Otitis media is an infection of the middle ear mainly caused by bacteria, and current treatments rely heavily on antibiotics. However, the emergence of antibiotic-resistant bacterial strains seriously affects their efficacy. In our study, we found that extracellular vesicles (EVs) derived from human natural killer cells (NKs) inhibit the proliferation of both standard and levofloxacin (LVX)-resistant strains of Staphylococcus aureus in a dose-dependent manner. Moreover, compared to LVX, EVs were more effective at reducing effusion and rescuing hearing thresholds in animal models. For LVX-sensitive strains, EVs were significantly more effective in terms of curative time but not curative rate. For LVX-resistant strains, EVs were significantly more effective in terms of both curative rate and curative time when applied alone or applied jointly with LVX. In summary, we found that NK EVs are highly effective in treating otitis media, providing an alternative approach for treating this common disease.
Killer Cells, Natural/metabolism* ; Exosomes/metabolism* ; Animals ; Humans ; Otitis Media/therapy* ; Staphylococcus aureus/drug effects* ; Disease Models, Animal ; Anti-Bacterial Agents/pharmacology* ; Levofloxacin/pharmacology*

Objective : To explore the relationship between single nucleotide polymorphisms ( SNPs) in D-loop region of mitochondrial DNA ( mtDNA) and mtDNA copy number and the risk of dermatomyositis ( DM) ，and its in- fluencing factors． Methods : 74 patients with DM and 92 healthy controls were included in the study． Genomic DNA was extracted from peripheral blood and the target fragment of mtDNA D-loop region was amplified by PCR technique，and the products were subsequently sequenced．Serum levels of ROS were assessed using a high-sensi- tivity reactive oxygen species detection kit．The expression levels of cytokines，interleukin ( IL) -5，IL-13，inter- feron-γ ( IFN-γ) ，IL-2，IL-6，IL-10，tumor necrosis factor-α ( TNF-α) and IL-4 were measured using Flow Fluo- rescence Immunmicrobeads Assay．Wilcoxon rank-sum test was used to assess the potential correlation between cy- tokines and SNPs associated with DM risk．The relative copy number of mtDNA was measured using quantitative re- al-time polymerase chain reaction ( qPCR) analysis． Results : Two SNPs ( 16304T / C，16519T / C) were found to be associated with the risk of developing DM，and alleles 16304C ( χ2 = 4. 937，P = 0. 026) and 16519C ( χ2 = 4. 405，P = 0. 036) in the mitochondrial D-loop region were confirmed to be associated with DM development risk． The DM risk-associated allele 16304C was significantly associated with lower IL-4 expression ( P = 0. 016) ．The mtDNA copy number was significantly higher in DM patients than in controls ( P ＜0. 001) ． Conclusion Mitochondrial D-loop SNPs can be potential biomarkers for DM risk，and SNPs may be involved in DM by influencing cytokines．DM shows high expression of mtDNA copy number，and the increase in mtDNA copy number may lead to mitochondrial dysfunction，which triggers the pathogenesis of DM．

This study was aimed at investigating the effectiveness of various host nucleic acid removal and non-specific amplifica-tion techniques in animal tissue samples,to increase the accuracy of pathogen identification in tissue samples.Simulated samples were prepared with a mixture of mouse lung tissue homogenates and Klebsiella pneumoniae fluids,and processed with six host nucleic acid removal kits and three non-specific amplification techniques.The effectiveness of each method in removing host DNA and enriching nucleic acids of pathogenic microorganisms was evaluated through real-time fluorescence quantitative PCR and high-throughput se-quencing.For host nucleic acid removal techniques,the method of selective cleavage and quantitative degradation of host DNA(Com-plete5 kit)effectively decreased the host nucleic acid content in tissue samples and increased the relative abundance of pathogen nucleic acids.In contrast,the magnetic bead method for host DNA removal(Next microbiome DNA enrichment Kit kit)was less effec-tive.At lower pathogen concentrations(77 CFU/mL),the Vazyme kit was more effective than the other kits in removing host nucleic acids.Non-specific amplification techniques(MALBAC whole genome amplification,MDA isothermal amplification,and random primer amplification)were not applicable to tissue samples and were not effective in increasing the relative abundance of pathogen nucleic acids.Selective lysis and quantitative degradation of host DNA were suitable for processing tissue samples with high host back-ground and low pathogenic microorganism levels,whereas non-specific amplification methods were not applicable to tissue samples for pre-processing of macro-genome high-throughput sequencing.

