To construct a recombinant fowl adenovirus 4(FAdV-4)expressing the Cap protein of goose astrovirus genotype 2(GoAstV-2),the expression cassette of Cap gene was inserted into the natural 1 966 bp deletion region of the FAdV-4 genome in the infectious clone p15A-cm-FAdV4-HNJZ.The resulted recombinant plasmid p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2 was linearized with restriction enzyme and transfected into chicken hepatoma cell line(LMH)to rescue the recombinant FAdV-4 expressing the Cap protein of GoAstV-2,rF Ad V4-Cap/GoAstV-2.After 15 passages in LMH cells,the recombinant rFAdV4-Cap/GoAstV-2 was identified by PCR using primers flanking the insertion site of the Cap gene expression cassette and using viral genome DNA extracted from rFAdV4-Cap/GoAstV-2 infected LMH cells as template.LMH cells were in-fected with 15th passage rFAdV4-Cap/GoAstV-2 and indirect immunofluorescence was performed with a polyclonal antibody against Cap protein as the primary antibody.Western blot was carried out with lysates of rFAdV4-Cap/GoAstV-2 infected LMH cells.The in vitro replication dynamic of the 15th passage of the rFAdV4-Cap/GoAstV-2 was also investigated in LMH cells.The results demonstrated that the Cap gene of GoAstV-2 was presented in the genome of the recombinant vi-rus rF AdV4-Cap/Go Ast V-2,and could be expressed stably.The prepared recombinant virus in this study will lay a foundation for developing inactivated bivalent vaccine candidate against co-in-fection of FAdV-4 and GoAstV-2 in goose.

Objective To investigate the application effect of biofeedback electrical stimulation combined with Kegel exercises using vaginal dumbbell in pelvic floor muscle strength rehabilitation of patients with postpartum pelvic floor dysfunction(PFD).Methods A prospective study was conducted on 70 PFD patients who were divided into control group(n=34)and combined group(n=36)according to random numbers generated by Excel.Control group received Kegel exercises using vaginal dumbbell,while combined group was given biofeedback electrical stimulation on this basis.Both groups underwent 3 months of continuous treatment.GRRUG method was adopted to assess pelvic floor muscle strength after treatment;a low-frequency neuromuscular stimulation therapy device was used to detect the pelvic floor muscle fiber electromyography value and fatigue value;and female sexual function index(FSFI)was utilized to evaluate the quality of sexual life.The pelvic floor function impairments in two groups were also recorded and compared.Results After treatment,compared with control group,combined group had greater proportions of above grade Ⅲ muscle strength of class Ⅰ and class Ⅱ muscle fibers(P<0.05),higher mean pelvic floor electromyography values of class Ⅰ and class Ⅱ muscle fibers(P<0.05),and lower fatigue value of pelvic floor muscle(P<0.05).In addition,combined group had higher FSFI scores for sexual desire,sexual arousal,vaginal lubrication,orgasm,satisfaction and dyspareunia,and the total score than control group(P<0.05),and the incidence rates of frequent and urgent urination,lumbosacral pain,and vaginal relaxation were lower in combined group as compared with control group(P<0.05).Conclusion The combination of biofeedback electrical stimulation and Kegel exercises using vaginal dumbbell in the treatment of patients with postpartum PFD can significantly improve pelvic floor muscle strength,alleviate symptoms such as frequent and urgent urination,lumbosacral pain and vaginal relaxation,and improve the quality of sexual life.

