ObjectiveTo investigate the protective effect and mechanism of verbenalin on mouse mononuclear macrophage leukemia cells （RAW264.7） damaged by human coronavirus （HCoV）-229E infection， thereby providing experimental evidence for its development and application. MethodsRAW264.7 macrophages were infected with different concentrations of HCoV-229E to establish a coronavirus-induced macrophage injury model using the cell counting kit-8 （CCK-8） assay for assessing cell proliferation and viability. Cells were randomly divided into four groups： normal control， verbenalin group （125 μmol·L^-1）， model group （HCoV-229E）， and HCoV-229E + verbenalin group （HCoV-229E + 125 μmol·L^-1 verbenalin）. Cell viability was measured using the CCK-8 assay， and the maximum non-toxic concentration （CC₀）， half-maximal cytotoxic concentration （CC₅₀）， half-maximal effective concentration （EC₅₀）， and selectivity index （SI） of verbenalin were calculated. Calcein/PI double staining was used to assess cell viability and cytotoxicity， and JC-1 staining was applied to evaluate changes in mitochondrial membrane potential （MMP）. mito-Keima adenovirus labeling was used to assess mitophagy levels in each group. ResultsA macrophage infection model was successfully established by infecting RAW264.7 cells with the original concentration of HCoV-229E for 36 h. The CC₀ of verbenalin was 125 μmol·L^-1. The CC₅₀ was 448.25 μmol·L^-1. The EC₅₀ against HCoV-229E-infected cells was 46.28 μmol·L^-1， and the SI was 9.68. Compared with the normal group， the model group showed significantly reduced cell survival rate （P<0.01）， increased cell death rate （P<0.01）， decreased MMP （P<0.01）， and suppressed mitophagy （P<0.01）. In contrast， verbenalin treatment significantly improved cell survival rate （P<0.01）， reduced cell death rate （P<0.01）， alleviated MMP loss （P<0.01）， and enhanced mitophagy levels （P<0.01） compared with the model group. ConclusionVerbenalin can enhance the survival rate of macrophages following HCoV-229E infection. The underlying mechanism may be associated with the activation of mitophagy， maintenance of MMP stability， and alleviation of mitochondrial damage.

ObjectiveTo construct an animal model of respiratory syncytial virus（RSV）-infected pneumonia suitable for preclinical studies. MethodsThe virulence of RSV to the four cell lines was observed by cytopathic effect （CPE）， and 50% tissue culture infective dose（TCID₅₀） was calculated. Twenty BALB/c mice were randomly divided into a normal group and a model group. Six BALB/c-hIGF1R mice served as the humanized IGF1R model group. Except for the normal group， the other groups received intranasal RSV infection on days 1 and 3 to establish a viral pneumonia model. The efficacy of establishing an RSV-induced pneumonia animal model based on humanized insulin-like growth factor 1 receptor （IGF1R） mice was evaluated by measuring organ indices， peripheral blood lymphocyte percentages， pulmonary pathology and imaging， and pulmonary viral load. Additionally， ten BALB/c mice served as normal group， and thirty-two BALB/c-hIGF1R mice were randomly assigned to humanized IGF1R model group， ribavirin group （82.5 mg·kg^-¹·d^-¹）， and high and low dose groups of Lianhua Qingwen （3.3 mg·kg^-¹·d^-¹ ， 1.65 mg·kg^-¹·d^-¹）， with 8 mice per group. The viral load in lung tissue was measured after ribavirin and Lianhua Qingwen intervention， and the model was applied to the evaluation of anti-RSV drugs. ResultsIn the lungs of the humanized IGF1R model group， large solid and diffuse ground-glass shadows were seen， and the lung volume was significantly increased （P<0.01）. The lung index was significantly increased （P<0.01）， and both the spleen index and thymus index were significantly decreased （P<0.01）. The percentages of CD3⁺ and CD4⁺T cells were significantly decreased （P<0.05）， and there was a large amount of inflammation and stasis in the perivascular area of the lung tissue， which was predominantly characterized by lymphocytes. The endothelium of blood vessels was partially detached， with a small number of eosinophils. After infecting BALB/c-hIGF1R mice with RSV， the expression of viral nucleic acids in the lung tissue of the mice was significantly increased， with significant differences compared with the normal group （P<0.01）. The expression of viral nucleic acids in the ribavirin group and the high and low dose groups of Lianhua Qingwen was significantly reduced， with significant differences compared with the normal group （P<0.01）. ConclusionHumanized IGF1R mice are more susceptible to respiratory SVC， and the animal model of RSV-infected pneumonia based on humanized IGF1R mice was successfully constructed， which is suitable for the evaluation of anti-RSV drugs.

