OBJECTIVE: To observe the effect of electroacupuncture at "Sishencong" (EX-HN1) and "Fengchi" (GB20) on hippocampal neuronal ferritinophagy mediated by the nuclear receptor coactivator 4 (NCOA4)/ferritin heavy chain 1 (FTH1) signaling pathway in vascular dementia (VD) rats, and to explore the potential mechanisms of electroacupuncture for VD. METHODS: A total of 60 male rats of SPF grade were randomly divided into a blank group (12 rats), a sham surgery group (12 rats) and a modeling group (36 rats). In the modeling group, the modified 4-vessel occlusion method was used to establish the VD model. The 24 successfully modeled rats were randomly divided into a model group and an electroacupuncture group, with 12 rats in each group. In the electroacupuncture group, electroacupuncture was applied at left and right "Sishencong" (EX-HN1), and bilateral "Fengchi" (GB20), with continuous wave, in frequency of 2 Hz and current intensity of 1 mA, 30 min a time, once daily for 21 consecutive days. The learning and memory abilities were assessed using the Morris water maze test before modeling, after modeling and after intervention, as well as the novel object recognition test after intervention. After intervention, the neuronal morphology in the hippocampus was observed by Nissl staining; the iron deposition was observed by Prussian blue staining; the reactive oxygen species (ROS) level was detected by dihydroethidium (DHE) fluorescence staining; the levels of iron, malondialdehyde (MDA) and superoxide dismutase (SOD) in the hippocampal tissue were measured by the colorimetric assay, TBA method, and WST-1 method, respectively; the positive expression of NCOA4, FTH1 and glutathione peroxidase 4 (GPX4) was detected by immunohistochemistry; the protein expression of NCOA4, FTH1, GPX4, and the ratio of microtubule-associated protein 1 light chain 3B (LC3B) Ⅱ/Ⅰ in the hippocampus were detected by Western blot. RESULTS: Compared with the sham surgery group, in the model group, the escape latency was prolonged, and the number of platform crossings reduced (P<0.01), the recognition index (RI) was decreased (P<0.01); the hippocampal neurons displayed a blurred laminar structure, disorganized cellular arrangement, and the number of Nissl bodies was decreased (P<0.01); the percentage of iron deposition area in the hippocampus was increased (P<0.01); in the hippocampus, the levels of ROS, iron, MDA, and the protein expression of NCOA4, as well as the LC3B Ⅱ/Ⅰ ratio were increased (P<0.01), the SOD level, and the protein expression of FTH1 and GPX4 were decreased (P<0.01). Compared with the model group, in the electroacupuncture group, the escape latency was shortened and the number of platform crossings was increased (P<0.01), the RI was increased (P<0.01); the hippocampal neurons exhibited more regular morphology, better-organized cellular structure, and the number of Nissl bodies was increased (P<0.05); the percentage of iron deposition area in the hippocampus reduced (P<0.01); in the hippocampus, the levels of ROS, iron, MDA, and the protein expression of NCOA4, as well as the LC3B Ⅱ/Ⅰ ratio were decreased (P<0.01, P<0.05), the SOD level, and the protein expression of FTH1 and GPX4 were increased (P<0.01). CONCLUSION Electroacupuncture at "Sishencong" (EX-HN1) and "Fengchi" (GB20) can improve learning and memory abilities in VD rats, and its mechanism may be associated with the regulation of the hippocampal NCOA4/FTH1 signaling pathway, inhibition of ferritinophagy, and alleviation of oxidative stress damage.
Animals ; Electroacupuncture ; Dementia, Vascular/genetics* ; Male ; Rats ; Signal Transduction ; Humans ; Memory ; Rats, Sprague-Dawley ; Nuclear Receptor Coactivators/genetics* ; Ferritins/genetics* ; Learning ; Hippocampus/metabolism* ; Acupuncture Points

Objective To explore the application effect of health education based on gain and loss message framing on the treatment behavior intention and self-management of patients with high-risk diabetic foot.Methods From July to September 2024,convenience sampling was used to select patients with high-risk diabetic foot who were hospitalized in the endocrinology department of a tertiary general hospital in Guiyang as the study subjects.They were divided into 3 groups according to the admission time,with 30 patients in each group.The experimental group adopted health education based on gain message framing or framing loss message,while in the control group,health education was provided in a conventional manner.Before and after intervention,the differences of intervention effects among the 3 groups were compared by using diabetic foot pre-hospital delay intention questionnaire,diabetic foot care knowledge questionnaire and Chinese version of Nottingham foot care assessment scale(CNAFF).Results Ultimately,29 cases in the gain framing group,29 cases in the loss framing group,and 29 cases in the control group completed the study.After intervention,the score of pre-hospital delay intention questionnaire of diabetic foot in the gain framing group was(21.48±4.32),and it was(24.31±2.49)in the loss framing group,and(17.76±5.03)in the control group.The difference among the 3 groups was statistically significant(F=18.725,P＜0.001);the loss framing group was superior to the gain framing group(P=0.01)and the control group(P＜0.001).After the intervention,the score of the CNAFF in the gain framing group was(55.83±3.06),and it was(59.14±2.90)in the loss framing group,and(48.66±2.58)in the control group.The difference between the 3 groups was statistically significant(F=102.245,P＜0.001).The loss framing group was superior to the gain framing group and the control group(all P＜0.001).Conclusion Health education based on the loss message framing is more conducive to improving patients' intention to delay diabetic foot visits,leading to good foot care behaviors,and may provide an effective means of pre-hospital prevention and control of diabetic foot.

