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.

[Electroacupuncture promotes expression of glutathione related regulatory enzymes in ovary tissue of rats with diminished ovarian reserve].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on ovarian function and expression of glutathione (GSH) related regulatory enzymes γ-glutamylcysteine synthetase (γ-GCS), glutathione reductase (GR) protein and gene in rats with diminished ovarian reserve (DOR), so as to explore its mechanisms underlying up-regulation of antioxidant stress ability. METHODS: A total of 30 female SD rats with normal estrous cycle were randomly divided into blank control, model and EA groups, with 10 rats in each group. The DOR model was established by gavage of tripterygium wilfordii polyglycoside suspension (50 mg·kg-1·d-1) for 14 consecutive days, while the rats in the blank group were given equal volume of 0.9% sodium chloride solution. One hour after daily gavage, EA (1.0 mA, 100 Hz) was applied alternately to bilateral "Shenshu"(BL23), and "Zhongwan"(CV12)+"Guanyuan"(CV4) for 10 min, for 14 consecutive days. Estrous cycles of rats in each group were observed and recorded daily during intervention.After the intervention, H.E.staining was used to observe histopathological changes of the ovarian tissue. The contents of serum sex hormones ï¼»follicle stimulating hormone (FSH), anti-mullerian hormone (AMH), estradiol (E2)ï¼½ and oxidative damage markers ï¼»8-hydroxydeoxyguanosine (8-OHDG) and nitrotyrosine (NTY)ï¼½ were determined by ELISA. The contents of GSH and oxidized glutathione (GSSG) in the liver tissue were determined by colorimetry, and their ratios were calculated. Immunohistochemistry and real-time fluorescence quantitative PCR were used to detect the immunoactivity and gene expression levels of γ-GCS and GR in the ovarian tissues, respectively. RESULTS: Compared with the blank group, the model group had a marked increase in the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents (P<0.01) and a considerable decrease in the levels of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number as well as the expression of γ-GCS and GR mRNAs (P<0.05, P<0.01). After EA intervention, the increase of the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents and the decrease of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number of γ-GCS and GR as well as the expression of γ-GCS genes were all reversed (P<0.01, P<0.05). H.E. staining showed degenerative changes of the ovarian tissue, fewer follicles at every level and increase of atretic follicles, disarrangement and layer number decrease of granulosa cells, and atrophy of corpus luteum in the model group, which were relatively milder in the EA group. CONCLUSION: EA can improve ovarian function, and reduce oxidative stress damage in DOR rats, which may be associated with its functions in up-regulating the expression of γ-GCS and GR protein and gene in the ovarian tissue.

Effects of acupuncture treatment on microRNAs expression in ovarian tissues from Tripterygium glycoside-induced diminished ovarian reserve rats.

Acupuncture is widely used to improve ovarian function. Previously, we demonstrated that acupuncture can improve oxidative stress in rats with tripterygium glycoside tablet suspension (TG)-induced diminished ovarian reserve (DOR). Herein, we aimed to explore the antioxidation mechanism of acupuncture for ameliorating the ovarian reserve in DOR rats. We performed microRNA sequencing and bioinformatics analysis to screen differentially expressed miRNAs (DE miRNAs) in ovarian tissues. In total, 1,172 miRNAs were identified by miRNA sequencing, of which 28 DE miRNAs were detected (including 14 upregulated and 14 downregulated) in ovarian tissues from the acupuncture group when compared with the DOR model rats. Based on functional enrichment analysis, the target genes of DE miRNAs were significantly enriched in GO-biological process (BP) terms associated with biological processes, positive regulation of transcription by RNA polymerase II, signal transduction, regulation of transcription, DNA-templated processes, and oxidation-reduction processes. In the Kyoto Encyclopedia of Genes and Genomes analysis, the main pathways were the MAPK signaling pathway, hepatitis B, proteoglycans in cancer, human cytomegalovirus infection, and the Ras signaling pathway. Finally, reverse transcription-quantitative PCR results confirmed that rno-miR-92b-3p, mdo-miR-26b-5p_R+1_1ss10TC, and bta-miR-7857-3p_R-1 were downregulated in the acupuncture group. The results revealed the impact of acupuncture on miRNA profiling of ovarian tissues from DOR rats, suggesting that rno-miR-92b-3p, mdo-miR-26b-5p_R+1_1ss10TC, and bta-miR-7857-3p_R-1 might provide relevant cues to relieve DOR-mediated oxidative stress.