Currently， viral pneumonia （VP） presents a major challenge to global public health. Traditional Chinese medicine （TCM） prevention and treatment of VP is guided by the core concept of strengthening vital energy and eliminating pathogenic factors rather than targeting specific pathogens， alongside a holistic approach of syndrome differentiation and treatment. By summarizing the clinical syndromes of patients， the core pathogenesis was clarified to achieve individualized therapy. Animal models for disease-syndrome combination integrate the etiology and pathogenesis of VP and simulate the individualized manifestations of patients at different disease stages， providing an experimental platform for elucidating the theoretical basis of TCM in treating VP and promoting the development of effective TCM formulations. However， there are limitations in the application and promotion of disease-syndrome combination animal models due to the lack of standardization and normalization of model construction systems， which arise from diverse species selection， compound modeling methods， and multidimensional evaluation indicators. This paper systematically reviewed the recent research on animal models for disease-syndrome combination in VP from the perspective of species selection， modeling methods， evaluation indicators， and application status. Furthermore， it summarized the advantages and limitations of existing models， identifies future directions for improvement， and proposes optimization strategies. This review provides a reference for establishing standardized and normalized animal models for disease-syndrome combinations in VP， supporting the theoretical modernization of TCM in preventing and controlling emerging respiratory infectious diseases， and contributing to the development of new TCM drugs.

ObjectiveTo investigate the protective effect and mechanism of verbenalin on mouse mononuclear macrophage leukemia cells （RAW264.7） damaged by human coronavirus （HCoV）-229E infection， thereby providing experimental evidence for its development and application. MethodsRAW264.7 macrophages were infected with different concentrations of HCoV-229E to establish a coronavirus-induced macrophage injury model using the cell counting kit-8 （CCK-8） assay for assessing cell proliferation and viability. Cells were randomly divided into four groups： normal control， verbenalin group （125 μmol·L^-1）， model group （HCoV-229E）， and HCoV-229E + verbenalin group （HCoV-229E + 125 μmol·L^-1 verbenalin）. Cell viability was measured using the CCK-8 assay， and the maximum non-toxic concentration （CC₀）， half-maximal cytotoxic concentration （CC₅₀）， half-maximal effective concentration （EC₅₀）， and selectivity index （SI） of verbenalin were calculated. Calcein/PI double staining was used to assess cell viability and cytotoxicity， and JC-1 staining was applied to evaluate changes in mitochondrial membrane potential （MMP）. mito-Keima adenovirus labeling was used to assess mitophagy levels in each group. ResultsA macrophage infection model was successfully established by infecting RAW264.7 cells with the original concentration of HCoV-229E for 36 h. The CC₀ of verbenalin was 125 μmol·L^-1. The CC₅₀ was 448.25 μmol·L^-1. The EC₅₀ against HCoV-229E-infected cells was 46.28 μmol·L^-1， and the SI was 9.68. Compared with the normal group， the model group showed significantly reduced cell survival rate （P<0.01）， increased cell death rate （P<0.01）， decreased MMP （P<0.01）， and suppressed mitophagy （P<0.01）. In contrast， verbenalin treatment significantly improved cell survival rate （P<0.01）， reduced cell death rate （P<0.01）， alleviated MMP loss （P<0.01）， and enhanced mitophagy levels （P<0.01） compared with the model group. ConclusionVerbenalin can enhance the survival rate of macrophages following HCoV-229E infection. The underlying mechanism may be associated with the activation of mitophagy， maintenance of MMP stability， and alleviation of mitochondrial damage.