Corynebacterium pseudotuberculosis(C.pseudotuberculosis)is a group of intracellular Gram-positive bacteria that can cause zoonotic diseases.This study investigated the mechanisms of inflammatory mediator secretion and the phagocytic and bactericidal functions of mouse peritoneal macrophages following C.pseudotuberculosis infection.Initially,transcriptomic sequencing was em-ployed to identify genes critical for C.pseudotuberculosis infection in macrophages.Subsequently,gene knockout mice were utilized to assess the impact of these key genes on inflammatory media-tor secretion,activation of inflammatory signaling pathways,and the phagocytic and bactericidal functions of macrophages infected with C.pseudotuberculosis.Techniques such as ELISA,Western blot,and immunofluorescence were employed in this analysis.Further,transcriptomic sequencing was conducted to identify key downstream genes.Following C.pseudotuberculosis infection,GO enrichment analysis was performed,and TLR2 was identified as the focal point of the study.Perito-neal macrophages from C57BL/6J and TLR2 knockout(TLR2-/-)mice were infected with C.pseudotuberculosis.ELISA results revealed that the levels of TNF-α,IL-1β,and IL-10 were signifi-cantly downregulated in TLR2-/-macrophages compared to C57BL/6J macrophages post-infec-tion.Western blot demonstrated that the absence of TLR2 led to a marked decrease in M APK(p38 and ERK)signaling pathway phosphorylation following C.pseudotuberculosis infection.Immuno-fluorescence results indicated that the phagocytic rate of TLR2-/-macrophages was significantly higher than that of C57BL/6J macrophages after infection.Subsequently,transcriptomic analysis of C57BL/6J and TLR2-/-macrophages infected with C.pseudotuberculosis was performed,followed by GO enrichment analysis of differential genes.IL-36a,Cx3cr1,TLR1,and TLR2 were identified as key differential genes.TLR2 plays a crucial role in the inflammatory response induced by C.pseudotuberculosis infection in mice,influencing the progression of the inflammatory response and host outcomes through the secretion of inflammatory mediators,activation of signaling pathways,and modulation of phagocytic and bactericidal functions.IL-36a and Cx3cr1 were identified as key downstream factors in this process.

The rationale for the design of control groups in tuina clinical trial is the foundation for rigorously validating the effectiveness and safety of this therapy. This article reviewed the current state of the design of tuina placebo in control groups of clinical trials， pointed out the necessity of setting up tuina placebo in clinical trials of tuina， analyzed the challenges in implementing blinding of tuina manipulation， and concluded that tuina placebo is still challenged by the placebo effect， the diversification of tuina manipulation but the lack of standardization， and the difficulty of implementing blinding due to the high level of public awareness of tuina. This article also summarized the design of placebo manipulation in three types of clinical trials， including spinal manipulation， acupressure， and paediatric tuina， and proposed four strategies for designing placebo tuina manipulation-controlling placebo effects， developing operational standards for placebo tuina manipulation， ensuring the rigor of blinding implementation， and applying new technologies to enhance the standardization and blinding capacity of placebo tuina methods. So the article is aimed at improving the methodological quality of tuina clinical trial designs， and promoting the standardization and scientificity of tuina clinical trial design.

