, ovarian follicles count, and levels of GSH, SOD, AMH, and estrogen were reduced with CP administration. All the aforementioned biochemical and histological abnormalities were considerably alleviated by the LCZ696 therapy compared to valsartan alone. SignificanceLCZ696 effectively mitigated CP-induced POF, offering promising protection that could be related to its suppression power on NLRP3-induced pyroptosis and TLR4/NF-B P65 pathway.

Adipose-derived stem cells promote the repair of chemotherapy-induced premature ovarian failure by inhibiting granulosa cells apoptosis and senescence

BackgroundChemotherapeutic drugs, particularly alkylating cytotoxics such as cyclophosphamide (CTX), play an important role to induce premature ovarian failure (POF). Hormone replacement therapy (HRT) is a widely used treatment to improve hormone secretion. However, the long-term HRT increases the risk of breast cancer and cardiovascular disease are attracting concerns. Therefore, there is an urgent need to develop a safe and effective treatment for POF.MethodAdipose-derived stem cells (ADSCs) were isolated and identified from human adipose tissue. For POF modeling, CTX were intraperitoneal injected into CTX-acute group, CTX-chronic group, CTX-acute + ADSCs group and CTX-chronic + ADSCs group rats; For transplantation, ADSCs were transplanted into POF rats through tail-vein. The control group rats were injected with PBS. The effects of POF modeling and transplantation were determined by estrous cycle analysis, histopathological analysis, immunohistochemical staining and apoptosis-related marker. To evaluate the effects of ADSC on granulosa cells in vitro, CTX-induced senescent KGN cells were co-cultured with ADSCs, and senescent-related marker expression was investigated by immunofluorescent staining.ResultsIn vivo studies revealed that ADSCs transplantation reduced the apoptosis of ovarian granulosa cells and secretion of follicle-stimulating hormone. The number of total follicles, primordial follicles, primary follicles, and mature follicles and secretion of anti-Müllerian hormone and estradiol (E2) were also increased by ADSCs. The estrous cycle was also improved by ADSC transplantation. Histopathological analysis showed that CTX-damaged ovarian microenvironment was improved by ADSCs. Furthermore, TUNEL staining indicated that apoptosis of granulosa cells was decreased by ADSCs. In vitro assay also demonstrated that ADSC markedly attenuated CTX-induced senescence and apoptosis of granulosa cell. Mechanistically, both in vivo and in vitro experiments proved that ADSC transplantation suppressed activation of the PI3K/Akt/mTOR axis.ConclusionOur experiment demonstrated that a single injection of high-dose CTX was a less damaging chemotherapeutic strategy than continuous injection of low-dose CTX, and tail-vein injection of ADSCs was a potential approach to promote the restoration of CTX-induced POF.

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

Ethnopharmacological relevantErxian 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 researchThe study aims to ascertain both in vivo and in vitro rewards of EXD. Materials and methodsEXD 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. ResultsUsing 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. ConclusionEXD 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.

Classical music restored fertility status in rat model of premature ovarian failure

Background:The restorative effect of classical music was assessed on the cyclophosphamide-induced animal model of premature ovarian failure (POF).Methods:Mozart’s piano classical music (K.448) was used for up to 4 and 8 weeks. Rats were exposed to music 6 h every day using a stereo system with a volume of 65–70 dB. Sera and ovarian tissue samples were collected for the evaluation of FSH, LH, and E2 and histopathological examination. At the same time points, samples were taken from the hypothalamus and hippocampus to monitor the expression of Ntrk2, Crh, and Pomc using real-time PCR. Mating trial was performed to evaluate the fertility status of POF rats.Results:Histopathological examination revealed a significant increase (p < 0.05) in the numbers of morphologically normal follicles at all the developmental stages in POF rats after music therapy compared to the POF group (p < 0.05). Music therapy decreased FSH and LH levels to near-to-normal levels conidied with elevation of E2 (p < 0.05). Ntrk2, Crh, and Pomc expressions were down-regulated in POF rats. Music therapy increasaed the expression of Ntrk2 in the hypothalamus of POF rats (p < 0.05). In contrast, Crh and Pomc failed to reach the detection limit before intervention and four weeks after the intervention however, these genes were expressed eight weeks after music therapy. Fertility status was increased (p < 0.05) in terms of litter size in POF rats after being exposed to music compared to the non-treated POF control group (p < 0.05).Conclusion:Results showed that music can exert therapeutic effects on POF rats via the alteration of sex-related hormones.

