Objective:To investigate the effect of mild moxibustion on transient receptor potential vanilloid type 1(TRPV1)channel expression in primary dysmenorrhea(PD)rats and explore its mechanism in alleviating central pain sensitization.Methods:Thirty-two female non-pregnant Wistar rats were randomized into a blank group,a model group,a mild moxibustion group,and a capsazepine group,with 8 rats in each group.Except for the blank group,the other three groups used estradiol benzoate,ice-water bath,and oxytocin to establish the rat PD model of cold-dampness stagnation pattern.The interventions began on day 1 of modeling,once a day,and lasted 10 d.The mild moxibustion group received mild moxibustion at Shenque(CV8)and Guanyuan(CV4),20 min/time;in the capsazepine group,capsazepine was injected at a dose of 2 mg/(kg·bw).The abdominal pain threshold was measured 10-30 min after oxytocin injection on day 11;enzyme-linked immunosorbent assay was used to detect serum prostaglandin F2α(PGF2α)level;the expression of TRPV1,cluster of differentiation 11B(CD11B),and proto-oncogene c-Fos in the spinal dorsal horn and hypothalamus was detected by immunofluorescence and Western blotting.Results:Compared to the blank group,the model group showed a decreased pain threshold(P<0.05)and an increased serum PGF2α level with elevated TRPV1,CD11B,and c-Fos protein expression in the spinal dorsal horn and hypothalamus(P<0.05).Compared to the model group,both the mild moxibustion group and capsazepine group showed significantly increased pain thresholds(P<0.05),along with decreased serum PGF2α levels and reduced protein expression levels of TRPV1,CD11B,and c-Fos in the spinal dorsal horn and hypothalamus(P<0.05).Rat pain threshold in the capsazepine group was higher than that in the mild moxibustion group(P<0.05).Serum PGF2α level,the expression levels of CD11B and c-Fos proteins in the spinal dorsal horn,as well as TRPV1,CD11B,and c-Fos proteins in the hypothalamus of the capsazepine group were lower than those in the mild moxibustion group(P<0.05).Conclusion:Mild moxibustion at Shenque(CV8)and Guanyuan(CV4)may alleviate the central pain sensitization in PD rats by down-regulating TRPV1 channel expression in the spinal dorsal horn and hypothalamus,thus playing an analgesic effect.

Objective To investigate the effects of indirect moxibustion on the expressions of DNA methyltransferases(DNMT)and methylation of the brain-derived neurotrophic factor(BDNF)promoter region in uterine tissues of rats with primary dysmenorrhea(PD);To explore the mechanism of epigenetic regulation of indirect moxibustion on PD model rats.Methods A total of 32 female SD rats were randomly divided into blank group,model group,indirect moxibustion group and Western medicine group,with 8 rats in each group.The PD model with cold dampness stagnation syndrome was established using ice-water baths combined with estradiol benzoate and oxytocin.Starting from the first day of modeling,the indirect moxibustion group received salt-partitioned moxibustion at"Shenque"and ginger-partitioned moxibustion at"Guanyuan"for 20 min,while the Western medicine group was gavaged ibuprofen solution.Both interventions were given once a day for 10 days.On day 11,writhing responses were observed and scored after oxytocin injection,Western blot and RT-qPCR were used to detect protein and mRNA expression of BDNF,DNMT3A and DNMT3B in uterine tissue,immunohistochemical staining was used to detect the positive expressions of DNMT3A and DNMT3B in uterine tissue.The DNA methylation of BDNF promoter region in uterine tissue was detected by sulfite sequencing.Results Compared with the blank group,the writhing latency was shortened and the writhing score increased in the model group(P<0.01);the protein and mRNA expressions of BDNF,DNMT3A and DNMT3B in uterine tissue increased(P<0.01),the positive expressions of DNMT3A and DNMT3B increased(P<0.01),and the DNA methylation rate in BDNF promoter region decreased(P<0.01).Compared with the model group,the writhing latency was lengthened and the writhing score decreased in the indirect moxibustion group and Western medicine group(P<0.05,P<0.01);the protein and mRNA expressions of BDNF,DNMT3A and DNMT3B in uterine tissue decreased(P<0.05,P<0.01),the positive expressions of DNMT3A and DNMT3B decreased(P<0.01),and the DNA methylation rate in BDNF promoter region increased(P<0.01).Conclusion Indirect moxibustion at"Shenque"and"Guanyuan"may inhibit the transcription of BDNF by increasing the DNA methylation level of BDNF promoter region,and reduce the expression of BDNF,so as to relieve the pain of PD rats.

