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.

Moxibustion prevents tripterygium glycoside-induced oligoasthenoteratozoospermia in rats via reduced oxidative stress and modulation of the Nrf2/HO-1 signaling pathway.

Oligoasthenoteratozoospermia (OAT) decreases male fertility, seriously affecting the production of offspring. This study clarified the preventive impact of different moxibustion frequencies on OAT and selected the optimal frequency to elucidate the underlying mechanism. An OAT rat model was constructed by gavage of tripterygium glycosides (TGS) suspension. Daily moxibustion (DM) or alternate-day moxibustion (ADM) was administered on the day of TGS suspension administration. Finally, we selected DM for further study based on sperm quality and DNA fragmentation index, testicular and epididymal morphology, and reproductive hormone level results. Subsequently, the oxidative stress (OS) status was evaluated by observing the OS indices levels; malondialdehyde (MDA), 8-hydroxy-deoxyguanosine (8-OHdG), total antioxidant capacity (T-AOC), and total superoxide dismutase (T-SOD) in testicular tissue using colorimetry and enzyme-linked immunosorbent assay. Furthermore, heme oxygenase 1 (HO-1) and nuclear factor erythropoietin-2-related factor 2 (Nrf2) were evaluated using Western blotting. Immunohistochemistry was employed to locate and assess the expression of HO-1 and Nrf2 protein, while quantitative real-time polymerase chain reaction was utilized to detect their mRNA expression. MDA and 8-OHdG levels decreased following DM treatment, while T-SOD and T-AOC increased, suggesting that DM may prevent TGS-induced OAT in rats by decreasing OS in the testis. Furthermore, protein and mRNA expression of Nrf2 and HO-1 in the testis were elevated, indicating that DM may reduce OS by activating the signaling pathway of Nrf2/HO-1. Therefore, DM could prevent OAT in rats via the Nrf2/HO-1 pathway, thereby presenting a promising therapeutic approach against OAT.

Magnoflorine Ameliorates Collagen-Induced Arthritis by Suppressing the Inflammation Response via the NF-κB/MAPK Signaling Pathways.

Objective: Magnoflorine (Mag) has been reported to have anxiolytics, anti-cancer, and anti-inflammatory properties. In this study, we aim to investigate the effects of Mag on the rheumatoid arthritis (RA) and explore the underlying mechanism using a collagen-induced arthritis (CIA) mouse model and a lipopolysaccharide (LPS)-stimulated macrophage inflammation model. Methods: The in vivo effects of Mag on CIA were studied by inducing CIA in a mouse model using DBA/1J mice followed by treatment with vehicle, methotrexate (MTX, 1 mg/kg/d), and Mag (5 mg/kg/d, 10 mg/kg/d, and 20 mg/kg/d), and the in vitro effects of Mag on macrophages were examined by stimulation of RAW264.7 cells line and peritoneal macrophages (PMs) by LPS in the presence of different concentrations of Mag. Network pharmacology and molecular docking was then performed to predict the the binding ability between Mag and its targets. Inflammatory mediators were assayed by quantitative real-time PCR and enzyme linked immunosorbent assay (ELISA). Signaling pathway changes were subsequently determined by Western blotting and immunohistochemistry (IHC). Results: In vivo experiments demonstrated that Mag decreased arthritis severity scores, joints destruction, and macrophages infiltration into the synovial tissues of the CIA mice. Network pharmacology analysis revealed that Mag interacted with TNF-α, IL-6, IL-1ß, and MCP-1. Consistent with this, analysis of the serum, synovial tissue of the CIA mice, and the supernatant of the cultured RAW264.7 cells and PMs showed that Mag suppressed the expression of TNF-α, IL-6, IL-1ß, MCP-1, iNOS, and IFN-ß. Furthermore, Mag attenuated the phosphorylation of p65, IκBα, ERK, JNK, and p38 MAPKs in the synovial tissues of the CIA mice and LPS-stimulated RAW 264.7 cells. Conclusion: Mag may exert anti-arthritic and anti-inflammatory effects by inhibiting the activation of NF-κB and MAPK signaling pathways.