MiRNA-190a-5p promotes primordial follicle hyperactivation by targeting PHLPP1 in premature ovarian failure.

We previously screened 6 differentially expressed miRNAs in ovarian tissues of 4-vinylcyclohexene diepoxide (VCD)-treated premature ovarian failure (POF) model in SD rats, including miRNA-190a-5p, miRNA-98-5p, miRNA-29a-3p, miRNA-144-5p, miRNA-27b-3p, miRNA-151-5p. In this study, to investigate the mechanisms causing the onset of POF, we first identified miRNAs with earlier differential expression at consecutive time points in the VCD-treated rat POF model and explored the mechanisms by which the target miRNAs promote POF. The SD rats were injected with VCD for 15days to induce POF. Additionally, we collected rat blood and ovaries at the same time every day for 15 consecutive days, and luteinizing hormone (LH), follicle-stimulating hormone (FSH), Anti-Mullerian hormone (AMH), and estradiol (E2) serum levels were detected by ELISA. Six miRNAs expression were measured in rat ovaries by qRT-PCR. Dual-luciferase reporter gene assays were employed to predict and verify the target gene (PHLPP1) of target miRNAs (miRNA-190a-5p). Western blot was examined to detect the expression levels of PHLPP1, AKT, p-AKT, FOXO3a, p-FOXO3a, and LHR proteins on the target gene PHLPP1 and its participation in the primordial follicular hyperactivation-related pathways (AKT-FOXO3a and AKT-LH/LHR). During the VCD modeling POF rat ovaries, miRNA-190a-5p was the first to show significant differential expression, i.e., 6th of VCD treating, and PHLPP1 was verified to be a direct downstream target of it. Starting from the 6th of VCD treatment, the more significant the up-regulation trend of miRNA-190a-5p expression, the more obvious the down-regulation trend of PHLPP1 and LHR mRNA and protein expression, accompanied by the more severe phosphorylation of AKT and FOXO3a proteins, thus continuously over-activating the rat primordial follicle to promote the development of POF. In conclusion, miRNA-190a-5p may become a potential biomarker for early screening of POF, and it can continuously activate primordial follicles in rats by targeting the expression of PHLPP1 and key proteins in the AKT-FOXO3a and AKT-LH/LHR pathways.

Jian-Pi-Yi-Shen decoction inhibits mitochondria-dependent granulosa cell apoptosis in a rat model of POF.

As a widely applied traditional Chinese medicine (TCM), Jian-Pi-Yi-Shen (JPYS) decoction maybe applied in curing premature ovarian failure (POF) besides chronic kidney disease (CKD). In vivo experiments, 40 female SD (8-week-old) rats were randomized into four groups, namely, control group (negative control), POF model group, JPYS treatment group, and triptorelin treatment group (positive control). JPYS group was treated with JPYS decoction (oral, 11 g/kg) for 60 days, and the triptorelin group was treated with triptorelin (injection, 1.5 mg/kg) for 10 days before the administration of cyclophosphamide (CTX) (50 mg/kg body weight) to establish POF model. We examined apoptosis, mitochondrial function, and target gene (ASK1/JNK pathway and mitochondrial fusion/fission) expression. In vitro experiments, the KGN human granulosa cell line was used. Cells were pretreated with CTX (20, 40, and 60 μg/mL) for 24 h, followed by JPYS-containing serum (2, 4, and 8 %) for 24 h. Thereafter, these cells were employed to assess apoptosis, mitochondrial function, and target gene levels of protein and mRNA. In vivo, JPYS alleviated injury and suppressed apoptosis in POF rats. In addition, JPYS improved ovarian function. JPYS inhibit apoptosis of granulosa cells through improving mitochondrial function by activating ASK1/JNK pathway. In vitro, JPYS inhibited KGN cell apoptosis through inhibited ASK1/JNK pathway and improved mitochondrial function. The effects of GS-49977 were similar to those of JPYS. During POF, mitochondrial dysfunction occurs in the ovary and leads to granulosa cell apoptosis. JPYS decoction improves mitochondrial function and alleviates apoptosis through ASK1/JNK pathway.

