This study aimed to investigate the protective effects of moxibustion on ovarian dysfunction in rats with cyclophosphamide (Cy)-induced premature ovarian insufficiency (POI). It also aimed at revealing its potential mechanisms and emphasizing its role in mitigating the mitochondrial dysfunction and NLRP3 inflammatory activation. POI models were established by the intraperitoneal administration of Cy using female Sprague-Dawley rats. Moxibustion (BL23 or CV4, CV8) was used to treat POI models for fifteen days. Vaginal smears, enzyme-linked immunosorbent assay, hematoxylin-eosin, tunnel staining, flow cytometry analysis, immunohistochemistry staining, qRT-PCR, and western blotting were conducted to evaluate the ovarian function, mitochondrial dysfunction, and NLRP3 inflammatory activation in this study. Moxibustion could improve the disorder of the estrous cycles and reproductive hormone levels, promote follicular growth, reduce the number of atresia follicles, and alleviate the apoptosis of ovarian granulosa cells (GCs) in rats with POI. Furthermore, moxibustion mitigated the mitochondrial damage, reversed the elevated serum levels of IL-18 and IL-1β, and decreased their protein expression in the ovaries of rats with POI. Moxibustion significantly inhibited the expression of the mRNAs and proteins of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase 1, and gasdermin D (GSDMD) in the ovaries of rats with POI. These results supported that moxibustion may ameliorate Cy-induced POI by mitigating the mitochondrial dysfunction and NLRP3 inflammatory activation. Targeted treatment of mitochondrial damage and NLRP3 inflammatory activation may be a novel therapeutic strategy for POI.

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. Graphical

Moxibustion, a common therapy in traditional Chinese medicine, has potential benefits for treating decreased ovarian reserve (DOR). The present study investigates the protective effect of moxibustion in a rat model of DOR and explores the possible mechanisms. Sixty-four female Sprague-Dawley rats were randomly divided into four groups: control, DOR, moxibustion (MOX), and hormone replacement therapy (HRT). The DOR rat model was established by intragastric administration of 50 mg/kg Tripterygium glycoside suspension (TGS), once daily for 14 days. MOX and HRT treatments were given from the day TGS administration was initiated. The ovarian reserve function was evaluated by monitoring the estrus cycle, morphological changes in ovaries, levels of serum estradiol (E₂), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and anti-Mullerian hormone (AMH), pregnancy rate and embryo numbers. Terminal-deoxynucleotidyl transferase-mediated nick-end-labeling staining was used to identify ovarian granulosa cell apoptosis, while the protein and mRNA expressions of Bax, B-cell lymphoma-2 (Bcl-2), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in ovarian tissues were examined by immunohistochemistry, Western blot and quantitative reverse transcription-polymerase chain reaction. Compared with the DOR group, MOX improved the disordered estrous cycle, promoted follicular growth, reduced the number of atresia follicles, increased the concentrations of serum E₂ and AMH, and decreased serum FSH and LH concentrations. More importantly, the pregnancy rate and embryo numbers in DOR rats were both upregulated in the MOX treatment group, compared to the untreated DOR model. Further, we found that the MOX group had reduced apoptosis of ovarian granulosa cells, increased Bcl-2 expression and reduced expression of Bax. Furthermore, the PI3K/AKT signaling pathway was triggered by the moxibustion treatment. Moxibustion improved ovarian function and suppressed apoptosis of ovarian granulosa cells in a rat model of DOR induced by TGS, and the mechanism may involve the PI3K/AKT signaling pathway.

Diminished ovarian reserve (DOR) is an increasingly emerging reproductive disorder that disturbs reproductive-aged women, which is closely linked with inflammation. In clinic, moxibustion has already been applied for reproductive problems. In the present study, we examined the involvement of inflammation in DOR and investigated the effect of moxibustion for its anti-inflammatory activities. Methods. DOR rat model was established using tripterygium glycosides A tablets (TGs) suspension by intragastric administration and was then treated with either moxibustion or hormone replacement therapy (HRT), respectively. Estrus cycles were observed through vaginal cytology. Ovarian morphological alterations were observed by HE staining. The serum levels of follicle-stimulating hormone (FSH), estradiol (E₂), anti-Müllerian hormone (AMH), tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) were measured through ELISA. The expression levels of Nrf2, HO-1, and NLRP3 were detected using immunohistochemistry. Nrf2, HO-1, and NLRP3 mRNA were examined by RT-PCR. Results. Moxibustion improved estrus cycles, FSH, E₂, and AMH levels relative to DOR rats as well as HRT, while also inhibiting ovarian tissue injury. Anti-inflammatory cytokine IL-10 in peripheral blood was upregulated, and proinflammatory factor TNF-α was decreased after treatment with moxibustion. Moxibustion enhanced the expression of mRNA and protein of nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1); in the mean time, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) was suppressed. Conclusions. We demonstrated that moxibustion could ameliorate the ovarian reserve in rats induced by TGs. Overall, the effect of moxibustion was comparable to that of HRT. The underlying mechanism could be attributed to the anti-inflammatory effects of moxibustion, which suppressed NLRP3 activation by upregulating Nrf2/HO-1 signaling pathway.

BACKGROUND:Mild moxibustion has been reported to effectively treat menopausal syndrome. This study investigated the efficacy and safety of using mild moxibustion at Shenshu for treating sub-health states in perimenopausal women. MATERIAL AND METHODS:...

Immunoinflammatory responses and macrophage polarisation are crucial for maintaining ovarian function. Moreover, electroacupuncture (EA) has been shown to protect ovarian function. However, the mechanisms by which EA improves ovarian function, including its effects on immunoinflammatory responses and macrophage polarisation, have not been determined. This study aimed to investigate the protective effects of EA on ovarian function in rats with diminished ovarian reserve (DOR) and to elucidate the regulatory mechanisms underlying inflammation and M1 and M2 macrophage polarisation. DOR models were established through the intragastric administration of 50 mg/kg Tripterygium glycoside suspension (TGs) for 14 consecutive days. The EA group received treatment at 2/100 Hz and 1.0 mA for 10 min using acupoints BL23, CV4 and CV12 for 14 days. Following the intervention, we employed various methodologies, including haematoxylin–eosin (H&E) staining, enzyme‐linked immunosorbent assay (ELISA), flow cytometry, immunohistochemical (IHC) staining, western blotting and quantitative reverse transcriptase‐polymerase chain reaction (PCR), to assess ovarian function, inflammatory factors and the expression levels of M1 and M2 macrophage‐related factors. EA intervention reduced the oestrous cycle disorder rate in the rats compared with that in the DOR group, leading to an increase in growing follicles, a reduction in atretic follicles (AFs) and an enhancement of both the capillary (Cap) network and corpus luteum (CL) structure. This intervention also resulted in decreased serum levels of follicle‐stimulating hormone (FSH), interferon‐ γ (IFN‐ γ ) and tumour necrosis factor‐ α (TNF‐ α ), along with increased levels of oestradiol (E ₂ ), interleukin‐4 (IL‐4) and interleukin‐10 (IL‐10). Furthermore, the number of M2 macrophages in the spleen increased, which was accompanied by elevated arginase 1 (Arg1) and decreased inducible nitric oxide synthase (iNOS) expression in the ovarian tissues. In summary, EA can restore the impaired ovarian function caused by TGs by promoting M2 macrophage polarisation and inhibiting inflammatory responses.