Huntingtin Decreases Susceptibility to a Spontaneous Seizure Disorder in FVN/B Mice.

Huntington disease (HD) is an adult-onset neurodegenerative disorder that is caused by a trinucleotide CAG repeat expansion in the HTT gene that codes for the protein huntingtin (HTT in humans or Htt in mice). HTT is a multi-functional, ubiquitously expressed protein that is essential for embryonic survival, normal neurodevelopment, and adult brain function. The ability of wild-type HTT to protect neurons against various forms of death raises the possibility that loss of normal HTT function may worsen disease progression in HD. Huntingtin-lowering therapeutics are being evaluated in clinical trials for HD, but concerns have been raised that decreasing wild-type HTT levels may have adverse effects. Here we show that Htt levels modulate the occurrence of an idiopathic seizure disorder that spontaneously occurs in approximately 28% of FVB/N mice, which we have called FVB/N Seizure Disorder with SUDEP (FSDS). These abnormal FVB/N mice demonstrate the cardinal features of mouse models of epilepsy including spontaneous seizures, astrocytosis, neuronal hypertrophy, upregulation of brain-derived neurotrophic factor (BDNF), and sudden seizure-related death. Interestingly, mice heterozygous for the targeted inactivation of Htt (Htt+/- mice) exhibit an increased frequency of this disorder (71% FSDS phenotype), while over-expression of either full length wild-type HTT in YAC18 mice or full length mutant HTT in YAC128 mice completely prevents it (0% FSDS phenotype). Examination of the mechanism underlying huntingtin's ability to modulate the frequency of this seizure disorder indicated that over-expression of full length HTT can promote neuronal survival following seizures. Overall, our results demonstrate a protective role for huntingtin in this form of epilepsy and provide a plausible explanation for the observation of seizures in the juvenile form of HD, Lopes-Maciel-Rodan syndrome, and Wolf-Hirschhorn syndrome. Adverse effects caused by decreasing huntingtin levels have ramifications for huntingtin-lowering therapies that are being developed to treat HD.

[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.

[Protective effect of moxibustion preconditioning in rats with premature ovarian insufficiency].

OBJECTIVE: To observe the effects of moxibustion preconditioning on ovarian function, fertility and ovarian granulosa cell apoptosis in rats with premature ovarian insufficiency (POI), so as to investigate its underlying mechanism in improving POI. METHODS: Forty-two female SD rats with two complete estrous cycles were randomly divided into control group, model group and pre-moxibustion group, with 14 rats in each group. The pre-moxibustion group was pretreated with mild moxibustion for 14 days before POI model establishment at 1) "Guanyuan" (CV4) and "Zhongwan" (CV12) and 2) bilateral "Shenshu" (BL23) as two sets of acupoints on alternate days, once each day, for 10 min each acupoint. After 14-day mild moxibustion intervention, 75 mg·kg-1·d-1 tripterygium glycoside tablet suspension was administered to rats in the pre-moxibustion group and the model group by gavage, for 14 consecutive days, while equivalent saline was given to rats in the control group in the same way. After modeling, the effect of moxibustion preconditioning on ovarian reserve function was evaluated by the estrous cycles, pregnancy rate and embryo number, morphological changes of ovaries, and serum sex hormone levels. TUNEL staining was used to detect the rate of granulosa cell apoptosis in ovaries. Immunohistochemistry and real time quantitative PCR were used to detect the relative expression of Caspase-3 and Caspase-9 proteins and mRNA levels in ovaries. RESULTS: Compared with the control group, the estrous cycles were disturbed; the pregnancy rate and number of embryos, the wet weight of ovary and ovarian index, the number of total follicles and different level of follicles, serum Estradiol (E2) and anti-mullerian hormone (AMH) levels were all significantly decreased (P<0.01,P<0.05), while the number of atretic follicles, serum follicule-stimulating hormone (FSH) and luteinizing hormone (LH) levels, the number of TUNEL-positive granulosa cells, the expression of ovarian Caspase-3 and Caspase-9 proteins and mRNAs were significantly increased (P<0.01) in the model group. Compared with the model group, the disordered estrous cycles were improved; the pregnancy rate, the embryo numbers, the wet weight of ovary, and the total follicle number and primary follicle number, serum AMH level were significantly increased (P<0.01，P<0.05), while the number of atretic follicles, serum FSH level, the number of TUNEL-positive granulosa cells, expression of ovarian Caspase-3 and Caspase-9 proteins and mRNAs were all significantly decreased (P<0.01,P<0.05) in the moxibustion group. CONCLUSION: Moxibustion preconditioning could improve ovarian function and improve fertility of POI rats, which may be associated with reducing the apoptosis of ovarian granulosa cells.