Objective To explore the anti-inflammatory properties of the ethyl acetate extract(ETCB)derived from Trollius chinensis Bge.using in vitro RAW264.7 cells stimulated with lipopolysaccharide and an in vivo mouse auricle model induced by xylene.Utilizing UHPLC-Q-TOF-MS(LC-MS)and network pharmacology,the components of ETCB were analyzed,and its anti-inflammatory mechanisms were preliminarily explored.Methods The anti-inflammatory activity of various solvent extracts of Trollius chinensis Bge.was assessed through the Griess assay.The impact of ETCB on the production of TNF-α and IL-6 in RAW264.7 cells induced by lipopolysaccharide was evaluated using ELISA.Real-time qPCR was conducted to determine the effect of ETCB on the expression levels of inflammatory factors such as TNF-α,IL-6,and iNOS in cells.The anti-inflammatory efficacy was further validated in a xylene-induced ear inflammation mouse model by measuring ear swelling and tissue levels of IL-6 and TNF-α.The composition of ETCB was analyzed using LC-MS.Network pharmacology was employed to screen for effective components,targets,and pathways involved in the anti-inflammatory effects of Trollius chinensis Bge.,followed by molecular docking verification between core components and targets.Results ETCB demonstrated the most potent inhibitory effect on NO production in RAW264.7 cells stimulated by lipopolysaccharide,indicating its primary role in the anti-inflammatory activity of Trollius chinensis Bge..ETCB significantly reduced TNF-α and IL-6 levels in inflammatory cells(P<0.01)and inhibited the mRNA expression of TNF-α,IL-6,and iNOS.In the xylene-induced mouse ear inflammation model,ETCB effectively alleviated ear swelling and decreased tissue levels of TNF-α and IL-6.LC-MS analysis identified 30 chemical components in ETCB,including 21 flavonoids,7 organic acids,1 polysaccharide,and 1 anthocyanin.Network pharmacology prediction and screening revealed TNF,Akt1,PTGS2,EGFR,SRC,and MMP9 as core targets,with hydroxyquercetin,lignin from fragrant leaves,zeaxanthin from willows,plantain,thistle,and sophora flavins as key anti-inflammatory active ingredients.The molecular docking analysis revealed positive interactions,characterized by favorable binding energy,between the active components and key targets.Conclusion ETCB demonstrates strong anti-inflammatory properties both inside and outside the body,functioning through various targets and pathways.This establishes a basis for deeper understanding of the anti-inflammatory mechanism of Trollius chinensis Bge.

Objective:To explore the possible mechanism of cognitive impairment in aged mice induced by sevoflurane and the role of the AMPK/GSK-3β/Nrf2 signaling pathway.Methods:Eighteen 16-month-old SPF male C57BL/6J mice were randomly assigned to either a control group or a sevoflurane group according to the random number table method( n=9 per group). Mice in the sevoflurane group were exposed to 3% sevoflurane for 2 h every day for three consecutive days.Cognitive function of mice was assessed by novel object recognition test and Morris water maze test.Golgi staining was used to analyze dendritic spine morphology and density in the hippocampus. Mitochondrial ultrastructure was examined by transmission electron microscopy. Oxidative stress in hippocampal tissue was evaluated by reactive oxygen species(ROS) and glutathione(GSH) assay kits. Western blot was performed to measure synaptic plasticity-related proteins and proteins involved in the AMPK/GSK-3β/Nrf2 signaling pathway, while Nrf2 expression was assessed by immunofluorescence. Statistical analysis was performed using GraphPad Prism 7 software with independent-samples t-test or Mann-Whitney U test for between-group comparisons. Results:(1) The novel object recognition test showed that the recognition index in the sevoflurane group was significantly lower than that in the control group ( Z=-3.256, P=0.001).The escape latency in the Morris water maze test was longer ((49.50±10.14) s, (18.62±6.59) s; t=-7.221, P<0.001) and the number of platform crossings was lower ( Z=-2.673, P=0.008) in the sevoflurane group compared with those of the control group. (2) Results from Western blot and Golgi staining showed that the expression levels of hippocampal synapse-related proteins in the sevoflurane group, including PSD95 ((0.38±0.07), (1.00±0.21); t=4.885) and SYN1 ((0.30±0.10), (1.00±0.10); t=8.575), were lower than those in the control group, as well as the number of dendritic spines ((10.3±2.5), (20.0±1.0); t=6.183)(all P<0.05). (3) Transmission electron microscopy showed mitochondrial damage in the hippocampus of the sevoflurane group, and the expression level of ROS ((3.05±0.90), (0.97±0.16); t=-4.555, P=0.004) was significantly higher than that of the control group, while the expression level of GSH ((0.71±0.07), (1.00±0.09); t=5.396, P=0.002) was significantly lower than that of the control group.(4) Western blot and immunofluorescence demonstrated that hippocampal p-AMPK, p-GSK-3β (Ser9), and HO-1 expression levels in the sevoflurane group were significantly lower than those in the control group ( t=2.845, 7.087, 4.551, all P<0.05), and Nrf2 fluorescence intensity was also markedly reduced ( P<0.05). Conclusion:The cognitive impairment induced by sevoflurane in aged mice may be caused by mitochondrial damage, oxidative stress response and synaptic damage, which maybe associated with the inhibition of the AMPK/GSK-3β/Nrf2 signaling pathway.