Efficacy and Clinical Significance of the Zuogui Pill on Premature Ovarian Failure via the GDF-9/Smad2 Pathway

Our study aims to investigate the efficacy and clinical significance of the Zuogui pill (ZGP) on premature ovarian failure (POF) via the GDF-9/Smad2 pathway. Changes in clinical symptoms in the control group (treated with Femoston alone) and the treatment group (treated with ZGP combined with Femoston) were assessed before and after treatment. Sex hormone levels, serum inflammatory cytokine levels, and ultrasound parameters were measured before and after treatment. POF rat models were established using cyclophosphamide and the POF rats were treated with Femoston, or ZGP combined with Femoston. GDF-9 and Smad2 expression levels were determined by RT-qPCR. The follicle-stimulating hormone (FSH), luteinizing hormone (LH), interleukin (IL)-6, and IL-21 levels, and the pulsatility index (PI) and resistance index (RI) values were decreased, while the estradiol (E2) and anti-Mullerian hormone (AMH) levels, antral follicle count (AFC), ovarian volume (OV), mean ovarian diameter (MOD), and peak systolic velocity (PSV) values were increased in the treatment group compared to the control group. After treatment with ZGP combined with Femoston, GDF-9 and Smad2 expression in the ovarian tissues of POF rats increased. ZGP has a therapeutic effect on POF via modulation of the GDF-9/Smad2 pathway.

MiR-126-3p containing exosomes derived from human umbilical cord mesenchymal stem cells promote angiogenesis and attenuate ovarian granulosa cell apoptosis in a preclinical rat model of premature ovarian failure

BackgroundIn our previous research, we found that overexpression of miR-126-3p in human umbilical cord MSCs (hucMSCs) promoted human umbilical vein endothelial cells angiogenic activities through exosome-mediated mechanisms. The present study aimed to investigate the role of miR-126-3p-modified hucMSCs derived exosomes (miR-126-3p-hucMSCs-exosomes) on the treatment of premature ovarian failure (POF).MethodsPrimary hucMSCs were isolated from human umbilical cords and identified by differentiation experiments and flow cytometry. miR-126-3p-hucMSCs were obtained by miR-126-3p lentivirus infection. miR-126-3p-hucMSCs-exosomes were purified by ultracentrifugation method and characterized by transmission electron microscopy and western blot analysis. Primary rat ovarian granulosa cells (OGCs) were collected from ovarian tissues and identified by cell immunohistochemistry. The effects of miR-126-3p-hucMSCs-exosomes and miR-126-3p on OGCs function were determined by cell proliferation and apoptosis assays in a cisplatin induced POF cell model. The levels of suitable target genes were analyzed by PCR and Western blot analysis and subsequent Dual-Luciferase reporter assay. The signal pathway was also analyzed by western blot analysis. A cisplatin-induced POF rat model was used to validate the therapeutic effects of miR-126-3p-hucMSCs-exosomes to treat POF. Ovarian function was evaluated by physical, enzyme-linked immunosorbent assay, and histological examinations in chemotherapy-treated rats. The angiogenesis and apoptosis of ovarian tissues were assessed by immunohistochemical staining and Western blots.ResultsPrimary hucMSCs and miR-126-3p-hucMSCs-exosomes and primary rat OGCs were successfully isolated and identified. The cellular uptake experiments indicated that miR-126-3p-hucMSC-exosomes can be internalized into OGCs in vitro. Annexin V-FITC/PI staining and EDU assays revealed that both miR-126-3p-hucMSCs-exosomes and miR-126-3p promoted proliferation and inhibited apoptosis of OGCs damaged by cisplatin. PCR and western blot analysis and subsequent dual-luciferase reporter assay verified that miR-126-3p targets the sequence in the 3' untranslated region of PIK3R2 in OGCs. Further analysis showed that PI3K/AKT/mTOR signaling pathway took part in miR-126-3p/PIK3R2 mediated proliferation and apoptosis in OGCs. In rat POF model, administration of miR-126-3p-hucMSCs-exosomes increased E2 and AMH levels, increased body and reproductive organ weights and follicle counts, and reduced FSH levels. But more importantly, immunohistochemistry results indicated miR-126-3p-hucMSCs-exosomes significantly promoted ovarian angiogenesis and inhabited apoptosis in POF rats. Additionally, the analysis of angiogenic-related factors and apoptosis-related factors showed miR-126-3p-hucMSCs-exosomes had pro-angiogenesis and anti-apoptosis effect in rat ovaries.ConclusionsOur findings revealed that hucMSCs-derived exosomes carrying miR-126-3p promote angiogenesis and attenuate OGCs apoptosis in POF, which highlighted the potential of exosomes containing miR-126-3p as an effective therapeutic strategy for POF treatment.