Moxibustion mitigates mitochondrial dysfunction and NLRP3 inflammatory activation in cyclophosphamide-induced premature ovarian insufficiency rats.

AIMS: 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. MATERIALS AND METHODS: 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. KEY FINDINGS: 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-1ß, 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. SIGNIFICANCE: 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.

Huntingtin Overexpression Does Not Alter Overall Survival in Murine Cancer Models.

A reduced incidence of various forms of cancer has been reported in Huntington's disease patients and may be due to pro-apoptotic effects of mutant huntingtin. We tested this hypothesis by assessing the effects of huntingtin protein overexpression on survival in two murine cancer models. We generated YAC HD mice containing human huntingtin transgenes with various CAG tract lengths (YAC18, YAC72, YAC128) on either an Msh2 or p53 null background which have increased cancer incidence. In both mouse models of cancer, the overexpression of either mutant or wild-type huntingtin had no significant effect on overall survival. These results do not support the hypothesis that mutant huntingtin expression is protective against cancer.

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.

[Effect of electroacupuncture on endometrial receptivity and IVF-ET pregnancy outcomes in patients with diminished ovarian reserve].

OBJECTIVE: To explore the effect of electroacupuncture on endometrial receptivity and the pregnancy outcomes of in vitro fertilization and embryo transfer (IVF-ET) in the patients with diminished ovarian reserve (DOR). METHODS: Sixty-eight patients of DOR undertaken IVF-ET were randomized into an observation group (34 cases, 2 cases dropped off) and a control group (34 cases, 1 case dropped off). In the control group, endometrial preparation was performed according to the routine protocol. In the observation group, on the base of the treatment as the control group, acupuncture was applied to Geshu (BL 17), Shenshu (BL 23), Mingmen (GV 4), Shiqizhui (EX-B 8), Ciliao (BL 32), Zhongliao (BL 33), Tianshu (ST 25), Qihai (CV 6) and Guanyuan (CV 4), etc. Electric stimulation was given at Ciliao (BL 32)-Zhongliao(BL 33), Tianshu (ST 25)-Zigong (EX-CA 1), with disperse-dense wave, 2 Hz/15 Hz in frequency and tolerable current in intensity. Electroacupuncture was given once every two days, 3 times weekly, lasing 3 menstrual cycles till 1 day before embryo transfer. The endometrial thickness and morphology were observed on the day of human chorionic gonadotropin (HCG) of egg retrieval cycle, the day of endometrial transformation in frozen-thawed embryo transfer (FET) cycle and the day of embryo transfer in both groups successively; as well as HCG positive rate, clinical pregnancy rate, embryo implantation rate and live birth rate. RESULTS: In the observation group, the proportion of type A endometrium on the embryo transfer day was higher than those on HCG day of the egg retrieval cycle and the endometrial transformation day of FET cycle (P<0.05), and also higher than that of the control group (P<0.01). In the observation group, HCG positive rate, clinical pregnancy rate, embryo implantation rate and live birth rate were 75.0% (24/32), 71.9% (23/32), 47.4% (27/57) and 56.3% (18/32) respectively, and all higher than 36.4% (12/33), 30.3% (10/33), 18.0% (11/61) and 15.2% (5/33) in the control group separately (P<0.01). CONCLUSION: Electroacupuncture improves the endometrial receptivity and IVF-ET pregnancy outcomes in the patients of diminished ovarian reserve.