Objective:To explore the possible mechanism of cognitive impairment in aged mice induced by sevoflurane and the role of the AMPK/GSK-3β/Nrf2 signaling pathway.Methods:Eighteen 16-month-old SPF male C57BL/6J mice were randomly assigned to either a control group or a sevoflurane group according to the random number table method( n=9 per group). Mice in the sevoflurane group were exposed to 3% sevoflurane for 2 h every day for three consecutive days.Cognitive function of mice was assessed by novel object recognition test and Morris water maze test.Golgi staining was used to analyze dendritic spine morphology and density in the hippocampus. Mitochondrial ultrastructure was examined by transmission electron microscopy. Oxidative stress in hippocampal tissue was evaluated by reactive oxygen species(ROS) and glutathione(GSH) assay kits. Western blot was performed to measure synaptic plasticity-related proteins and proteins involved in the AMPK/GSK-3β/Nrf2 signaling pathway, while Nrf2 expression was assessed by immunofluorescence. Statistical analysis was performed using GraphPad Prism 7 software with independent-samples t-test or Mann-Whitney U test for between-group comparisons. Results:(1) The novel object recognition test showed that the recognition index in the sevoflurane group was significantly lower than that in the control group ( Z=-3.256, P=0.001).The escape latency in the Morris water maze test was longer ((49.50±10.14) s, (18.62±6.59) s; t=-7.221, P<0.001) and the number of platform crossings was lower ( Z=-2.673, P=0.008) in the sevoflurane group compared with those of the control group. (2) Results from Western blot and Golgi staining showed that the expression levels of hippocampal synapse-related proteins in the sevoflurane group, including PSD95 ((0.38±0.07), (1.00±0.21); t=4.885) and SYN1 ((0.30±0.10), (1.00±0.10); t=8.575), were lower than those in the control group, as well as the number of dendritic spines ((10.3±2.5), (20.0±1.0); t=6.183)(all P<0.05). (3) Transmission electron microscopy showed mitochondrial damage in the hippocampus of the sevoflurane group, and the expression level of ROS ((3.05±0.90), (0.97±0.16); t=-4.555, P=0.004) was significantly higher than that of the control group, while the expression level of GSH ((0.71±0.07), (1.00±0.09); t=5.396, P=0.002) was significantly lower than that of the control group.(4) Western blot and immunofluorescence demonstrated that hippocampal p-AMPK, p-GSK-3β (Ser9), and HO-1 expression levels in the sevoflurane group were significantly lower than those in the control group ( t=2.845, 7.087, 4.551, all P<0.05), and Nrf2 fluorescence intensity was also markedly reduced ( P<0.05). Conclusion:The cognitive impairment induced by sevoflurane in aged mice may be caused by mitochondrial damage, oxidative stress response and synaptic damage, which maybe associated with the inhibition of the AMPK/GSK-3β/Nrf2 signaling pathway.

OBJECTIVE To quantify pulmonary vascular endothelial subpopulations during bleomycin-induced pulmonary fibrosis in mice.METHODS Sixty male C57BL/6 mice were randomly divided into five groups(n=12 per group)corresponding to distinct observation timepoints:0,1,2,3,and 4 weeks.A model was established via intratracheal instillation of bleomycin(3 mg·kg-1).Lung tissues were harvested at 0,1,2,3 and 4 weeks post-bleomycin induction.Pathological staining was performed to assess lung histoarchitecture and collagen fiber deposition.Single-cell suspensions were analyzed by flow cytometry to quantify temporal changes in pulmonary vascular endothelial subpopulations,including pulmonary macrovascular endothelial cells,general capillaries,and aerocyte capillaries.Immunofluo-rescence staining was performed to validate the expressions of endothelial markers(CD31,APLN,APLNR,CD93).Single-cell transcriptomic data from the Tabula Muris database was analyzed to evalu-ate gene expression profiles of vascular endothelial subpopulations.RESULTS Pathological staining revealed progressive destruction of lung histoarchitecture and collagen deposition during bleomycin-induced pulmonary fibrosis.Flow cytometry demonstrated three-phase dynamics in vascular endothelial cells(CD45-CD31+CD90.2-):a significant decrease during the acute inflammatory phase,stabilization in the fibrotic phase,and partial recovery during the resolution phase.The proportion of von Willebrand factor-positive(VWF+)vascular endothelial cells significantly decreased during the resolution phase,whereas VWF-vascular endothelial cells increased.Single-cell transcriptomics identified Cd93 asa specific gene for general capillary endothelial cells,with a negative correlation with"aerocyte"genes enriched in gas-exchange alveolar capillary endothelial cells.Immunofluorescence confirmed CD93 localization to general capillary endothelial cells.A flow sorting strategy based on CD45-CD31+CD90.2-VWF-CD93-effectively enriched alveolar capillary endothelial cells.This subpopulation trended upward in pulmonary vascular endothelial composition during bleomycin induction.CONCLUSION During bleo-mycin-induced pulmonary fibrosis in mice,pulmonary vascular endothelial subpopulations exhibit dynamic compositional heterogeneity across fibrotic injury and repair phases.