BACKGROUND:With the increasing attention of scholars at home and abroad to children's cervical spine-related diseases,the demand for exploring the anatomical indicators and changes of cervical spine morphology and development in children of different ages is increasing.OBJECTIVE:To explore and analyze the morphological changes of children with different ages and vertebral sequences by measuring the anatomical position indexes of C2-C7 neurocentral synchondrosis in children aged 1-6 years.METHODS:Normal cervical spine CT images were retrospectively collected from 160 children aged 1-6 years at provincial tertiary hospitals.They were divided into six groups according to an age group of 1 year.The raw data of consecutively scanned cervical spine tomography images were imported into Mimics 16.0 software.The positional anatomical indexes of cervical spine segments C2-C7 in coronal and transverse planes were measured and analyzed under the two-dimensional image window by choosing the measurement tools under the toolbar of Measurements.RESULTS AND CONCLUSION:(1)The distance between the two sides of C2-C7 neurocentral synchondrosis and the distance between the left and right sides of neurocentral synchondrosis and the transverse process gradually increased with age.The overall development of vertebrae in each cervical vertebral segment was faster than the ossification of the neurocentral synchondrosis.(2)The cross-sectional angles on both sides of C2-C7 neurocentral synchondrosis gradually increased with age,and the angles between the left and right sides of neurocentral synchondrosis and the anterior and posterior edges of the vertebral body gradually decreased.Both sides of the neurocentral synchondrosis in cervical vertebral segments tended to grow toward the arch site,which mainly promoted the growth and development of the arch.(3)Except for C7,the angle between the coronal planes on both sides of the cervical spine changed little with the descending neurocentral synchondrosis of the cervical spine,and the neurocentral synchondrosis of the cervical spine was more inclined to longitudinal growth and ossification.(4)The neurocentral synchondrosis position changes in C7 were significantly different from those in the rest of the cervical vertebrae.(5)The anatomical indexes of C2-C7 neurocentral synchondrosis position in children have obvious development rules among different ages and vertebral bodies,and these rules are helpful for the clinical diagnosis and treatment of cervical spine diseases in children.

This study presents prenatal diagnosis and genetic analysis of a pedigree with X-linked intellectual disability. A gravida at approximately 20 gestational weeks underwent prenatal diagnosis following non-invasive prenatal testing suggested sex chromosome abnormalities. Copy number variation (CNV) sequencing identified a 5.7 Mb duplication at Xp22.2p22.11 in the fetus, which initially classified as a variant of uncertain clinical significance. This duplication was inherited from the phenotypically normal mother, while paternal CNV results were normal. Genetic testing of four intellectually disabled family members revealed the identical 5.7 Mb duplication. Through expanded pedigree analysis, the pathogenicity classification of the Xp22.2p22.11 microduplication was upgraded to likely pathogenic. After comprehensive genetic counseling, the family elected pregnancy termination with informed consent.