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.

[Mechanism of Huanglian Wendan Decoction in improving impaired glucose tolerance based on skeletal muscle NLRP3/caspase-1/IL-1ß, IL-18 pathway].

This study investigated the mechanism of improving impaired glucose tolerance(IGT) of rats by Huanglian Wendan Decoction from the perspective of the skeletal muscle Nod-like receptor protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1)/interleukin-1ß(IL-1ß), interleukin-18(IL-18) pathway. Healthy male SD rats were fed with the diet containing 45% fat for 20 weeks, accompanied by a high-temperature and high-humidity environment and an inactive lifestyle, for the establishment of the IGT rat model. The rats were divided into the blank control group, model control group, metformin hydrochloride group(positive drug group, 0.05 g·kg~(-1)·d~(-1)) and Huanglian Wendan Decoction group(7.8 g·kg~(-1)·d~(-1)). After continuous intragastric administration for 4 weeks, the obesity and glycemic indexes of all the rats were measured. The fasting serum insulin(FINS) level was determined by ELISA, with the insulin sensitivity index(ISI) and insulin resistance index(IRI) calculated. The mRNA and protein expression le-vels of nuclear factor kappaB(NF-κB), NLRP3, caspase-1, IL-1ß and IL-18 in skeletal muscle tissue were detected by real-time polymerase chain reaction(PCR), Western blot and immunofluorescence. Compared with the blank control group, the model control group witnessed significantly increased mRNA and protein expression of NF-κB, NLRP3, caspase-1, IL-1ß and IL-18. As revealed by the comparison with the model control group, Huanglian Wendan Decoction could effectively regulate the obesity status, reduce body weight, correct the abnormal levels of 2-hour plasma glucose(2 hPG), insulin resistance index(IRI), insulin sensitivity index(ISI), and lower the mRNA and protein expression of NF-κB, NLRP3, caspase-1, IL-1ß and IL-18 in the skeletal muscle tissue of IGT rats. Combined with previous studies, the above results showed that the occurrence and development of IGT was closely related to inflammatory response and the classic pyroptosis pathway in skeletal muscle, such as NLRP3/caspase-1/IL-1ß, IL-18. It is inferred that the mechanism of Huanglian Wendan Decoction was to alleviate insulin resistance(IR) and then reverse the course of IGT lies in the regulation of the abnormal insulin receptor signaling pathway based on the NLRP3 inflammasome 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.

Pan-Genome Analysis of Vibrio cholerae and Vibrio metschnikovii Strains Isolated From Migratory Birds at Dali Nouer Lake in Chifeng, China.

Migratory birds are recently recognized as Vibrio disease vectors, but may be widespread transporters of Vibrio strains. We isolated Vibrio cholerae (V. cholerae) and Vibrio metschnikovii (V. metschnikovii) strains from migratory bird epidemic samples from 2017 to 2018 and isolated V. metschnikovii from migratory bird feces in 2019 from bird samples taken from the Inner Mongolia autonomous region of China. To investigate the evolution of these two Vibrio species, we sequenced the genomes of 40 V. cholerae strains and 34 V. metschnikovii strains isolated from the bird samples and compared these genomes with reference strain genomes. The pan-genome of all V. cholerae and V. metschnikovii genomes was large, with strains exhibiting considerable individual differences. A total of 2,130 and 1,352 core genes were identified in the V. cholerae and V. metschnikovii genomes, respectively, while dispensable genes accounted for 16,180 and 9,178 of all genes for the two strains, respectively. All V. cholerae strains isolated from the migratory birds that encoded T6SS and hlyA were non-O1/O139 serotypes without the ability to produce CTX. These strains also lacked the ability to produce the TCP fimbriae nor the extracellular matrix protein RbmA and could not metabolize trimetlylamine oxide (TMAO). Thus, these characteristics render them unlikely to be pandemic-inducing strains. However, a V. metschnikovii isolate encoding the complete T6SS system was isolated for the first time. These data provide new molecular insights into the diversity of V. cholerae and V. metschnikovii isolates recovered from migratory birds.