Objective·To comprehensively evaluate the efficiency of different kits and methods for DNA and RNA extraction from blood samples.Methods·A total of 145 blood samples were collected,including those from patients with Alzheimer's disease(20 cases),fibrosis(5 cases),colorectal cancer(108 cases),and healthy individuals(12 cases).A column-based kit(Kit A)and a nucleic acid extraction instrument were used to extract genomic DNA(gDNA)from leukocytes in the blood.Cell-free DNA(cfDNA)and cell-free RNA(cfRNA)in plasma were extracted using five different kits(Kit B?F),which employed either column-based(Kit B,E)or magnetic bead-based methods(Kit C,D,F).The extraction process of Kit B was optimized by increasing the plasma sample volume and extending the elution incubation time.Furthermore,this protocol was applied to extracting cfDNA from plasma samples of 100 colorectal cancer patients.Quantitative real-time PCR(qPCR)was used to quantify the extracted DNA and RNA,and the molecular yields were compared to evaluate the extraction efficiency.A comprehensive assessment was conducted,considering factors such as cost and operation time.Results·In gDNA extraction,although the the operation time was shortened by using the nucleic acid extraction instrument,the median number of DNA molecules extracted using Kit A(column-based method)was 25.36-fold higher than that obtained with the instrument(P<0.05).For cfDNA extraction,while the overall efficiency of the three kits(Kit B?D)was similar,Kit B(column-based method)showed superior performance in low-concentration samples,with average DNA yields 4.24-fold and 1.18-fold higher than those of Kit D and Kit C(both magnetic bead-based).Optimization of Kit B's extraction protocol further improved cfDNA yield.When comparing three samples,the cfDNA yields from larger plasma input volumes was 3.98-fold,2.38-fold,and 3.82-fold higher than those from smaller input volumes,respectively.The results of cfDNA extraction from 100 colorectal cancer patients indicated that this extraction protocol reliably extracted sufficient amounts of cfDNA from clinical samples.For cfRNA extraction,Kit E(column-based method)was widely recommended due to its high efficiency,convenience,and cost-effectiveness.The median RNA content extracted using Kit E was 5.01-fold higher than that of Kit F(magnetic bead-based method).Lastly,a comparison of the copy numbers of cfDNA and cfRNA in plasma revealed that the average copy number of cfRNA per milliliter of plasma was 27.65-fold higher than that of cfDNA.Conclusion·Kit A,Kit B,and Kit E show outstanding performance in leukocyte gDNA extraction,plasma cfDNA extraction,and plasma cfRNA extraction,respectively.However,although Kit E has advantages in extraction efficiency and cost,its safety requires further evaluation.

Objective To investigate the relationship between systemic lupus erythematosus(SLE)and hypothyroidism using bidirectional two-sample Mendelian randomization(MR)method.Methods The Genome-Wide Association Study data of SLE and hypothyroidism were obtained online.Independent single nucleotide polymorphisms closely related to SLE were screened as instrumental variable(Ⅳ),and outlier values were tested and eliminated by MR-PRESSO tool of R 4.3.1 software.The inverse variance weighted(IVW),MR-Egger,weighted mode(WM),weighted median(WME)and simple mode(SM)were used for MR analysis,and the values of odds ratio(OR)and 95％confidence interval(95％CI)were used to evaluate whether there was an association between SLE and hypothyroidism.The Cochran's Q heterogeneity test was performed for the results of IVW and MR-Egger,the pleiotropy test was performed by Egger-intercept method,and the sensitivity analysis was performed by elimination test one by one.F value was calculated to evaluate whether there was a weak Ⅳ bias.Results MR analysis results showed that there was a positive causal relationship between SLE and hypothyroidism in the overall population,and the results calculated by IVW,MR-Egger,WM and WME were statistically significant,with OR(95％CI)being 1.004(1.002-1.005),1.004(1.001-1.008),1.004(1.002-1.007),and 1.004(1.002-1.006),respectively.The heterogeneity test results for IVW and MR-Egger were P=0.086 and P=0.098,respectively,indicating no heterogeneity;the Egger-intercept result was P=0.295,indicating no pleiotropy;sensitivity analysis showed MR results were stable;and all F values were greater than 10,indicating no weak Ⅳ bias.Conclusion Compared with healthy people,the risk of hypothyroidism in patients with SLE is significantly higher.