ObjectiveTo construct an animal model of respiratory syncytial virus（RSV）-infected pneumonia suitable for preclinical studies. MethodsThe virulence of RSV to the four cell lines was observed by cytopathic effect （CPE）， and 50% tissue culture infective dose（TCID₅₀） was calculated. Twenty BALB/c mice were randomly divided into a normal group and a model group. Six BALB/c-hIGF1R mice served as the humanized IGF1R model group. Except for the normal group， the other groups received intranasal RSV infection on days 1 and 3 to establish a viral pneumonia model. The efficacy of establishing an RSV-induced pneumonia animal model based on humanized insulin-like growth factor 1 receptor （IGF1R） mice was evaluated by measuring organ indices， peripheral blood lymphocyte percentages， pulmonary pathology and imaging， and pulmonary viral load. Additionally， ten BALB/c mice served as normal group， and thirty-two BALB/c-hIGF1R mice were randomly assigned to humanized IGF1R model group， ribavirin group （82.5 mg·kg^-¹·d^-¹）， and high and low dose groups of Lianhua Qingwen （3.3 mg·kg^-¹·d^-¹ ， 1.65 mg·kg^-¹·d^-¹）， with 8 mice per group. The viral load in lung tissue was measured after ribavirin and Lianhua Qingwen intervention， and the model was applied to the evaluation of anti-RSV drugs. ResultsIn the lungs of the humanized IGF1R model group， large solid and diffuse ground-glass shadows were seen， and the lung volume was significantly increased （P<0.01）. The lung index was significantly increased （P<0.01）， and both the spleen index and thymus index were significantly decreased （P<0.01）. The percentages of CD3⁺ and CD4⁺T cells were significantly decreased （P<0.05）， and there was a large amount of inflammation and stasis in the perivascular area of the lung tissue， which was predominantly characterized by lymphocytes. The endothelium of blood vessels was partially detached， with a small number of eosinophils. After infecting BALB/c-hIGF1R mice with RSV， the expression of viral nucleic acids in the lung tissue of the mice was significantly increased， with significant differences compared with the normal group （P<0.01）. The expression of viral nucleic acids in the ribavirin group and the high and low dose groups of Lianhua Qingwen was significantly reduced， with significant differences compared with the normal group （P<0.01）. ConclusionHumanized IGF1R mice are more susceptible to respiratory SVC， and the animal model of RSV-infected pneumonia based on humanized IGF1R mice was successfully constructed， which is suitable for the evaluation of anti-RSV drugs.

Currently， viral pneumonia （VP） presents a major challenge to global public health. Traditional Chinese medicine （TCM） prevention and treatment of VP is guided by the core concept of strengthening vital energy and eliminating pathogenic factors rather than targeting specific pathogens， alongside a holistic approach of syndrome differentiation and treatment. By summarizing the clinical syndromes of patients， the core pathogenesis was clarified to achieve individualized therapy. Animal models for disease-syndrome combination integrate the etiology and pathogenesis of VP and simulate the individualized manifestations of patients at different disease stages， providing an experimental platform for elucidating the theoretical basis of TCM in treating VP and promoting the development of effective TCM formulations. However， there are limitations in the application and promotion of disease-syndrome combination animal models due to the lack of standardization and normalization of model construction systems， which arise from diverse species selection， compound modeling methods， and multidimensional evaluation indicators. This paper systematically reviewed the recent research on animal models for disease-syndrome combination in VP from the perspective of species selection， modeling methods， evaluation indicators， and application status. Furthermore， it summarized the advantages and limitations of existing models， identifies future directions for improvement， and proposes optimization strategies. This review provides a reference for establishing standardized and normalized animal models for disease-syndrome combinations in VP， supporting the theoretical modernization of TCM in preventing and controlling emerging respiratory infectious diseases， and contributing to the development of new TCM drugs.

