Objective To investigate the role and mechanism of afzelin(AFZ)in treating Crohn's disease-like colitis.Methods A mouse model of 2,4,6-trinitrobenzene sulfonic acid-induced colitis was established to assess the effect of AFZ on experimental colitis in vivo.A Caco-2 cell model of tumor necrosis factor(TNF)-α-induced inflammation was established to evaluate the effects of AFZ on the intestinal barrier function,intestinal epithelial cell apoptosis,and mitochondrial function in vitro.The animal and cell experiments were performed to validate the regulatory role of the adenosine monophosphate-activated protein kinase(AMPK)/silent information regulater 1(SIRT1)/peroxisome proliferator-activated receptor gamma coactivator(PGC)-1α pathway in the treatment of colitis with AFZ.Results AFZ reduced the disease activity index(P=0.003),weight loss(P<0.001),colon shortening(P<0.001),inflammation score(P=0.002),pro-inflammatory cytokine release(interleukin-6:P<0.001;TNF-α:P=0.010),and intestinal barrier permeability(fluorescein isothiocyanate dextran 4:P<0.001;intestinal-type fatty acid-binding protein:P=0.013).Meanwhile,AFZ increased the colonic transepithelial electric resistance(P=0.001),reduced bacterial translocation(P<0.001),and promoted the localization and up-regulated the expression of tight junction proteins [zonula occluden-1(P=0.005) and Claudin-1(P=0.024)].AFZ exerted a protective effect on the Caco-2 cells exposed to TNF-α in terms of intestinal epithelial cell permeability(P=0.017),transepithelial electric resistance(P=0.014),and tight junction protein[zonula occluden-1(P=0.014) and Claudin-1(P=0.006)] localization and expression.Furthermore,the cell and animal experiments confirmed that AFZ reduced the percentage of apoptosis(P<0.001,P=0.013)and the expression of cleaved-caspase 3(P=0.028,P=0.004)and Bax(P=0.004,P=0.020),and upregulated the Bcl2(P=0.020,P=0.006)level in intestinal epithelial cells.Additionally,AFZ increased the number of mitochondria,mitochondrial membrane potential,and copy number of mitochondrial DNA(P=0.007)in intestinal epithelial cells,while enhancing the activities of mitochondrial respiratory chain complex Ⅰ(P=0.005)and complex Ⅳ(P=0.001).The activation of the AMPK/SIRT1/PGC-1α pathway was involved in the protective effects of AFZ on mitochondrial function and apoptosis in intestinal epithelial cells.Conclusion AFZ alleviates mitochondrial dysfunction and apoptosis in intestinal epithelial cells by activating the AMPK/SIRT1/PGC-1α pathway,thereby ameliorating intestinal barrier dysfunction and experimental colitis.
Animals ; Colitis/drug therapy* ; Humans ; Caco-2 Cells ; Mice ; Trinitrobenzenesulfonic Acid ; Apoptosis/drug effects* ; Disease Models, Animal ; AMP-Activated Protein Kinases/metabolism* ; Sirtuin 1/metabolism*

Objective To investigate the effect of the small molecule inhibitor C42 of kallikrein-related peptidase 7(KLK7)on ovarian cancer with elevated expression of KLK7 and evaluate the feasibility of C42 as a new therapeutic strategy for ovarian cancer.Methods The CCK-8 assay,flow cytometry,cell scratch assay,Transwell assay,and Western blotting were employed to assess the effects of C42 on the proliferation,migration,and invasion of the ovarian cancer cell line SKOV3,which was characterized by high KLK7 expression.Additionally,a subcutaneous xenograft model of ovarian cancer was established with SKOV3 cells in nude mice to evaluate the effects of C42 on the tumor growth and metastasis.The expression levels of proteins associated with tumor metastasis and invasion in the tumor tissue were examined by immunohistochemical techniques.Results The cellular experiment showed that C42 suppressed the proliferation,migration,and invasion(all P<0.001)of SKOV3 cells,compared with the control group.The animal experiment showed that compared with the control group,the 10.2 mg/kg C42 group exhibited a decreased tumor weight(P=0.009) and attenuated liver metastases.Immunohistochemical staining revealed that the 10.2 mg/kg C42 group demonstrated down-regulated expression of the tumor proliferation marker Ki-67(P=0.002)and the tumor metastasis and invasion-associated proteins such as matrix metalloproteinase-9(P=0.027)and Vimentin(P=0.039).Conclusion The small molecule inhibitor C42 of KLK7 effectively suppresses the proliferation,migration,and invasion of ovarian cancer SKOV3 cells.