OBJECTIVE To quantify pulmonary vascular endothelial subpopulations during bleomycin-induced pulmonary fibrosis in mice.METHODS Sixty male C57BL/6 mice were randomly divided into five groups(n=12 per group)corresponding to distinct observation timepoints:0,1,2,3,and 4 weeks.A model was established via intratracheal instillation of bleomycin(3 mg·kg-1).Lung tissues were harvested at 0,1,2,3 and 4 weeks post-bleomycin induction.Pathological staining was performed to assess lung histoarchitecture and collagen fiber deposition.Single-cell suspensions were analyzed by flow cytometry to quantify temporal changes in pulmonary vascular endothelial subpopulations,including pulmonary macrovascular endothelial cells,general capillaries,and aerocyte capillaries.Immunofluo-rescence staining was performed to validate the expressions of endothelial markers(CD31,APLN,APLNR,CD93).Single-cell transcriptomic data from the Tabula Muris database was analyzed to evalu-ate gene expression profiles of vascular endothelial subpopulations.RESULTS Pathological staining revealed progressive destruction of lung histoarchitecture and collagen deposition during bleomycin-induced pulmonary fibrosis.Flow cytometry demonstrated three-phase dynamics in vascular endothelial cells(CD45-CD31+CD90.2-):a significant decrease during the acute inflammatory phase,stabilization in the fibrotic phase,and partial recovery during the resolution phase.The proportion of von Willebrand factor-positive(VWF+)vascular endothelial cells significantly decreased during the resolution phase,whereas VWF-vascular endothelial cells increased.Single-cell transcriptomics identified Cd93 asa specific gene for general capillary endothelial cells,with a negative correlation with"aerocyte"genes enriched in gas-exchange alveolar capillary endothelial cells.Immunofluorescence confirmed CD93 localization to general capillary endothelial cells.A flow sorting strategy based on CD45-CD31+CD90.2-VWF-CD93-effectively enriched alveolar capillary endothelial cells.This subpopulation trended upward in pulmonary vascular endothelial composition during bleomycin induction.CONCLUSION During bleo-mycin-induced pulmonary fibrosis in mice,pulmonary vascular endothelial subpopulations exhibit dynamic compositional heterogeneity across fibrotic injury and repair phases.

Objective To explore the anti-inflammatory properties of the ethyl acetate extract(ETCB)derived from Trollius chinensis Bge.using in vitro RAW264.7 cells stimulated with lipopolysaccharide and an in vivo mouse auricle model induced by xylene.Utilizing UHPLC-Q-TOF-MS(LC-MS)and network pharmacology,the components of ETCB were analyzed,and its anti-inflammatory mechanisms were preliminarily explored.Methods The anti-inflammatory activity of various solvent extracts of Trollius chinensis Bge.was assessed through the Griess assay.The impact of ETCB on the production of TNF-α and IL-6 in RAW264.7 cells induced by lipopolysaccharide was evaluated using ELISA.Real-time qPCR was conducted to determine the effect of ETCB on the expression levels of inflammatory factors such as TNF-α,IL-6,and iNOS in cells.The anti-inflammatory efficacy was further validated in a xylene-induced ear inflammation mouse model by measuring ear swelling and tissue levels of IL-6 and TNF-α.The composition of ETCB was analyzed using LC-MS.Network pharmacology was employed to screen for effective components,targets,and pathways involved in the anti-inflammatory effects of Trollius chinensis Bge.,followed by molecular docking verification between core components and targets.Results ETCB demonstrated the most potent inhibitory effect on NO production in RAW264.7 cells stimulated by lipopolysaccharide,indicating its primary role in the anti-inflammatory activity of Trollius chinensis Bge..ETCB significantly reduced TNF-α and IL-6 levels in inflammatory cells(P<0.01)and inhibited the mRNA expression of TNF-α,IL-6,and iNOS.In the xylene-induced mouse ear inflammation model,ETCB effectively alleviated ear swelling and decreased tissue levels of TNF-α and IL-6.LC-MS analysis identified 30 chemical components in ETCB,including 21 flavonoids,7 organic acids,1 polysaccharide,and 1 anthocyanin.Network pharmacology prediction and screening revealed TNF,Akt1,PTGS2,EGFR,SRC,and MMP9 as core targets,with hydroxyquercetin,lignin from fragrant leaves,zeaxanthin from willows,plantain,thistle,and sophora flavins as key anti-inflammatory active ingredients.The molecular docking analysis revealed positive interactions,characterized by favorable binding energy,between the active components and key targets.Conclusion ETCB demonstrates strong anti-inflammatory properties both inside and outside the body,functioning through various targets and pathways.This establishes a basis for deeper understanding of the anti-inflammatory mechanism of Trollius chinensis Bge.