BACKGROUND:Currently,there have been studies on three-dimensional digitalization and visualization systems for adult acupoints,but there are not many reports on the visualization of pediatric acupoints based on real pediatric digital sectional anatomical datasets. OBJECTIVE:To design and develop a digital three-dimensional visualization system for children's neck acupoints,to provide a basis for acupuncture and moxibustion,meridian and acupoint science teaching,clinical practice,acupuncture manipulation practice,and acupuncture safety research,and to provide a basis for the development of children's acupoint simulation system. METHODS:Based on a real cross-sectional anatomical dataset of pre-school boys,a three-dimensional digital virtual anatomical model of the neck region of children and internal multi-organ three-dimensional reconstruction were completed using PhotoShop 2021 and Digihuman Reconstruction System software.A database of 11 acupoints was compiled,including Fengfu and Fengchi,using the Unity database language.A three-dimensional model of children's neck anatomy,acupoint database,and writing acupuncture operation codes were integrated in Unity3D software.A three-dimensional digital visualization system for children's neck acupoints was successfully created,which integrated simulation acupoint positioning,three-dimensional acupoint anatomy,acupuncture training,clinical teaching,and acupuncture safety research. RESULTS AND CONCLUSION:(1)This study was based on real child specimens.Manual layer by layer segmentation of cross-sectional images was used to ensure the accuracy of the three-dimensional model to the greatest extent possible.The 3D software Digihuman Reconstruction System was utilized to extract and save independent segmentation data.PhotoShop 2021 software was collaborated with to complete dozens of three-dimensional reconstruction anatomical models of the outer skin of the neck and its internal bone structure,cervical spinal cord,blood vessels and nerves,muscles,and ligaments in children.The basic morphology and overall contour integrity verification of each independent structure were completed in MeshLab software.The 3-material research 13.0 software was applied for final fine tuning and anatomical position confirmation,successfully simulating and restoring the true anatomical morphology of the neck of preschool children.(2)Based on and referring to the national standards of the People's Republic of China,a database of commonly used acupoints in children's neck region was collected and organized,including their names,meridians,positioning,local anatomy,needle insertion levels,acupuncture methods,acupuncture accidents and prevention,acupoint indications,and two-dimensional anatomical sectional images.(3)Unity3D software was employed to integrate the three-dimensional model of children's neck,acupuncture simulation operation,and acupoint database,and a three-dimensional digital children's neck acupoint acupuncture visualization system was successfully constructed.The system displayed information on children's neck acupoints,two-dimensional and three-dimensional anatomical structures,and achieved two-dimensional and three-dimensional acupuncture simulation functions and acupuncture safety research functions for children's neck acupoints.Based on the ultra-thin sectional anatomical dataset of real child specimens,the first three-dimensional digital and visualization system for acupoints in the neck region of children had been constructed.Compared with previous acupoint acupuncture systems,it is more in line with the anatomical and morphological development characteristics of Asian children and has high application value in the fields of acupuncture safety research,clinical teaching,and acupuncture simulation training.

Objective: To construct a Family with sequence similarity 50 member A(FAM50A) gene knockout mouse insulinoma pancreatic β-cell line Beta-TC-6 using CRISPR/Cas9 gene editing technology and to prepare polyclonal antibodies specifically recognizing FAM50A. Methods: Two guide RNAs(sgRNAs) targeting the FAM50A gene were designed,and a recombinant plasmid expressing blue fluorescent protein(BFP) was constructed for gene knockout.The successfully constructed plasmid was transfected into Beta-TC-6 cells,and BFP-positive single cells were isolated for clonal expansion.The expanded monoclonal cell lines were genotyped by Sanger sequencing,and FAM50A protein expression was assessed by Western blot.Purified human recombinant FAM50A protein was used to immunize New Zealand rabbits for the preparation of a polyclonal antibody.The specificity of the prepared antibody was then validated using the successfully established FAM50A knockout cell line. Results: A monoclonal cell line with a successful knockout of the FAM50A gene was identified.Sanger sequencing confirmed base deletions at the target site.Western blot analysis showed a complete absence of FAM50A protein expression in this cell line.The prepared polyclonal antibody successfully recognized endogenous murine FAM50A protein in wild-type Beta-TC-6 cells and in hTERT-RPE1 cells overexpressing human FAM50A-GFP fusion protein,while no signal was detected in the FAM50A knockout cells. Conclusion This study successfully established a FAM50A gene knockout Beta-TC-6 cell model and generated a FAM50A polyclonal antibody,providing powerful tools for future research.