Female ; Humans ; Ovarian Neoplasms/drug therapy* ; Kallikreins/antagonists & inhibitors* ; Animals ; Mice, Nude ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Mice ; Cell Movement/drug effects* ; Xenograft Model Antitumor Assays ; Mice, Inbred BALB C

Objective To explore the regulatory effects of cytokines interleukin(IL)-1β,IL-6,tumor necrosis factor alpha(TNF-ɑ),gamma interferon(IFN-γ),granulocyte colony-stimulating factor(G-CSF),and granulocyte-macrophage colony-stimulating factor(GM-CSF)on spontaneous pyroptosis in neutrophils.Methods Neutrophils isolated from mouse bone marrow by density-gradient centrifugation were cultured in vitro for 20 h with or without 10,50 or 100 ng/mL IL-1β,IL-6,IFN-γ,G-CSF or GM-CSF,or for 12 h with or without 1,10 or 50 ng/mL TNF-α.After incubation,cells were stained with annexin Ⅴ(AV)/propidium iodide(PI),and the proportions and absolute number of neutrophils undergoing different forms of cell death were determined by fluorescence microscopy combined with manual counting.Pyroptotic neutrophils were identified by cell morphology in conjunction with AV/PI staining.Flow cytometry with counting beads was employed to measure the proportions and number of AV/PI-stained Ly6g⁺neutrophils in different forms of cell death.Western blotting was employed to assess the cleavage and activation levels of cysteinyl aspartate-specific proteinase-3(caspase-3)and gasdermin E(GSDME).Results Treatment with IL-1β or IL-6 had no significant effect on the proportion or number of neutrophils undergoing spontaneous pyroptosis.After 12 h of treatment with TNF-α at 1,10,and 50 ng/mL,the proportions of pyroptotic neutrophils were(14.79±0.45)%,(19.99±3.02)%,and(20.66±1.99)%,respectively,higher than that[(10.22±1.12)%]in the untreated control(P=0.024,P<0.001,and P<0.001,respectively).Treatment with 10,50,and 100 ng/mL IFN-γ for 20 h reduced the proportion of pyroptotic neutrophils from(17.43±1.88)%to 12.00%(all P<0.001).G-CSF at 10,50,and 100 ng/mL reduced the proportion of pyroptotic cells to around 6.00%and greatly inhibited the cleavage of both caspase-3 and GSDME.After 20 h of treatment with 10,50,and 100 ng/mL GM-CSF,the proportions of pyroptotic neutrophils decreased to(7.52±0.53)%,(5.27±2.30)%,and(0.64±1.11)%,respectively.Conclusions Neither IL-1β nor IL-6 affects GSDME-mediated spontaneous pyroptosis in neutrophils.TNF-ɑ induces spontaneous pyroptosis in neutrophils,whereas IFN-γ,G-CSF,and GM-CSF demonstrate inhibitory effects.