Study on the Mechanism of Action of Different Acupuncture Regimens on Premature Ovarian Failure Model Rats.

To study the mechanism of acupuncture on premature ovarian failure (POF) through the apoptosis pathway mediated by Bcl-2/Bax. POF rats were successfully obtained by cyclophosphamide. They were divided into five groups. After that, acupuncture was performed. The blank group and model group were not treated. The routine acupuncture group was acupuncture at Guanyuan, Sanyinjiao, Zhongji, and Guilai four points. The Neck-seven-acupuncture group was selected from Fengchi, Fengfu, Tianzhu, and Wangu four acupoints; the three-viscera simultaneous treatment group selected Guanyuan, Shenshu, Sanyinjiao, Taichong, and Baihui five points; and the data mining group selected Guanyuan and Sanyinjiao two points for 14 days of treatment. During the treatment, some rats were shed one after another due to the side effects of bone marrow suppression caused by mold-making. After treatment, serum estradiol (E2), follicle forming hormone (FSH), and luteinizing hormone (LH) were detected by radioimmunoassay, Bcl-2 and Bax proteins were analyzed by Western blot method, and Bcl-2 and Bax RNA were analyzed by PCR method. Bcl-2 increased and Bax decreased in rats with premature ovarian failure treated with acupuncture. It shows that acupuncture can affect the secretion of ovarian-related hormones and the expression of apoptosis-related proteins, which is more significant in the conventional acupuncture point group. Acupuncture can inhibit the apoptosis of granulosa cells in ovarian tissue of rats with premature ovarian failure and improve ovarian function. The mechanism of its effect is to promote Bcl-2 gene expression and protein synthesis and inhibit Bax gene expression and protein synthesis. The conventional treatment group works best. This provides an experimental basis for the clinical use of acupuncture to intervene in the treatment of premature ovarian failure.

Melatonin Protects Against Cyclophosphamide-induced Premature Ovarian Failure in Rats.

This study was designed to understand the efficacy and molecular cues of melatonin in cyclophosphamide(CTX)-induced premature ovarian failure (POF) in rats. Female SD rats were used to evaluate the potential effects of melatonin on the ovarian hormonal status, follicular development, and granulosa cells in CTX-treated rats. Here, we found that pretreatment with melatonin before CTX administration preserved the normal sex hormone levels, improved follicular morphology, and granulosa cell proliferation, and reduced apoptosis, as compared to the CTX treatment alone. Additionally, melatonin also up-regulated CYR6 and CTGF at the mRNA and protein levels. A potential mechanism is that melatonin inhibits LATS1, Mps1-One binder (MOB1), and YAP phosphorylation, thereby activating the Hippo signal pathway to promote its downstream targets, CYR61 and CTGF. In conclusion, pretreatment with melatonin effectively protected the ovaries against CTX-induced damage by activating the Hippo pathway. This study lay the foundation for the clinical application of melatonin for cancer patients with CTX treatment.