Moxibustion alleviates decreased ovarian reserve in rats by restoring the PI3K/AKT signaling pathway.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Moxibustion Ameliorates Ovarian Reserve in Rats by Mediating Nrf2/HO-1/NLRP3 Anti-Inflammatory 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 (E 2), 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, E 2, 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.

[Effect of moxibustion on Nrf2/HO-1 signaling pathway in rats with diminished ovarian reserve].

OBJECTIVE: To observe the effect of moxibustion on Nrf2/HO-1 signaling pathway in rats with diminished ovarian reserve (DOR), and to explore the protective mechanism of moxibustion on ovarian reserve function. METHODS: Forty SD rats were randomly divided into a blank group, a model group, a moxibustion group and a hormone group, 10 rats in each group. The rats in the model group, moxibustion group and hormone group were treated with intragastric administration of tripterysium glycosides turbid liquid to prepare DOR model. The rats in the blank group were treated with intragastric administration of sodium chloride solution with the same volume, once a day for 14 days. The rats in the hormone group were treated with hormone sequential therapy for 14 days from the day of modeling; the rats in the moxibustion group were treated with moxibustion at bilateral "Shenshu" (BL 23) or "Guanyuan" (CV 4) and "Zhongwan" (CV 12) from the day of modeling, and the two groups acupoints were alternated every other day, 10 min each time, for 14 consecutive days. The estrus cycle was observed every day by vaginal exfoliated cell smear, and the estrus cycle disorder rate in each group was calculated. After the intervention, the HE staining was used to observe the histological morphology of ovaries; ELISA was used to detect the contents of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), anti Mullerian hormone (AMH), superoxide dismutase (SOD) and malondialdehyde (MDA); the protein levels of Nrf2 and HO-1 in ovarian tissue were detected by immunohistochemistry; real-time PCR (TaqMan probe method) was used to detect the expression of Nrf2 and HO-1 mRNA. RESULTS: Compared with the blank group, the rate of estrus cycle disorder in the model group was increased (P<0.01); compared with the model group, the rate of estrus cycle disorder in the moxibustion group and hormone group was decreased (P<0.01). Compared with the blank group, the serum contents of FSH, LH and MDA in the model group were increased (P<0.01), and the serum contents of E2, AMH and SOD were decreased (P<0.01). Compared with the model group, the serum contents of FSH, LH and MDA in the moxibustion group and hormone group were decreased (P<0.01, P<0.05), and the serum contents of E2, AMH and SOD were increased (P<0.01). Compared with the blank group, the protein and mRNA expression of Nrf2 and HO-1 in the model group were decreased (P<0.01); compared with the model group, the protein and mRNA expressions of Nrf2 and HO-1 in the moxibustion group and hormone group were increased (P<0.01). CONCLUSION: Moxibustion could reduce the rate of estrus cycle disorder, improve the level of serum sex hormones and antioxidant stress in DOR rats, and the mechanism may be related to the regulation of Nrf2/HO-1 signaling pathway.

Ultrasound-Enhanced Protective Effect of Tetramethylpyrazine via the ROS/HIF-1A Signaling Pathway in an in Vitro Cerebral Ischemia/Reperfusion Injury Model.

Reactive oxygen species-induced oxidative stress is an important pathophysiological process during cerebral ischemia/reperfusion (I/R) injury. It has been reported that the protective effect of tetramethylpyrazine (TMP) against cerebral I/R injury can be significantly improved by its combination with ultrasound exposure. However, the molecular mechanisms and signaling pathways underlying the synergistic protective effect remain unclear. In the present work, the damage induced by I/R injury was modeled by glutamate-induced toxicity to pheochromocytoma (PC12) cells. The ultrasound-enhanced protective effect of TMP was systemically investigated by measuring variations in cell viability, cell migration and levels of intracellular reactive oxygen species, the oxidative stress-related protein glutathione, apoptosis-related proteins (caspase-8, -9 and -3), as well as expression of related genes (hypoxia-inducible factor-1a, p53, murine double minute2). The results suggest that the ultrasound-enhanced protective effect of TMP against cerebral I/R injury might act via the reactive oxygen species/hypoxia-inducible factor-1a signaling pathway, and an appropriate ultrasound intensity should be selected to achieve an optimal synergistic neuroprotective effect.

[Effects of moxibustion at Shenshu (BL 23) on level of sex hormones and AMH in sub-health peri-menopausal women].