Pyroptosis/drug effects* ; Animals ; Neutrophils/cytology* ; Mice ; Cytokines/pharmacology* ; Interleukin-1beta/pharmacology* ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology* ; Cells, Cultured ; Granulocyte Colony-Stimulating Factor/pharmacology* ; Tumor Necrosis Factor-alpha/pharmacology* ; Interferon-gamma/pharmacology* ; Interleukin-6/pharmacology*

Objective To investigate the effects of folate deficiency on changes in histone H3 lysine 4 (H3K4) mono-methylation (me1)-marked enhancers and the molecular mechanism underpinning the folate deficiency-induced neural tube defects (NTD). Methods Mouse embryonic stem cells (mESCs) were cultured in the folate-free DMEM medium (folate-deficient group) and the DMEM medium containing 4 mg/L folate (normal control group),respectively.Chromatin immunoprecipitation sequencing (ChIP-seq) was performed for H3K4me1. The mouse model of folate-induced NTD was established,and transcriptome sequencing (RNA-seq) was performed for the brain tissue of fetal mice to reveal the differential expression profiles.The results were validated through real-time quantitative polymerase chain reaction (RT-qPCR).The activity of the differential peak regions of H3K4me1 was verified through the luciferase reporter assay. Results The folate content in the mESCs cultured in the folate-free medium reduced compared with that in the normal control group (P=0.008).The H3K4me1-maked enhancers in the mESCs cultured in the folate-free medium induced significant changes in intronic regions,and these changes were concentrated in metabolic and energy metabolism processes (q=9.56×10^-48,P=1.28×10^-47).The differentially expressed genes harboring H3K4me1-marked enhancers in mESCs were mainly enriched in the Wnt signaling pathway (q=0.004,P=0.004 7).ChIP-qPCR results confirmed that H3K4me1 binding decreased in the differential peak regions of the Ldlrap1 gene (P=0.008),Camta1 gene (P=0.002),and Apc2 gene (P=0.012).The H3K4 demethylase inhibitor T-448 effectively reversed the H3K4me1 binding in the differential peak regions of the aforementioned genes (P=0.01).The results of RNA-seq for the brain tissue of NTD fetal mice showed significant enrichment of the differentially expressed genes in the Wnt signaling pathway (P=1.52×10^-5).The enrichment of differential peak regions of H3K4me1-marked enhancers in Apc2,Ldlrap1,and Camta1 genes in the brain tissue also showed significant changes.The differential peak region in Apc2 exhibited transcription factor activity (P=0.020). Conclusion Folate deficiency may affect changes in H3K4me1-marked enhancers to participate in the regulation of neural tube closure genes,thereby inducing the occurrence of NTD.
Neural Tube Defects/genetics* ; Animals ; Mice ; Folic Acid Deficiency/complications* ; Histones/metabolism* ; Folic Acid/metabolism* ; Methylation ; Mouse Embryonic Stem Cells/metabolism* ; Wnt Signaling Pathway ; Lysine/metabolism* ; Chromatin Immunoprecipitation Sequencing

OBJECTIVE: To observe the effects of the "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture on key regulatory factors during mitochondrial apoptosis of testicular tissue in asthenozoospermia mice, and explore the potential mechanism of the protective effect of acupuncture on reproductive function. METHODS: Thirty C57BL/6 male mice were randomly divided into a blank group, a model group and an acupuncture group, 10 mice in each group. In the model and the acupuncture groups, the intraperitoneal injection of cyclophosphamide (30 mg•kg^-1•d^-1) was delivered for 7 days to prepare the asthenozoospermia model. After the success of modeling, the modeled mice in the acupuncture group were intervened with "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture, once daily and the needles were retained for 20 min. The duration of the intervention was 2 weeks. The general condition of each mouse was observed, and the body mass was recorded before modeling, after modeling and after intervention completion. After intervention, the testicular mass was recorded and the weight coefficient was calculated, and the mouse sperm quality was examined; the serum contents of testosterone (T), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were detected using ELISA, the morphology of testicular tissue was observed using HE, the mitochondrial ultra-microstructure of testicular tissue was observed under transmission electrone microscopy, the mitochondrial membrane potential level of testicular tissue was detected using JC-1 staining, the positive rate of apoptosis cell of testicular tissue was observed using TUNEL; and the mRNA and protein expression of b-cell lymphocytoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cytochrome c (Cyt C), apoptotic protease-activating factor1 (Apaf-1), Caspase-9 and Caspase-3 of testicular tissue was detected using real-time quantitative fluorescence PCR and Western blot methods separately; and the positive expression of Cleaved Caspase-3 of the testicular tissue was detected using immunohistochemistry. RESULTS: Compared with the blank group, the mice were in listless spirits, had shaggy hairs, the reduced appetite and movement, and weight loss in the model group (P<0.01); the testicular mass and the weight coefficient decreased (P<0.01); the total number of sperms, sperm motility, and sperm viability were declined (P<0.01); while the levels of serum T, FSH, and LH were dropped (P<0.01). The morphology of seminiferous tubules in testicular tissue was abnormal, the number of spermatogenic cells and the number of mitochondria decreased, the inner mitochondrial crest was fractured and lost, and vacuoles appeared. The level of mitochondrial membrane potential was reduced (P<0.01); and the positive rate of apoptosis cell in testicular tissue increased (P<0.01). The mRNA and protein expression of Bax, Cyt C, Apaf-1, Caspase-9 and Caspase-3 was elevated (P<0.01, P<0.05), the mRNA and protein expression of Bcl-2 was dropped (P<0.01), and the average absorbance value of Cleaved Caspase-3 increased (P<0.01). When compared with the model group, in the acupuncture group, the general condition of mice was improved, the testicular mass and the weight coefficient elevated (P<0.01); the total number of sperms, sperm motility, and sperm viability increased (P<0.01); while the levels of serum T, FSH, and LH rose (P<0.01). The pathological morphology of testicular tissue and the inner mitochondrial ultra-microstructure were ameliorated, the level of mitochondrial membrane potential was elevated (P<0.01); the positive rate of apoptosis cell was reduced (P<0.01). The mRNA and protein expression of Bax, Cyt C, Apaf-1, Caspase-9 and Caspase-3 was dropped (P<0.01, P<0.05), the mRNA and protein expression of Bcl-2 elevated (P<0.05), and the average absorbance value of Cleaved Caspase-3 declined (P<0.01). CONCLUSION "Zhibian" (BL54)-toward- "Shuidao" (ST28) acupuncture may ameliorate mouse reproductive function by inhibiting mitochondrial apoptosis pathway, alleviating testicular tissue damage in the asthenospermia mice induced by cyclophosphamide.