Capsaicin prevents radiotherapy-induced premature ovarian failure in rats.

Ionising radiation exposure of 5-10gray (Gy) to the pelvic area induces premature ovarian failure (POF). Twenty-four young adult Wistar albino female rats were were treated with subcutaneous capsaicin 0.5mg/kg per day or placebo for 10days then exposed to whole body irradiation. Rats were randomly divided into four groups: (1) control; (2) capsaicin; (3) radiation only (IR): rats were injected with placebo before exposure to a single dose of 8.3-Gy whole body irradiation; (4) radiation-capsaicin (IR+CAP): rats were injected with capsaicin prior to whole body irradiation. Radiation triggered oxidative stress, increased ovarian inflammation, increased follicular apoptosis and diminished ovarian follicle pool. Capsaicin significantly ameliorated oxidative stress by decreasing serum total oxidant status, oxidative stress index, disulphide, and malondialdehyde levels (P ≤0.001); ovarian inflammatory status by decreasing expressions of TNF-α, IL-1β, PARP-1 (P =0.002); apoptosis by decreasing expressions of active caspase-3 and p53 (P =0.015, P =0.002); and follicle counts by increasing primordial follicles and decreasing apoptotic follicles (P ≤0.001) in rats when administered before radiation exposure. The beneficial effects of capsaicin are demonstrated for the first time on ionising radiation exposed rat ovaries. Capsaicin pre-treatment before radiotherapy restores the primordial follicle pool, inhibits atresia of ovarian follicles and may be an acceptable therapeutic modality to prevent radiation-induced POF.

Exploration of the Danggui Buxue Decoction Mechanism Regulating the Balance of ESR and AR in the TP53-AKT Signaling Pathway in the Prevention and Treatment of POF

Objective The purpose of this study was to explore the molecular mechanism of Danggui Buxue Decoction (DBD) intervening premature ovarian failure (POF). Methods The active compounds-targets network, active compounds-POF-targets network, and protein-protein interaction (PPI) network were constructed by a network pharmacology approach: Gene Ontology (GO) function and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis by DAVID 6.8 database. The molecular docking method was used to verify the interaction between core components of DBD and targets. Then, High-Performance Liquid Chromatography (HPLC) analysis was used to determine whether the DBD contained two key components including quercetin and kaempferol. Finally, the estrous cycle, organ index, ELISA, and western blot were used to verify that mechanism of DBD improved POF induced by cyclophosphamide (CTX) in rats. Results Based on the network database including TCMSP, Swiss Target Prediction, DisGeNET, DrugBank, OMIM, and Malacard, we built the active compounds-targets network and active compounds-POF-targets network. We found that 2 core compounds (quercetin and kaempferol) and 5 critical targets (TP53, IL6, ESR1, AKT1, and AR) play an important role in the treatment of POF with DBD. The GO and KEGG enrichment analysis showed that the common targets involved a variety of signaling pathways, including the reactive oxygen species metabolic process, release of Cytochrome C from mitochondria and apoptotic signaling pathway, p53 signaling pathway, the PI3K-Akt signaling pathway, and the estrogen signaling pathway. The molecular docking showed that quercetin, kaempferol, and 5 critical targets had good results regarding the binding energy. Chromatography showed that DBD contained quercetin and kaempferol compounds, which was consistent with the database prediction results. Based on the above results, we found that the process of DBD interfering POF is closely related to the balance of ESR and AR in TP53-AKT signaling pathway and verified animal experiments. In animal experiments, we have shown that DBD and its active compounds can effectively improve estrus cycle of POF rats, inhibit serum levels of FSH and LH, protein expression levels of Cytochrome C, BAX, p53, and IL6, and promote ovary index, uterine index, serum levels of E2 and AMH, and protein expression levels of AKT1, ESR1, AR, and BCL2. Conclusions DBD and its active components could treat POF by regulating the balance of ESR and AR in TP53-AKT signaling pathway.