OBJECTIVE: To compare the effects between moxibustion at Shenshu (BL 23) and oral administration of Vitamin E on quality of life and mechanism in sub-health perimenopausal women, aiming to provide clinical evidence of moxibustion for health care of sub-health female. METHODS: Sixty participants were randomly divided into a moxibustion group and a medication group, 30 cases in each one. The volunteers in the moxibustion group were treated with mild moxibustion at bilateral Shenshu (BL 23) for 15 min, once a day; five treatments were considered as a course of treatment, and totally 4 courses were given with an interval of 2 days between courses. The volunteers in the medication group were treated with oral administration of soft capsule of Vitamin E, once a day, continuously for 28 days. The scores of quality of life and serum levels of sex hormones, anti-Mullerian hormone (AMH) and inhibin B (INHB) were measured before and after treatment in the two groups. RESULTS: ① Compared before treatment, the scores of quality of life in the two groups were both significantly increased after treatment (both P<0.01), which was more superior in the moxibustion group (P<0.05). ② Compared before treatment, the serum levels of estradiol (E2) and progesterone in the moxibustion group were significantly increased after treatment, especially for premenopausal volunteers (both P<0.01). ③ After moxibustion, the serum level of AMH was significantly improved (P<0.01), however, the effect on INHB had no statistical difference (P>0.05). CONCLUSIONS: Moxibustion could effectively improve the quality of life in sub-health perimenopausal women, regulate the levels of sex hormones and AMH, improve ovarian reserve function, and delay ovarian aging.

An Antagomir to MicroRNA-106b-5p Ameliorates Cerebral Ischemia and Reperfusion Injury in Rats Via Inhibiting Apoptosis and Oxidative Stress.

We previously observed that microRNA miR-106b-5p significantly increased in serum of patients with acute ischemic stroke. The present study was to determine whether miR-106b-5p antagomir can protect against cerebral ischemia/reperfusion (I/R) injury and elucidate its underlying mechanisms. Middle cerebral artery occlusion (MCAO) was operated on male Sprague Dawley rats. MiR-106b-5p antagomir significantly decreased neurological deficit scores, infarct volumes, and neuronal injury. Furthermore, miR-106b-5p antagomir markedly reduced malondialdehyde (MDA) content, restored superoxide dismutase (SOD) activity, increased the expression of myeloid cell leukemia-1 (Mcl-1) and B cell lymphoma-2 (Bcl-2), and decreased the expression of Bax in the ischemic cortex. In PC12 cells, miR-106b-5p inhibitor increased the Mcl-1 and Bcl-2 expression, which provided protection against glutamate-induced apoptosis and oxidative damage, as evidenced by decreased lactate dehydrogenase (LDH) release, and enhanced SOD activity. Notably, luciferase reported assay proved Mcl-1 was the target gene of miR-106b-5p. In conclusion, our data indicates that the neuroprotective effects of miR-106b-5p antagomir on cerebral I/R injury are associated with its inhibition of apoptosis and oxidative stress, suggesting a potential therapeutic target for ischemic stroke.

Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway.

Electroacupuncture (EA) has anti-oxidative and anti-inflammatory actions, but whether the neuroprotective effect of EA against cerebral ischemia-reperfusion (I/R) injury involves modulation of the extracellular regulated kinase 1/2 (ERK1/2) signaling pathway is unclear. Middle cerebral artery occlusion (MCAO) was performed in Sprague-Dawley rats for 2 hours followed by reperfusion for 24 hours. A 30-minute period of EA stimulation was applied to both Baihui (DU20) and Dazhui (DU14) acupoints in each rat (10 mm EA penetration depth, continuous wave with a frequency of 3 Hz, and a current intensity of 1-3 mA) when reperfusion was initiated. EA significantly reduced infarct volume, alleviated neuronal injury, and improved neurological function in rats with MCAO. Furthermore, high mRNA expression of Bax and low mRNA expression of Bcl-2 induced by MCAO was prevented by EA. EA substantially restored total glutathione reductase (GR), glutathione (GSH) and glutathione peroxidase (GSH-Px) levels. Additionally, Nrf2 and glutamylcysteine synthetase (GCS) expression levels were markedly increased by EA. Interestingly, the neuroprotective effects of EA were attenuated when ERK1/2 activity was blocked by PD98059 (a specific MEK inhibitor). Collectively, our findings indicate that activation of the ERK1/2 signaling pathway contributes to the neuroprotective effects of EA. Our study provides a better understanding of the regulatory mechanisms underlying the therapeutic effectiveness of EA.