Animals ; Male ; Mice ; Apoptosis ; Acupuncture Therapy ; Mitochondria/metabolism* ; Asthenozoospermia/genetics* ; Humans ; Testis/metabolism* ; Mice, Inbred C57BL ; Spermatozoa/metabolism* ; Acupuncture Points ; Sperm Motility ; Testosterone/blood* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Caspase 3/genetics* ; Follicle Stimulating Hormone/blood* ; Reproduction ; Cytochromes c/genetics* ; bcl-2-Associated X Protein/genetics* ; Apoptotic Protease-Activating Factor 1/genetics*

OBJECTIVE: To investigate the underlying mechanism of "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture technique for improving reproductive function in mice with asthenospermia by regulating ferroptosis pathway. METHODS: Sixty male C57BL/6 mice were randomly divided into a blank group, a model group, an acupuncture group and a Fer-1 group, 15 mice in each one. Except the blank group, the intraperitoneal injection with cyclophosphamide (50·kg^-1·d^-1) was administered to establish the asthenospermia model in the mice of the rest 3 groups for 5 consecutive days. In the acupuncture group, "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture technique was operated in the mice, for 20 min each time; and in the Fer-1 group, Fer-1 solution (1 mg/kg) was injected intraperitoneally. The interventions of these two groups were delivered once daily and for 2 consecutive weeks. The testicular wet weight was measured and the testicular coefficient was calculated. Using sperm quality detection system, the sperm quality was detected. With ELISA used, the contents of testosterone (T), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the serum were detected. With HE staining, testicular and epididymal morphology was observed. Immunofluorescence was used to detect the expression of reactive oxygen species (ROS) in the testes. Biochemical assay was conducted to determine the contents of malondialdehyde (MDA), reduced glutathione (GSH), and total iron ion (TFe) in the testicular tissue. Transmission electron microscopy was used to examine mitochondrial structure of the testis, while JC-1 staining was used to assess mitochondrial membrane potential in the testicular tissue. Fluorescence quantitative PCR and Western blot analyses were employed to measure the mRNA and protein expression of solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), and acyl-CoA synthetase long-chain family member 4 (ACSL4) in the testicular tissue. RESULTS: Compared with the blank group, in the model group, the testicular wet weight and testicular coefficient decreased (P<0.01); the sperm concentration and sperm motility reduced (P<0.01), and the contents of T, FSH, and LH decreased in the serum (P<0.01); and the seminiferous tubules in the testis showed loose structure and deformed lumen, sperm cells were disorganized and the sperm numbers reduced; the tubular walls became thinner, and sperm numbers in the lumen less; the expression of ROS in testicular tissue, as well as the contents of MDA and TFe increased (P<0.01), and the content of GSH decreased (P<0.01); and the numbers of mitochondria reduced, the structure of cristae was serious damaged, and mitochondrial membrane potential level declined (P<0.01); the mRNA and protein expression of SLC7A11, GPX4, and FTH1 decreased (P<0.01), while the mRNA and protein expression of ACSL4 increased (P<0.01). In comparison with the model group, the acupuncture and Fer-1 groups showed the increase of testicular wet weight and coefficient (P<0.01), sperm concentration and motility (P<0.01), and the serum contents of T, FSH, and LH (P<0.01); and the improvements in testicular and epididymal histopathology; ROS expression and the contents of MDA and TFe decreased (P<0.01), and the content of GSH elevated (P<0.05); the mitochondrial structure and numbers were ameliorated and mitochondrial membrane potential rose (P<0.01). Besides, in comparison with the model group, the mRNA expression of SLC7A11 was higher (P<0.05, P<0.01), the mRNA and protein expression of GPX4 and FTH1 increased (P<0.01, P<0.05), and the mRNA and protein expression of ACSL4 decreased (P<0.01) in the acupuncture and the Fer-1 groups; and the protein expression of SLC7A11 was higher in the Fer-1 group (P<0.05). CONCLUSION "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture technique may improve the reproductive capacity in the mice with asthenospermia by alleviating ferroptosis-induced cellular damage and ameliorating testicular function.