ObjectiveTo evaluate the pharmacological effects of verbenalin on both in vitro and in vivo infection models of human coronavirus 229E （HCoV-229E） and to preliminarily explore the antiviral mechanism of verbenalin through proteomic analysis. MethodsIn vitro， the cell counting kit-8 （CCK-8） for cell proliferation and viability assessment was used to establish a model of HCoV-229E-induced injury in human lung adenocarcinoma cells（A549）. A549 cells were divided into five groups： normal group， model group， and three verbenalin treatment groups （125， 62.5， and 31.25 μmol·L^-1）. The cell protective activity of verbenalin was evaluated through cell viability assay and immunofluorescence staining. In vivo， 30 BALB/c mice were randomly divided into normal group， model group， chloroquine group， and high-dose， low-dose verbenalin groups （40 and 20 mg·kg^-1）， with six mice per group. An HCoV-229E-induced mouse lung injury model was established to evaluate the therapeutic effects of verbenalin. Lung injury was assessed by detecting the lung index and lung inhibition rate. The severity of pulmonary inflammation cytokines was measured by enzyme-linked immunosorbent assay （ELISA）， while the lung morphology and structure were analyzed by micro-computed tomography （Micro-CT）. Hematoxylin and eosin （HE） staining was used to assess histopathological changes in lung tissue. Additionally， four-dimensional data-independent acquisition （4D-DIA） proteomics was employed to preliminarily explore the potential mechanisms of verbenalin in treating HCoV-229E-induced lung injury in mice， through differential protein expression screening， functional annotation， enrichment analysis， and protein-protein interaction network analysis. ResultsThe A549 cells were infected with HCoV-229E at the original viral titer for 36 hours to establish an in vitro infection model. The maximum non-toxic concentration of verbenalin was 125 μmol·L^-1， and the half-maximal cytotoxic concentration （CC₅₀） was 288.8 μmol·L^-1. Compared with the normal group， the model group showed a significant decrease in cell viability （P<0.01）， a significant increase in the proportion of dead cells （P<0.01）， mitochondrial damage， and a significant reduction in mitochondrial membrane potential （P<0.01）. After treatment with different concentrations of verbenalin （125， 62.5， and 31.25 μmol·L^-1）， cell viability was significantly increased （P<0.01）， and the proportion of dead cells was reduced （P<0.01）， with mitochondrial membrane potential restored （P<0.01）. In vivo experiments further confirmed the therapeutic effect of verbenalin on HCoV-229E-infected mice. Compared to the normal group， the model group showed a significant increase in the lung index （P<0.01）， severe lung tissue injury， lung volume enlargement， and a significant increase in the expression of inflammatory cytokines， including interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） （P<0.01）. In contrast， in the verbenalin treatment groups， these pathological changes were significantly improved， with a reduction in the lung index （P<0.01）， alleviation of lung tissue injury， reduced lung volume enlargement， and a significant decrease in inflammatory cytokine expression （P<0.01）. Proteomics analysis revealed that， compared to the normal group， the model group showed enrichment in several antiviral immune-related signaling pathways， including the nuclear factor-κB （NF-κB） signaling pathway （P<0.05）. Compared to the model group， the verbenalin treatment group showed enrichment in several signaling pathways related to inflammatory response and autophagy （P<0.05）， suggesting that verbenalin may exert its antiviral and anti-inflammatory effects by regulating these pathways. ConclusionVerbenalin demonstrates significant therapeutic effects in both in vitro and in vivo HCoV-229E infection models， with its mechanism likely related to the NOD-like receptor protein 3 （NLRP3） inflammasome pathway and mitochondrial autophagy.