[Effect of electroacupuncture on expression of gamma-glutamylcysteine synthetase protein and mRNA in cerebral cortex in rats with focal cerebral ischemia-reperfusion].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Baihui" (GV 20) and "Dazhui" (GV 14) on the expression of gamma-glutamylcysteine synthetase (gamma-GCS) protein and gene in the cerebral cortex in rats with cerebral ischemia-reperfusion (CI/R) injury, so as to explore its molecular biological mechanism underlying anti-oxidative stress. METHODS: A total of 30 male Sprague-Dawley rats were randomized into sham operation (sham, n = 10), model (n = 10), and EA (n = 10) groups. CI/R model was established by right middle cerebral artery occlusion (modified Longa's thread occlusion method) for 2 hours and reperfusion for 24 hours. EA (3 Hz, 1-3 mA) was applied to "Dazhui" (GV 14) and "Baihui" (GV 20) for 30 min. gamma-GCS protein expression of the parietotemporal region of cerebral cortex was detected by immunohistochemistry and gamma-GCS heavy subunit (gamma-GCSh) mRNA and gamma-GCS light subunit (gamma-GCSI) mRNA expression levels were assayed by real-time quantitative polymerase chain reaction (RT-PCR). RESULTS: Compared with the sham group, the expression levels of gamma-GCS protein in the pyramidal cell layer of the cerebral cortical parietotemporal region, and y-GCSh mRNA and gamma-GCSI mRNA, and the number of gamma-GCS immuno-reaction positive cells had no remarkable changes in the model group (P > 0.05), while in comparison with the model group, the expression levels of cerebral cortical gamma-GCS protein, and gamma-GCSh mRNA and gamma-GCSI mRNA, and the number of gamma-GCS immuno-reaction positive cells were increased considerably in the EA group (P < 0.01, P < 0.05). CONCLUSION: EA of GV 20 and GV 14 can upregulate expression levels of gamma-GCS protein, gamma-GCSh mRNA and gamma-GCSI mRNA of the cerebral cortical parietotemporal region in CI/R rats, which may contribute to its effect in protecting cerebral cortical cells from injury by clearing away excessive oxygen free radicals.

[Involvement of melatonin in the adjusting effect of electroacupuncture in resisting oxygen stress in cerebral ischemia-reperfusion injury rats].

OBJECTIVE: To explore the effect of electroacupuncture (EA) in resisting acute cerebral ischemia-reperfusion injury (CI-RI) via anti-oxidation of melatonin (MT). METHODS: A total of 52 male SD rats were randomized into Sham-operation (sham, n=8), CI-RI (model, n=8), EA (n=8), MT (3.2 mg/Kg, i.p., n=8), EA+MT (n=8), EA+Luzindole (L, a blocker of MT, 1 mg/100 microl/rat, n=6), EA+PD 98059 (a blocker of mitogen-activated protein kinase, MAPK, 1 mg 100 microl/rat, n=6) groups. EA (3 Hz,1-3 mA, continuous waves) was applied to "Dazhui" (GV 14) and "Baihui" (GV 20) for 30 min. CI-RI model was induced by right middle cerebral artery occlusion (MCAO) for 60 min and reperfusion for 24 h except sham-operation group. MT content of pineal gland tissue was determined by high performance liquid chromatography (HPLC) and neuroethological scores of the rats were evaluated by Kuluz's and Julio's methods. Bax and Bcl-2 gene protein expression of the striate body was detected by immunohistochemistry. RESULTS: In comparison with sham group, the number of Bax immunoreactive (IR) positive cells and Bax/Bcl-2 in striate body increased significantly (P<0.001), and neuroethological score and Bcl-2 IR-positive cells decreased considerably in model group (P<0.01). Compared with model group, pineal MT content, neuroethological score and Bcl-2 IR-positive cells in EA group, neuroethological scores in EA+L and EA+P groups, and Bcl-2 expression in MT and EA+MT groups increased considerably (P<0.01, 0.001); while Bax IR-positive cells and Bax/Bcl-2 in EA, MT and EA+MT groups decreased very significantly (P<0.001). It showed that EA could obviously increase pineal MT content, improve CI-RI rats' activity score, upregulate Bcl-2 expression and down-regulate Bax expression in striate body. In comparison with simple EA group, neuroethological scores of EA+L and EA+P groups, Bax IR-positive cells and Bax/Bcl-2 of EA+MT group were significant lower (P<0.05, 0.01). No significant differences were found between EA+L and EA+P groups in behavior scores, and between MT and EA+MT groups in Bax and Bcl-2 IR-positive cell number and Bax/Bcl-2 (P>0.05). CONCLUSION: EA has a good effect of anti-oxygen stress, protecting the brain from ischemic damage directly or indirectly, which may be related to its effect in upregulating the synthesis and release of MT.