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*

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.

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.

Ubiquitination, a diverse post-translational modification, is carried out by enzymes including E1-activating enzymes, E2-conjugating enzymes, E3 ligases, and deubiquitinating enzymes (DUBs). Ubiquitin itself possesses 7 lysine residues and N-terminal methionine, allowing for the formation of polyubiquitin chains with different lengths and linkages. These chains exhibit various topologies that can be recognized by proteins containing ubiquitin-binding domain, thereby transmitting distinct cellular signals. To unravel the physiological mechanisms associated with ubiquitin, numerous ubiquitin probes have been developed. This review provides an overview of recent advancements in the field of ubiquitin probes, focusing on activity-based and affinity-based probes. Activity-based probes are designed to covalently bind to DUBs, E1s, or E3s, enabling the identification and characterization of these enzymes. Affinity-based probes, on the other hand, selectively bind to ubiquitin-binding domains, facilitating the identification of proteins that interact with ubiquitin. Moreover, this review comprehensively discusses the synthetic methodologies employed for the acquisition of ubiquitin probes. These includes meticulous discussions on the synthesis of individual monomeric modules, the establishment of isopeptide linkages, as well as the incorporation of reactive functional groups. Additionally, the review explores the emerging area of cell-penetrating ubiquitin probes and highlights their latest applications in living cells. These probes incorporate cell-penetrating peptides to enable their internalization into cells, allowing for direct visualization and manipulation of ubiquitin-modified proteins within their native environment. Overall, this review offers insights into the design, synthesis, and applications of ubiquitin probes, highlighting their significance in elucidating ubiquitin-mediated cellular processes.