Objective We aimed to investigate the effects of different non-surgical embryo transfer devices,number of transferred embryos,embryo stage,and embryos obtained from different mouse strains on the efficiency of non-surgical embryo transfer in mice,and to compare the efficiencies of surgical and non-surgical embryo transfer,in order to establish a stable non-surgical embryo transfer technology system.Methods Mouse embryo transfer was carried out using non-surgical method.Results The pregnancy rates using two different non-surgical transfer devices were(75.00±0.00)％and(66.67±14.43)％,and the birth rates were(46.11±6.31)％and(18.89±0.96)％,respectively.Transfer of 10,15,and 20 embryos resulted in pregnancy rates of(66.67±11.55)％,(80.00±0.00)％,and(66.67±23.09)％,and birth rates of(29.33±4.16)％,(38.67±4.81)％,and(17.00±3.46)％,respectively.When blastocysts and morulae were transferred non-surgically,the resulting pregnancy rates were(80.00±0.00)％and(46.67±11.55)％and the birth rates were(38.67±4.81)％and(10.22±2.77)％,respectively.Four strains(C57BL/6J,ICR,genetically modified mice A,genetically modified mice B)were used as donors for non-surgical embryo transfer,with resulting pregnancy rates of(66.67±11.55)％,(80.00±0.00)％,(73.33±11.55)％,and(80.00±0.00)％,and birthrates of(26.67±2.67)％,(38.67±4.81)％,(32.00±3.53)％,and(29.34±2.31)％,respectively.Fifteen pseudo-pregnant mice were transplanted surgically and 15 were transplanted non-surgically,with pregnancy rates of(80.00±0.00)％and(86.67±11.55)％,and birth rates of(38.67±4.81)％and(36.00±5.82)％,respectively.Conclusions Transfer device A resulted in a higher birth rate in this study.The embryo transfer efficiency was higher when 15 embryos were transferred into unilateral uterine horns of pseudo-pregnant 2.5-day recipients.Blastocyst-stage embryo transfer was more efficient than morula-stage transfer.There was no significant difference in efficiency between surgical and non-surgical embryo transfer procedures.

As an important and fundamental resource of scientific research,laboratory animals have become essential tools for the continuous advancements in life sciences,medical research,drug development,and other fields.With the development of related laws and regulations,the welfare of laboratory animals is increasingly valued by the general public and international research communities alike.To ensure the welfare of laboratory animals,the ethical review and conduct of laboratory animal practitioners should be standardized,incorporating and adapting advanced international practices with those in China.This article primarily outlines the process for reviewing and approving animal use protocols,along with the standards for evaluation,with the aim of providing a reference for researchers writing animal use protocols and for International Animal Care and Use Committee members conducting ethical reviews of laboratory animal welfare.

Background and Aims:Laparoscopic hiatal hernia repair(LHHR)is the gold-standard surgical treatment for hiatal hernia(HH),but postoperative recurrence remains a challenge due to hiatal enlargement and disruption of the phrenoesophageal ligament.This study aimed to assess the feasibility,safety,and short-term efficacy of a novel three-dimensional small intestinal submucosa(3D-SIS)patch designed for circumferential crural reinforcement and ligament-like reconstruction in a porcine LHHR model.Methods:Twelve healthy pigs(35-40 kg)were equally randomized into non-mesh group,SIS flat patch group,and 3D-SIS patch group.All animals underwent LHHR and Nissen fundoplication under general anesthesia.In the non-mesh group,the hiatal defect was closed with interrupted sutures.In the SIS flat patch group,a U-shaped SIS patch was placed posterior to the esophagus to reinforce the crura and fixed with medical glue.In the 3D-SIS patch group,an intraoperatively assembled three-dimensional patch was applied,consisting of an upper keyhole-shaped layer for circumferential diaphragmatic reinforcement,a tubular middle part encircling the abdominal esophagus,and a lower small keyhole-shaped patch covering the gastroesophageal junction,all fixed with medical glue.After 3 months,laparotomy was performed to assess recurrence,patch integration,and complications,followed by biomechanical and histological evaluations.Results:All procedures were completed successfully with no deaths or major complications.Operative time was slightly longer in the patch groups,while blood loss was similar.No hernia recurrence or patch migration was observed at 3 months.Biomechanical testing revealed higher ultimate load and Young's modulus in both SIS groups than in the non-mesh group.Histological analysis demonstrated neovascularization and collagen deposition in the patch groups,with the 3D-SIS patch showing more complete circumferential integration and ligament-like tissue formation.Conclusion:The 3D-SIS patch is feasible and safe in porcine LHHR.It provides circumferential diaphragmatic reinforcement and promotes phrenoesophageal ligament-like regeneration,offering a new concept for reducing postoperative recurrence and reconstructing the anti-reflux barrier.