Objective To investigate the regulatory effect and mechanism of Yiqi Jiedu Decoction(YQJD)on ionizing radiation-induced macrophage polarization and its correlation with the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor kappa-B(NF-κB)signaling pathway.Methods Fifty-five specific-pathogen-free male Sprague-Dawley rats were randomly divided into blank(n=30),anduolin(n=10),and YQJD groups(n=15).They were respectively gavaged with deionized water,anduolin suspension(0.345 6 g/kg),and YQJD high-dose(20.88 g/kg)at a dose of 0.01 mL/g body weight once a day for seven consecutive days.2 hours after the last gavage,blood was collected from the abdominal aorta to prepare the control rat,andolin rat,and YQJD high-dose sera.Appropriate amounts of YQJD high-dose and control sera were mixed in a ratio of 1∶1 and 1∶3,respectively,to obtain YQJD medium-and low-dose rat serum.RAW264.7 cells were divided into blank(10%blank rat serum),model(10%blank rat serum),anduolin(10%anduolin rat serum),and YQJD-L,YQJD-M,YQJD-H groups(10%YQJD low-,medium-,and high-dose rat serum).Except for the blank group,the cells in other groups were irradiated with 12 Gy60 Co γ-rays once to establish the macrophage radiation injury model.At 24 h after irradiation,cell viability was detected using the CCK-8 method,and the cell migration rate was measured using the scratch test.Cell morphology was observed using phalloidin staining,tumor necrosis factor-alpha(TNF-α)and interleukin-10(IL-10)levels in the cell supernatant were quantified using enzyme-linked immunosorbent assay,and the proportion of M1 macrophages was detected using flow cytometry.TLR4,MyD88,and NF-κB protein expression were detected using Western blotting.Results Twenty-four hours after irradiation,compared with the blank group,the model group exhibited significantly reduced cell viability and migration rate(P<0.01),increased cell volume and pseudopodia formation,elevated TNF-α and IL-10 levels,an increased proportion of M1 macrophages,and upregulated TLR4,MyD88,and NF-κB protein expression(P<0.05,P<0.01).Compared with the model group,each drug-treated group showed improved cell viability and migration rate(P<0.05,P<0.01),decreased cell volume,more regular cell shape,reduced TNF-α levels,lower M1-type macrophage proportion,and downregulated TLR4,MyD88,and NF-κB protein expression(P<0.05,P<0.01).IL-10 level showed an upward trend.Conclusion YQJD can partially inhibit M1 macrophage polarization and suppress inflammatory responses,which may be related to the TLR4/MyD88/NF-κB signaling pathway.