Animals ; Male ; Ferroptosis ; Mice ; Acupuncture Therapy ; Mice, Inbred C57BL ; Asthenozoospermia/metabolism* ; Humans ; Acupuncture Points ; Testis/metabolism* ; Luteinizing Hormone/metabolism* ; Malondialdehyde/metabolism* ; Reproduction ; Testosterone/metabolism*

OBJECTIVE: To observe the effect of "Zhibian" (BL54)-to-"Shuidao" (ST28) needling technique on the reproductive function of the mice with poor ovarian response (POR) and explore the molecular mechanism of acupuncture on early embryos after in vitro fertilization-embryo transfer (IVF-ET) in POR mice. METHODS: Of 70 female C57BL/6 mice, 60 mice with regular estrous cycle were screened and 30 of them were randomly divided into a blank group, a model group, and an acupuncture group, 10 mice in each one. Mice in the model and acupuncture groups were administered with tripterygium glycosides suspension (50 mg·kg^-1·d^-1) via gavage for 2 weeks to prepare POR models; while in the blank group, the mice received an equal volume of 0.9% sodium chloride solution via gavage. After successful modeling, mice in the acupuncture group underwent "Zhibian" (BL54)-to-"Shuidao" (ST28) needling technique, once daily, for 20 min each time and lasting 2 consecutive weeks. After intervention completion, subsequently, all the three groups underwent ovulation induction, orbital blood collection, and ovary extraction and fresh denuded oocyte collection. Denuded oocytes, after incubated, together with the sperms from 15 male C57BL/6 mice, were transferred into the oviducts of 30 donor pseudopregnant C57BL/6 female mice via IVF-ET; and the embryonic tissue was collected on day 7 of implantation. After successful modeling and intervention completion, the general conditions of mice in each group were observed, and the estrous cycle disorder rate was compared among the groups. After intervention completion, the numbers of oocyte, ovarian wet weight and ovarian index were recorded in each group; the levels of anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) in the serum were measured using ELISA; and ovarian morphology and mitochondrial ultrastructure were examined using HE staining and transmission electron microscopy. The transcriptome sequencing technology was employed to identify differentially expressed genes (DEGs) in early embryos of each group, followed by bioinformatics analysis; and the reversed DEGs with significant difference were verified using quantitative real-time PCR (qRT-PCR). RESULTS: After intervention, compared with the blank group, the mice in the model group presented poor spirits and declined water and food intake, reduced activity, the higher rate of estrous cycle disorder (P< 0.01); the decrease of the numbers of oocyte, ovarian wet weight, and ovarian index (P<0.01), reduced AMH and E2 in the serum (P<0.01), elevated FSH and LH in the serum (P<0.01); more atresia follicles in ovarian tissue, disorganized granulosa cells, reduced the numbers of mitochondria, vacuoles appeared and cristae breakage. When compared with the model group, in the acupuncture group, the spirits of mice, food intake and activity were improved, the rate of estrous cycle disorder was reduced (P<0.01); the numbers of oocyte, ovarian wet weight, and ovarian index were higher (P<0.01, P<0.05), the levels of AMH and E2 in the serum increased (P<0.01), the levels of FSH and LH in the serum decreased (P<0.01); the atresia follicles were reduced, the numbers of follicle increased at all levels, the numbers of mitochondria got higher, vacuoles and cristae breakage were declined. In comparison between the blank group and the model group, and between the model group and the acupuncture group, a total of 258 intersecting DEGs were identified, and 194 DEGs of them exhibited a trend of reversion before and after intervention. The top 5 reversed DEGs with significant difference included dexamethasone-induced Ras-related protein 1 (Rasd1), gene regulated by estrogen in breast cancer 1 (Greb1), leucine-rich repeat-containing G protein-coupled receptor 6 (Lgr6), Fraser syndrome 1 (Fras1), and apolipoprotein D (Apod). Compared with the blank group, the