AIM:To establish a cell model of visceral hypersensitivity and nerve hyperplasia in irritable bowel syndrome(IBS)by conducting an in vitro co-culture of mouse P815 mast cells and N2a nerve cells and explore its possible mechanism.METHODS:Enzyme-linked immunosorbent assay(ELISA)with three replicates was used to confirm the C48/80-induced P815 degranulation.The length of neurites was observed under bright field microscopy to determine the number of differentiated neurons,thereby selecting the concentration of retinoic acid(RA)for stimulating the differentia-tion of N2a cells,with four replicates.A co-culture system of P815 and N2a cells was established using Transwell cham-bers with four replicates.The following groups were established:N2a cells cultured alone,N2a cells co-cultured with P815 cells,N2a cells co-cultured with P815 cells plus C48/80,and N2a cells plus RA group.After co-culturing,the num-ber of differentiated N2a cells was observed under bright field.The expression of nerve growth factor(NGF),tyrosine ki-nase receptor A(TRKA),growth-associated protein-43(GAP43),neuron-specific enolase(NSE),synapsin(SYN),and postsynaptic density protein-95(PSD-95)at both protein and gene levels in N2a cells was detected using Western blot and polymerase chain reaction(PCR),with four replicates.RESULTS:The best condition for N2a differentiation was stimulation with 10 μmol/L RA for 24 hours,whereas the best condition for degranulation was stimulation of P815 cells with 20 mg/L C48/80 for 24 hours.Compared with N2a cells cultured alone,the differentiation ratio of the N2a+P815+C48/80 and N2a+RA groups was significantly increased(P<0.01),and the protein and mRNA expressions of NGF,TRKA,GAP43,NSE,SYN,and PSD-95 were significantly increased(P<0.05).CONCLUSION:Our results revealed that mast cell degranulation enhances the level of nerve hyperplasia in enteric nerve cells and promotes changes in nerve structure and function.Synaptic remodeling regulated by abnormal expression of key proteins such as NGF,TRKA,and GAP43 is involved in the nerve hyperplasia induced by mast cell degranulation.

Objective To establish a rapid and accurate droplet digital PCR(ddPCR)detection method for detecting Staphylococcus aureus(SA)in laboratory animals and the environment.Methods Using the heat-stable nuclease gene(nuc)of SA as the target gene,a pair of specific primers and probes are designed within its conserved region.Optimize the reaction conditions,test the dynamic range,and evaluate the specificity and stability of the method.Using the same template,test reactions were performed with both ddPCR and real-time quantitative PCR(qPCR)method to assess the interchangeability between the two approaches.Finally,the method is applied to the detection of various clinical samples.Results The kinetic range of the established SA ddPCR method is 100～15 000 copies/μL,with a detection limit of 2.5 copies and a quantification limit of 10 copies;The specificity of this method was tested,and only SA showed positive droplets,while no positive droplets were found for other pathogens;After measuring three parallel samples,the standard deviation and relative standard deviation were calculated.It was found that within the dynamic detection interval of ddPCR,as the target copy number gradually decreased,the relative standard deviation showed an upward trend,but remained below 25％.This result indicates that the detection method has good stability.Conclusions The established ddPCR method for detecting SA has the advantages of high sensitivity,strong specificity,good stability,and good reproducibility.This method can be applied for the detection of SA in laboratory animals.

Corynebacterium pseudotuberculosis(C.pseudotuberculosis)is a group of intracellular Gram-positive bacteria that can cause zoonotic diseases.This study investigated the mechanisms of inflammatory mediator secretion and the phagocytic and bactericidal functions of mouse peritoneal macrophages following C.pseudotuberculosis infection.Initially,transcriptomic sequencing was em-ployed to identify genes critical for C.pseudotuberculosis infection in macrophages.Subsequently,gene knockout mice were utilized to assess the impact of these key genes on inflammatory media-tor secretion,activation of inflammatory signaling pathways,and the phagocytic and bactericidal functions of macrophages infected with C.pseudotuberculosis.Techniques such as ELISA,Western blot,and immunofluorescence were employed in this analysis.Further,transcriptomic sequencing was conducted to identify key downstream genes.Following C.pseudotuberculosis infection,GO enrichment analysis was performed,and TLR2 was identified as the focal point of the study.Perito-neal macrophages from C57BL/6J and TLR2 knockout(TLR2-/-)mice were infected with C.pseudotuberculosis.ELISA results revealed that the levels of TNF-α,IL-1β,and IL-10 were signifi-cantly downregulated in TLR2-/-macrophages compared to C57BL/6J macrophages post-infec-tion.Western blot demonstrated that the absence of TLR2 led to a marked decrease in M APK(p38 and ERK)signaling pathway phosphorylation following C.pseudotuberculosis infection.Immuno-fluorescence results indicated that the phagocytic rate of TLR2-/-macrophages was significantly higher than that of C57BL/6J macrophages after infection.Subsequently,transcriptomic analysis of C57BL/6J and TLR2-/-macrophages infected with C.pseudotuberculosis was performed,followed by GO enrichment analysis of differential genes.IL-36a,Cx3cr1,TLR1,and TLR2 were identified as key differential genes.TLR2 plays a crucial role in the inflammatory response induced by C.pseudotuberculosis infection in mice,influencing the progression of the inflammatory response and host outcomes through the secretion of inflammatory mediators,activation of signaling pathways,and modulation of phagocytic and bactericidal functions.IL-36a and Cx3cr1 were identified as key downstream factors in this process.