Objective To investigate the regulatory effect and mechanism of Yiqi Jiedu Decoction(YQJD)on ionizing radiation-induced macrophage polarization and its correlation with the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor kappa-B(NF-κB)signaling pathway.Methods Fifty-five specific-pathogen-free male Sprague-Dawley rats were randomly divided into blank(n=30),anduolin(n=10),and YQJD groups(n=15).They were respectively gavaged with deionized water,anduolin suspension(0.345 6 g/kg),and YQJD high-dose(20.88 g/kg)at a dose of 0.01 mL/g body weight once a day for seven consecutive days.2 hours after the last gavage,blood was collected from the abdominal aorta to prepare the control rat,andolin rat,and YQJD high-dose sera.Appropriate amounts of YQJD high-dose and control sera were mixed in a ratio of 1∶1 and 1∶3,respectively,to obtain YQJD medium-and low-dose rat serum.RAW264.7 cells were divided into blank(10%blank rat serum),model(10%blank rat serum),anduolin(10%anduolin rat serum),and YQJD-L,YQJD-M,YQJD-H groups(10%YQJD low-,medium-,and high-dose rat serum).Except for the blank group,the cells in other groups were irradiated with 12 Gy60 Co γ-rays once to establish the macrophage radiation injury model.At 24 h after irradiation,cell viability was detected using the CCK-8 method,and the cell migration rate was measured using the scratch test.Cell morphology was observed using phalloidin staining,tumor necrosis factor-alpha(TNF-α)and interleukin-10(IL-10)levels in the cell supernatant were quantified using enzyme-linked immunosorbent assay,and the proportion of M1 macrophages was detected using flow cytometry.TLR4,MyD88,and NF-κB protein expression were detected using Western blotting.Results Twenty-four hours after irradiation,compared with the blank group,the model group exhibited significantly reduced cell viability and migration rate(P<0.01),increased cell volume and pseudopodia formation,elevated TNF-α and IL-10 levels,an increased proportion of M1 macrophages,and upregulated TLR4,MyD88,and NF-κB protein expression(P<0.05,P<0.01).Compared with the model group,each drug-treated group showed improved cell viability and migration rate(P<0.05,P<0.01),decreased cell volume,more regular cell shape,reduced TNF-α levels,lower M1-type macrophage proportion,and downregulated TLR4,MyD88,and NF-κB protein expression(P<0.05,P<0.01).IL-10 level showed an upward trend.Conclusion YQJD can partially inhibit M1 macrophage polarization and suppress inflammatory responses,which may be related to the TLR4/MyD88/NF-κB signaling pathway.

This study was aimed at investigating the effectiveness of various host nucleic acid removal and non-specific amplifica-tion techniques in animal tissue samples,to increase the accuracy of pathogen identification in tissue samples.Simulated samples were prepared with a mixture of mouse lung tissue homogenates and Klebsiella pneumoniae fluids,and processed with six host nucleic acid removal kits and three non-specific amplification techniques.The effectiveness of each method in removing host DNA and enriching nucleic acids of pathogenic microorganisms was evaluated through real-time fluorescence quantitative PCR and high-throughput se-quencing.For host nucleic acid removal techniques,the method of selective cleavage and quantitative degradation of host DNA(Com-plete5 kit)effectively decreased the host nucleic acid content in tissue samples and increased the relative abundance of pathogen nucleic acids.In contrast,the magnetic bead method for host DNA removal(Next microbiome DNA enrichment Kit kit)was less effec-tive.At lower pathogen concentrations(77 CFU/mL),the Vazyme kit was more effective than the other kits in removing host nucleic acids.Non-specific amplification techniques(MALBAC whole genome amplification,MDA isothermal amplification,and random primer amplification)were not applicable to tissue samples and were not effective in increasing the relative abundance of pathogen nucleic acids.Selective lysis and quantitative degradation of host DNA were suitable for processing tissue samples with high host back-ground and low pathogenic microorganism levels,whereas non-specific amplification methods were not applicable to tissue samples for pre-processing of macro-genome high-throughput sequencing.

Objective To construct a recombinant poxvirus vector vaccine, rVTTδTK-RBD, and to evaluate its safety and immunogenicity. Methods The receptor-binding domain (RBD) gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE, to construct the recombinant poxvirus shuttle vector pSTKE-RBD. This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain (VTT). The recombinant poxvirus rVTTδTK-RBD was successfully obtained after several rounds of fluorescence phage screening. The effect of rVTTδTK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination. The levels of specific and neutralizing antibodies produced by rVTTδTK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly. The effect of rVTTδTK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry. Results Through homologous recombination, enhanced green fluorescent protein (EGFP) screening marker, and multiple rounds of fluorescent phosphorescence phage screening, a recombinant poxvirus rVTTδTK-RBD, expressing RBD with deletions in the thymidine kinase (TK) gene, was successfully obtained, which was validated by PCR. The in vivo experiments on BALB/c mice showed that rVTTδTK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT. Conclusion The recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2 is successfully constructed and obtained, with its safety and immunogenicity confirmed through various experiments.
Animals ; Mice ; SARS-CoV-2/genetics* ; COVID-19 ; Vaccines, Synthetic/genetics* ; Genes, Reporter ; Bacteriophages ; Mice, Inbred BALB C