mRNA expression of Rasd1, Greb1, Lgr6, Fras1 and Apod in embryonic tissues decreased in the model group (P<0.01, P<0.05); and when compared with the model group, the mRNA expression of Rasd1, Greb1, Lgr6, Fras1 and Apod in embryonic tissues increased in the acupuncture group (P<0.05, P<0.01). These findings were consistent with the DEGs screening results. Gene ontology (GO) analysis revealed that the reversed genes of co-expression were primarily participated in the biological processes such as myoblast differentiation, endocardial cushion development, and cardiac morphogenesis. Kyoto encyclopedia of genes and genomes (KEGG) analysis indicated that the reversed genes of co-expression are enriched in the Wnt signaling pathway, and they were associated with various types of cardiac diseases. CONCLUSION The "Zhibian" (BL54)-to-"Shuidao" (ST28) needling technique may ameliorate the decline of ovarian response in mice with POR, promote normal follicle development and ovulation, thereby improve embryo quality and reduce the risk of developmental defects and deformity in the organs such as the embryonic heart. The underlying mechanism may be related to the regulation of the expression pattern of embryonic key genes through the Wnt signaling pathway.
Animals ; Female ; Mice ; Mice, Inbred C57BL ; Acupuncture Therapy ; Ovary/physiopathology* ; Humans ; Fertilization in Vitro ; Transcriptome ; Embryo Transfer ; Infertility, Female/physiopathology*

OBJECTIVE: To explore the peripheral neural mechanism underlying representation along the distribution of stomach meridian induced by intestinal inflammatory reaction using diarrhea predominant-irritable bowel syndrome (IBS-D) mice. METHODS: Among 62 healthy male C57BL/6 mice of clean grade, 12 mice were randomly selected and divided into a control group and a model group, 6 mice in each group, additionally, 12 mice were randomly selected and divided into a Tianshu group, a Liangqiu group and a Zusanli group, 4 mice in each group. In the model group, citrobacter was administered orally to establish IBS-D model. In the control group and the model group, the visceral pain threshold was observed using fecal colorectal distension (fCRD) induced electromyography of external oblique muscle, the positive cell number of neutrophil in the colonic muscularis was detected by myeloperoxidase (MPO) staining, the number, location and distribution rule of Evans blue (EB) extravasation points were observed by injection of EB staining solution into the tail vein. In the Tianshu group, the Liangqiu group and the Zusanli group, fluorescent dye Dil was injected at bilateral "Tianshu" (ST25), "Liangqiu" (ST34) and "Zusanli" (ST36) respectively, to observe the dye-positive cell number in different dorsal root ganglion (DRG) segments. In the control group and the model group, the activation of satellite glial cells (SGCs) in different DRG segments was observed by immunofluorescence. RESULTS: Compared with the control group, in the model group, the area under curve of electromyography of external oblique muscle was increased at fCRD of 25, 50 and 75 μL distilled water (P<0.001, P<0.01); the MPO-positive cell number of neutrophil in the colonic muscularis was increased (P<0.01). Few EB extravasation points could be found in the control group, while there were much more EB extravasation points observed in the model group, which was specially distribution in the area of stomach meridian, from "Huaroumen" (ST24) to "Zusanli" (ST36), as well as the surface area dominated by L₂-L₅ segment of the spinal cord. The Dil-positive cells were mainly exhibited in the DRG of T₁₁, L₅ and L₄ segments in the Tianshu group, the Liangqiu group and the Zusanli group, respectively. Compared with the control group, the ratio of glial fibrillary acidic protein (GFAP)/glutamine synthetase (GS) co-expression was increased in the DRG of T₁₁, L₄ and L₅ segments in the model group (P<0.05, P<0.01). CONCLUSION The activation of SGCs within DRG of T₁₁, L₄ and L₅ segments may relate closely to the occurrence of the representation along the stomach meridian distribution in IBS-D mice.