To investigate the effects of phosphorus fertilizer on the morphological traits, active ingredients and rhizosphere soil microbial community of Polygala tenuifolia. The phosphorus fertilizer was calculated in terms of P_2O_5. Five treatments were set up: 0(CK), 17(P1), 34(P2), 51(P3), and 68(P4) kg per Mu(1 Mu≈667 m~2). A randomized block design was adopted. Samples of P. tenuifolia and its rhizosphere soil were collected under different superphosphate fertilizer treatments. Illumina high-throughput sequencing was used to analyze the rhizosphere soil microbial community, 9 morphological traits were measured and the content of 11 active ingredients were determined. The results showed that the whole plant weight, shoot fresh weight, root weight, and root peel thickness were the highest under P1 treatment, increasing by 34.41%, 38.80%, 39.21%, and 3.17% respectively compared to CK. Under P2 treatment, the plant height, stem diameter, root thickness, and core thickness were significantly higher than CK. Phosphorus fertilizer had a significant impact on the content of tenuifolin, sibiricose A5, sibiricose A6, arillanin A, 3,6'-disinapoyl sucrose, and polygalaxanthone Ⅲ. Correlation analysis results showed that the relative abundance of Arthrobacter, Bacillus, norank＿f＿Vicinamibacteraceae, norank＿o＿Vicinamibacterales, MND1 and other bacteria, as well as the relative abundance of Neocosmospora, Paraphoma and other fungi were positively correlated with root diameter, wood core diameter, the whole plant weight, root weight, shoot fresh weight of P. tenuifolia. Bacillus, Neocosmospora, Subulicystidium were significantly positively correlated with oligosaccharides such as 3,6'-disinapoyl sucrose, sibiricose A5、sibiricose A6、glomeratose A、arillanin A and tenuifoliside C. Arthrobacter, Humicola, Aspergillus, Paraphoma were positively correlated with tenuifolin and norank＿f＿Vicinamibacteraceae, norank＿o＿Vicinamibacterales, Fusarium were positively correlated with polygalaxanthone Ⅲ. Evidently, appropriate phosphorus application is conducive to the growth and quality improvement of P. tenuifolia, and can increase the abundance of beneficial microorganisms in the soil.
Rhizosphere ; Phosphorus/pharmacology* ; Soil Microbiology ; Polygala/anatomy & histology* ; Fertilizers/analysis* ; Bacteria/metabolism* ; Soil/chemistry* ; Microbiota/drug effects* ; Plant Roots/metabolism*

Octapeptin has strong antibacterial activity against Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii, while it also has activity against some Gram-positive bacteria. This study used natural octapeptin A3 and B3 as lead compounds for structural modification. Twenty-one peptide derivatives (including A3 and B3) containing eight amino acid residues were prepared by solid-phase synthesis, and evaluated for antibacterial activity and renal cytotoxicity. Among them, three compounds 6, 7 and 17 exhibited broad-spectrum antibacterial activity and significantly enhanced the activity for Gram-positive bacteria while maintaining the activity of Gram-negative bacteria. Several compounds improved the activity for Pseudomonas aeruginosa. Compound 7 was active against all test strains and had relatively low renal cytotoxicity. The results provide a basis for the further development of novel polypeptide antibiotics.

Objective: To explore the pre-exposure prophylaxis (PrEP) awareness, willingness, uptake patterns, barriers and needs among Chinese student men who have sex with men (MSM), so as to provide relevant evidence for targeted interventions with PrEP. Methods: A proportional sampling method was used to conduct a cross sectional survey of MSM aged 16 years and older residing in 21 provinces, municipalities, and autonomous regions in mainland China between October 20 and December 20, 2021, to collect information on demographic and sexual behavioral characteristics, and 923 students of them were selected as the subjects of this study. The chi-square test and Fisher s test were used for univariate analysis, followed by multivariate Logistic regression to analyze the influencing factors of PrEP awareness and uptake. Results: According to the cascade analysis method, the awareness, willingness, uptake and adherence rates of PrEP among the student MSM were obtained as 88.95%, 67.36%, 13.20% and 45.21 %, respectively. HIV testing more than once in the last 3 months, and all of them were aware of the HIV test results of their sexual partners, and those with high frequency of condom use had a higher rate of awareness ( OR =2.32, 1.79, 1.69, P <0.05). Willingness rates were higher for those from the pilot city, using substances, and HIV testing more than once in the last 3 months ( OR =2.13, 1.65, 1.69, P <0.05). Higher rates of uptake were found among those from high risk and pilot areas, presence of commercial sex, substance use, and high literacy ( OR =5.60, 3.54,2.81, 1.92, 4.54, P <0.05). Adherence rates were higher among those who used one pill per day or both ( OR =12.77, 13.26, P <0.05). The barriers and needs faced by student MSM were primarily personal concerns about medication side effects, preventative effects, and confidence in sexual behavioral styles, and the high cost of medication and related service costs. Conclusions The student MSM population in China is characterized by high awareness, low willingness, low uptake, and low adherence to PrEP. Targeted interventions should be considered and tailored by the departments to facilitate PrEP promotion among student MSM.