Intracerebroventricular Administration of AAV9-PHP.B SYN1-EmGFP Induces Widespread Transgene Expression in the Mouse and Monkey Central Nervous System.

Viral vectors made from adeno-associated virus (AAV) have emerged as preferred tools in basic and translational neuroscience research to introduce or modify genetic material in cells of interest. The use of viral vectors is particularly attractive in nontransgenic species, such as nonhuman primates. Injection of AAV solutions into the cerebrospinal fluid is an effective method to achieve a broad distribution of a transgene in the central nervous system. In this study, we conducted injections of AAV9-PHP.B, a recently described AAV capsid mutant, in the lateral ventricle of mice and rhesus macaques. To enhance the expression of the transgene (the tag protein emerald green fluorescent protein [EmGFP]), we used a gene promoter that confers high neuron-specific expression of the transgene, the human synapsin 1 (SYN1) promoter. The efficacy of the viral vector was first tested in mice. Our results show that intracerebroventricular injections of AAV9-PHP.B SYN1-EmGFP-woodchuck hepatitis virus posttranscriptional regulatory element resulted in neuronal EmGFP expression throughout the mice and monkey brains. We have provided a thorough characterization of the brain regions expressing EmGFP in both species. EmGFP was observed in neuronal cell bodies over the whole cerebral cortex and in the cerebellum, as well as in some subcortical regions, including the striatum and hippocampus. We also observed densely labeled neuropil in areas known to receive projections from these regions. Double fluorescence studies demonstrated that EmGFP was expressed by several types of neurons throughout the mouse and monkey brain. Our results demonstrate that a single injection in the lateral ventricle is an efficient method to obtain transgene expression in many cortical and subcortical regions, obviating the need of multiple intraparenchymal injections to cover large brain areas. The use of intraventricular injections of AAV9-PHP.B SYN1-EmGFP could provide a powerful approach to transduce widespread areas of the brain and may contribute to further development of methods to genetically target-specific populations of neurons.

Human progranulin-expressing mice as a novel tool for the development of progranulin-modulating therapeutics.

The granulin protein (also known as, and hereafter referred to as, progranulin) is a secreted glycoprotein that contributes to overall brain health. Heterozygous loss-of-function mutations in the gene encoding the progranulin protein (Granulin Precursor, GRN) are a common cause of familial frontotemporal dementia (FTD). Gene therapy approaches that aim to increase progranulin expression from a single wild-type allele, an area of active investigation for the potential treatment of GRN-dependent FTD, will benefit from the availability of a mouse model that expresses a genomic copy of the human GRN gene. Here we report the development and characterization of a novel mouse model that expresses the entire human GRN gene in its native genomic context as a single copy inserted into a defined locus (Hprt) in the mouse genome. We show that human and mouse progranulin are expressed in a similar tissue-specific pattern, suggesting that the two genes are regulated by similar mechanisms. Human progranulin rescues a phenotype characteristic of progranulin-null mice, the exaggerated and early deposition of the aging pigment lipofuscin in the brain, indicating that the two proteins are functionally similar. Longitudinal behavioural and neuropathological analyses revealed no significant differences between wild-type and human progranulin-overexpressing mice up to 18 months of age, providing evidence that long-term increase of progranulin levels is well tolerated in mice. Finally, we demonstrate that human progranulin expression can be increased in the brain using an antisense oligonucleotide that inhibits a known GRN-regulating micro-RNA, demonstrating that the transgene is responsive to potential gene therapy drugs. Human progranulin-expressing mice represent a novel and valuable tool to expedite the development of progranulin-modulating therapeutics.

[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.