Animals ; Male ; Mice ; Irritable Bowel Syndrome/therapy* ; Mice, Inbred C57BL ; Meridians ; Stomach/physiopathology* ; Humans ; Acupuncture Points ; Disease Models, Animal

OBJECTIVE: To explore the possible mechanism by which the "Zhibian" (BL54) toward "Shuidao" (ST28) acupuncture improves ovarian function in mice with poor ovarian response (POR) by observing its effect on gut microbiota. METHODS: A total of 35 SPF-grade C57BL/6 female mice were screened for normal estrous cycles using vaginal smears, and 30 mice were selected. Ten mice were assigned to the blank group, while the remaining mice were used to establish the POR model by intragastric administration of tripterygium wilfordii suspension. The successfully modeled mice were randomly divided into a model group and an acupuncture group, with 10 mice in each group. After modeling, the acupuncture group received the "Zhibian" (BL54) toward "Shuidao" (ST28) acupuncture method once daily for 20 minutes per session. Ovulation induction began the day after the intervention, and samples were collected after ovulation induction. Vaginal cytology was used to observe estrous cycle changes, and the number of oocytes obtained, ovarian wet weight, and ovarian index were recorded. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and anti-Müllerian hormone (AMH) were detected using ELISA. HE staining was used to observe ovarian histology. Gut microbiota was analyzed using 16S rRNA gene sequencing technology. Western blot was used to detect the relative protein expression levels of Occludin and zonula occludens-1 (ZO-1) in colonic tissue. Correlation analysis was conducted among serum hormone indexes, the number of oocytes obtained, ovarian index and gut microbiota. RESULTS: Compared with the blank group, the model group showed a higher estrous cycle disorder rate (P<0.01), increased serum FSH and LH levels, and a higher LH/FSH ratio (P<0.01), while the number of oocytes obtained, ovarian wet weight, ovarian index, and serum E2 and AMH levels were significantly reduced (P<0.01). Compared with the model group, the acupuncture group showed a lower estrous cycle disorder rate (P<0.01), decreased serum FSH and LH levels, and a lower LH/FSH ratio (P<0.01), along with an increased number of oocytes obtained, higher ovarian wet weight, ovarian index, and elevated serum AMH and E2 levels (P<0.01, P<0.05). The blank group had a large number of well-developed primordial follicles, with abundant and closely arranged follicles at various stages. In the model group, there was a significant increase in the number of atretic follicles, a reduction in the number of follicles at various stages, and loosely arranged ovarian tissue. Compared with the blank group, the model group showed a significant decrease in the number of normal follicles (P<0.01) and an increase in the number of atretic follicles (P<0.01). The acupuncture group showed a reduction in atretic follicles and an increase in the number of follicles at various stages compared with the model group, with a significant increase in normal follicles (P<0.01) and a decrease in atretic follicles (P<0.01). Compared with the blank group, the model group exhibited reduced gut microbiota diversity and richness, with significantly lower Chao1 and Shannon indices (P<0.01), and a greater clustering distance from the blank group. The model group also showed an increase in the relative abundance of Firmicutes_D, Verrucomicrobiota, Paramuribaculum, Dubosiella, and Muribaculum (P<0.01, P<0.05), while the relative abundance of Firmicutes_A and the relative protein expression of Occludin and ZO-1 in colonic tissue were decreased (P<0.01). Compared with the model group, the acupuncture group showed improved gut microbiota diversity and richness, with increased Chao1 and Shannon indices (P<0.05), and a clustering distance closer to the blank group. The acupuncture group exhibited reduced relative abundance of Firmicutes_D, Verrucomicrobiota, and Muribaculum (P<0.05, P<0.01), while the relative abundance of Firmicutes_A and the relative protein expression of Occludin and ZO-1 were significantly increased (P<0.01, P<0.05). Correlation analysis indicated a relationship between gut microbiota and serum hormone indicators, as well as the ovarian index. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the metabolic pathways of the intersecting species were related to amino acid biosynthesis and nucleotide metabolism. CONCLUSION The "Zhibian" (BL54) toward "Shuidao" (ST28) acupuncture method improves ovarian function in POR mice, and its mechanism may be related to regulating gut microbiota structure and maintaining intestinal barrier homeostasis.