Objective: To understand the influencing factors underlying physical exercise behavior among children and adolescents aged 9-18 years in Ningxia, China, in 2019, and to provide suggestions for physical exercise among children and adolescents by adopting a social ecological model. Methods: Based on data related to 12 018 children and adolescents in Ningxia aged 9-18 years old which were obtained from the National Survey on Students Constitution and Health in 2019, the survey questions scored based on the four levels of the social ecological model. Results: The proportion of children and adolescents who engaged in physical exercise for less than 1 hour a day accounted for 55.97% of the total number of people in Ningxia. Regardless of whether they were able to engage in enough physical activity, individual factors played a dominant role in influencing the behavior of the children and adolescents, and an interaction was found between influencing factors at different levels( r=-0.01, 0.01, -0.08, 0.04, P <0.01). The results of the stepwise regression analysis showed that the personal factors e.g. sports makes me healthy, I don t have enough time, muscle strength exercise, I have no movement of any relationship partner, sports make me know partner, policy in the number of physical education, number of sports meeting in a school year, the average daily lesson hold number all affect children s sports activities in time( B =-0.05-0.16, P <0.01). Conclusions The lack of physical exercise among children and adolescents in Ningxia is the result of multiple factors. Comprehensive and diversified intervention should be administered from a socio ecological framework to promote the formation of physical exercise habits among children and adolescents.

Objective:To investigate the mediating effect of inflammatory factors in the association between low density lipoprotein-cholesterol(LDL-C) and thyroid associated ophthalmopathy(TAO).Methods:This study was a prospective study, which icluded a total of 86 patients with Graves′ disease who attended the Department of Endocrinology of the First Affiliated Hospital of Zhengzhou University from January 2021 to June 2022. Among them, there were 56 patients with Graves′ disease accompanied by TAO, including 30 cases in the inactive group and 26 in the active group. Additionally, there were 30 cases having Graves′ disease alone. The relationship between LDL-C, inflammatory factors, and the onset and activity of TAO were analyzed using binary logistic regression. Mediation analysis was used to explore the mediating effect of inflammatory factors in the association between LDL-C and TAO onset and activity.Results:Interleukin(IL) -6 was a potential mediator that linking the association between LDL-C and TAO onset: LDL-C had a direct effect on TAO(Total effect value=0.274, 95% CI 0.161-0.386), while IL-6(mediated effect=0.067, 95% CI 0.011-0.123) and IL-17(mediated effect=0.042, 95% CI 0.007-0.077) partially mediated the effect of LDL-C on TAO, accounting for 24.45% and 15.33% of the total effect, respectively. IL-6 was a potential mediator of the association between LDL-C and TAO activity: LDL-C had a direct effect on TAO activity(Total effect value=0.320, 95% CI 0.204-0.435), and IL-6(mediated effect=0.103, 95% CI 0.021-0.185) partially mediated the effect of LDL-C on TAO activity, with a mediation effect of 32.19%. Conclusion:IL-6 plays a partiall mediating role in the association of LDL-C with TAO onset and activity.

Astaxanthine (AST) has important biological activities including antioxidant and anti-inflammatory effects that could alleviate neurological and heart diseases, but its role in the prevention of cisplatin-induced hearing loss (CIHL) is not yet well understood. In our study, a steady interaction between AST and the E3 ligase adapter Kelch-like ECH-associated protein 1, a predominant repressor of nuclear factor erythroid 2-related factor 2 (NRF2), was performed and tested via computer molecular docking and dynamics. AST protected against cisplatin-induced ototoxicity via NRF2 mediated pathway using quantitative PCR and Western blotting. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential revealed that AST reduced ROS overexpression and mitochondrial dysfunction. Moreover, AST exerted anti-apoptosis effects in mouse cochlear explants using immunofluorescence staining and HEI-OC1 cell lines using quantitative PCR and Western blotting. Finally, AST combined with poloxamer was injected into the middle ear through the tympanum, and the protection against CIHL was evaluated using the acoustic brain stem test and immunofluorescent staining in adult mice. Our results suggest that AST reduced ROS overexpression, mitochondrial dysfunction, and apoptosis via NRF2-mediated pathway in cisplatin-exposed HEI-OC1 cell lines and mouse cochlear explants, finally promoting cell survival. Our study demonstrates that AST is a candidate therapeutic agent for CIHL.