Repair mechanism of human umbilical cord mesenchymal stem cell-derived exosome on neuronal ischemia and hypoxia injury / 中华急诊医学杂志

Objective:To investigate the effects of human umbilical cord mesenchymal stem cell-derived exosome (hucMSC-ex) on proliferation, migration, apoptosis and autophagy in ischemia-anoxia neurons, and to provide a theoretical study for clinical research on stroke.Methods:Primary glial cells were cultured and OGD model was established. Then, these cells were incubated with huMSC-exosome. The inhibition rate of proliferation was detected by MTT assay. Apoptosis was observed by flow cytometry. The expressions of apoptosis related proteins were confirmed by RT-PCR and Western blot. The expressions of autophagy related proteins and PI3K/Akt signal were observed by Western blot. The data were analyzed using SPSS 17.0 software, multiple-group comparisons were performed using one-way ANOVA, and SNK- q test was used for pairwise comparison between groups. Results:MTT assay showed that OGD could inhibit cell proliferation of primary glial cells. After incubation with hucMSC-ex for 2 h, the inhibition rate of cell proliferation was lower than that of the control. The flow cytometry technology showed that hucMSC-ex reduced cell apoptosis. The cell migration experiments showed that OGD reduced cell migration capacity, but cell migration increased after exosomal incubation. RT-PCT and Western blot showed that OGD induced autophagy and apoptosis, hucMSC-ex activated PI3K/Akt signaling pathway, inhibited the expression of Bax and Caspase-3 (both P<0.05), and promoted the expression of Bcl-2 ( P<0.05). hucMSC-ex inhibited the expression of Beclin-1, Atg3 and LC3-Ⅱ(al l P<0.01). Conclusions:huMSC-exosome promote the proliferation and migration in ischemia-anoxia-injured neurons and inhibit the apoptosis and autophagy. The mechanism that hucMSC-ex repaired the injured nerve cells might be associated with PI3K/Akt signaling pathway.

Isolating cells from adult murine brain for validation of cell-type specific cre-mediated deletion.

BACKGROUND: TheCre/loxP system allows for the temporal and spatial investigation of the expression of a single gene in the nervous system. Current methods of validating conditional knock-out mouse models rely on heterogeneous brain tissue or primary culture. These methods may assess the extent of genetic knockdown in the brain but do not provide age-appropriate, cell-type specific information. NEW METHOD: We isolated specific cell types from adult murine brain using FACS to assess cell type-specific gene expression in conditional mouse models. RESULTS: We identified robust but incomplete genetic knockdown in microglia isolated from two separate microglia-specific knockout models. COMPARISONWITH EXISTING METHODS(S): Genetic knockdown in isolated adult microglia differed significantly from cultured primary microglia. CONCLUSIONS: Differences observed in primary cultured microglia compared to isolated adult microglia suggest that current methods used to validate microglia-specific gene deletion over-estimate deletion efficiency. Assessment of gene expression in isolated adult microglia provides a more accurate assessment of Cre-mediated gene deletion.

Mutant huntingtin expression in microglia is neither required nor sufficient to cause the Huntington's disease-like phenotype in BACHD mice.

Huntington's disease (HD) is caused by a CAG repeat expansion in the HTT gene and is characterized by early and selective striatal neurodegeneration. The huntingtin (HTT) protein is ubiquitously expressed in many tissues and the cellular pathogenesis of the disease is not fully understood. Immune cell dysfunction due to mutant HTT (mHTT) expression and aberrant immune system activation in HD patients suggests that inflammatory processes may contribute to HD pathogenesis. Here we used the BACHD mouse model of HD, which carries a conditional transgene expressing full-length human mHTT, to selectively deplete mHTT expression in myeloid lineage cells, including microglia, and evaluated the effects on HD-related behavior and neuropathology. In the converse experiment, we depleted mHTT expression in the majority of cells in the brain but specifically excluding microglia and again evaluated behavior and neuropathology. In mice with myeloid-specific mHTT-depletion, we observed no significant rescue of any behavioral or neuropathological outcome measures, while neural-specific knockout mice showed significant rescue of body weight, rotarod performance and striatal volume. We conclude that mHTT expression in microglia, though clearly affecting specific aspects of microglia function, does not alter disease pathogenesis in the BACHD mouse model. This may have implications for current or future therapeutic trials testing immune-modulating drugs in HD patients.