Animals ; Female ; Gastrointestinal Microbiome ; Mice ; Acupuncture Therapy ; Mice, Inbred C57BL ; Humans ; Ovary/physiopathology* ; Acupuncture Points ; Follicle Stimulating Hormone/metabolism* ; Luteinizing Hormone/metabolism* ; Estrous Cycle ; Anti-Mullerian Hormone/blood*

OBJECTIVE: To explore the improvement effect of electroacupuncture (EA) based on Xingnao Kaiqiao acupuncture (acupuncture for regaining consciousness and opening orifices) on cognitive impairment in mice with Parkinson's disease (PD), and to explore its regulatory mechanisms on the kinesin family member 5A (KIF5A)/mitochondrial Rho GTPase 1 (Miro1) pathway and mitophagy in prefrontal cortical neurons. METHODS: A total of 70 male C57BL/6J mice of clean grade were randomly divided into a normal group (12 mice), a sham operation group (12 mice), and a model pre-screening group (46 mice). Unilateral stereotaxic injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle was adopted to establish the PD model in the model pre-screening group. Twenty-four mice after successful modeling were randomly selected and divided into a model group and an EA group, 12 mice in each one. In the EA group, acupuncture was applied at "Shuigou" (GV26) and bilateral "Sanyinjiao" (SP6) and "Neiguan" (PC6), ipsilateral "Sanyinjiao" (SP6) and "Neiguan" (PC6) were connected to EA respectively, with disperse-dense wave, 5 Hz/20 Hz in frequency, 0.5 mA in current intensity, 20 min a time, 6 times a week for 30 days. Cognitive function was assessed by Y-maze and Morris water maze tests; morphology of prefrontal cortex was observed by H.E. staining; reactive oxygen species (ROS) level in prefrontal cortex was detected by fluorescence probe method; mitochondrial morphology and autophagosome ultrastructure were observed by transmission electron microscopy; the mRNA expression of tyrosine hydroxylase (TH) was detected by quantitative real-time PCR; the protein expression of TH, KIF5A, Miro1, p62, Parkin and PTEN induced kinase 1 (PINK1) was detected by Western blot. RESULTS: Compared with the sham operation group, both the model group and the EA group exhibited increased rotation number of per minute (P<0.001). Compared with the sham operation group, in the model group, the novel arm exploration time of Y-maze test was shortened (P<0.001), the escape latency of Morris water maze test was prolonged (P<0.05) and the platform crossing number of Morris water maze test was reduced (P<0.01); in the prefrontal cortex, the number of cellular vacuole and neurons with karyopyknosis was increased (P<0.001), and mitochondrial autophagosomes could be observed; in the prefrontal cortex, the relative expression of ROS was increased (P<0.001), the protein and mRNA expression of TH was decreased (P<0.001), the protein expression of Miro1, PINK1, Parkin was increased (P<0.001, P<0.01), the protein expression of KIF5A and p62 was decreased (P<0.001). Compared with the model group, in the EA group, the novel arm exploration time of Y-maze test was prolonged (P<0.01), the escape latency of Morris water maze test was shortened (P<0.05) and the platform crossing number of Morris water maze test was increased (P<0.05); in the prefrontal cortex, the number of cellular vacuole and neurons with karyopyknosis was decreased (P<0.001), and the number of mitochondrial autophagosomes reduced and the mitochondrial morphology was improved; in the prefrontal cortex, the relative expression of ROS was decreased (P<0.01), the protein and mRNA expression of TH was increased (P<0.001, P<0.01), the protein expression of Miro1, PINK1, Parkin was decreased (P<0.001, P<0.01, P<0.05), the protein expression of KIF5A and p62 was increased (P<0.01, P<0.05). CONCLUSION Xingnao Kaiqiao electroacupuncture effectively alleviates cognitive impairment and damage of neuronal function in PD mice, its mechanism may be related to the regulation of KIF5A/Miro1 pathway, hence reducing the mitophagy in prefrontal cortical neurons.
Animals ; Electroacupuncture ; Male ; Mice ; Parkinson Disease/physiopathology* ; Cognitive Dysfunction/psychology* ; Kinesins/genetics* ; Humans ; Mitophagy ; Mice, Inbred C57BL ; rho GTP-Binding Proteins/genetics* ; Mitochondria/genetics* ; Prefrontal Cortex/metabolism*