Two new coumarin glycosides were isolated and purified from the dichloromethane fraction of Angelica biserrata 75% ethanol extract using silica gel, Sephadex LH-20 column chromatography, and semi-preparative high-performance liquid chromatography. Through the comprehensive use of modern spectroscopic methods, their structures were identified as angelmanetin A (1) and angelmanetin B (2), respectively.

ObjectiveTo understand the epidemiological distribution and gene evolutionary variation of influenza A (H3N2) viruses in Fengxian District, Shanghai, in the surveillance year of 2023, and to provide a reference basis for influenza prevention and control. MethodsThe prevalence of influenza virus in Fengxian District in the 2023 influenza surveillance year (April 2023‒March 2024) was analyzed. The hemagglutinin (HA) gene, neuraminidase (NA) gene, and amino acid sequences of 75 strains of H3N2 influenza viruses were compared with the vaccine reference strain for similarity matching and phylogenetic evolutionary analysis, in addition to an analysis of gene characterization and variation. ResultsIn Fengxian District, there was a mixed epidemic of H3N2 and H1N1 in the spring of 2023, with H3N2 being the predominant subtype in the second half of the year, and Victoria B becoming the predominant subtype in the spring of 2024. A total of 75 influenza strains of H3N2 with HA and NA genes were distributed in the 3C.2a1b.2a.2a.2a.3a.1 and B.4 branches, with overall similarity to the reference strain of the 2024 vaccine higher than that of the reference strain of the 2022 and 2023 vaccine. Compared with the 2023 vaccine reference strain, three antigenic sites and one receptor binding site were changed in HA, with three glycosylation sites reduced and two glycosylation sites added; where as in NA seven antigenic sites and the 222nd resistance site changed with two glycosylation sites reduced. ConclusionThe risk of antigenic variation and drug resistance of H3N2 in this region is high, and it is necessary to strengthen the publicity and education on the 2024 influenza vaccine and long-term monitoring of influenza virus prevalence and variation levels.

ObjectiveTo study the effect and mechanism of Shentong Zhuyutang in treating intervertebral disc degeneration （IDD） in rats. MethodsIn the cell experiment， male rats were administrated with normal saline or low-， medium-， and high-dose （3.38， 6.75，13.5 g·kg^-1， respectively） Shentong Zhuyutang by gavage， respectively， and serum samples were collected after 7 days of continuous administration. Another 10 male rats were selected for the isolation of nucleus pulposus cells. The cell model of IDD was established by treatment with interleukin （IL）-1β. The modeled cells were then treated with Shentong Zhuyutang-containing serum and the ferroptosis inhibitor ferrostatin-1 （Fer-1）， respectively， to investigate the effects of Shentong Zhuyutang-containing serum on the proliferation and ferroptosis of nucleus pulposus cells. To study the role of silent information regulator 1 （SIRT1）/nuclear factor erythroid 2-related factor 2 （Nrf2） in the regulation of ferroptosis in nucleus pulposus cells by Shentong Zhuyutang-containing serum， this study treated the cells with the SIRT1 inhibitor Ex 527 and the Nrf2 inhibitor ML385， respectively， in addition to the treatment with IL-1β and high-dose Shentong Zhuyutang-containing serum. The cell-counting kit-8 （CCK-8） assay and EdU staining were employed to measure the cell viability and proliferation， respectively. The Fe²⁺， glutathione （GSH）， and malondiadehyde （MDA） levels were measured by colorimetric assay. Western blot was employed to determine the protein levels of glutathione peroxidase 4 （GPX4）， acyl-CoA synthetase long-chain family 4 （ACSL4）， Collagen Ⅱ， Aggrecan， SIRT1， and Nrf2. Immunofluorescence was used detect SIRT1 expression. In the animal experiment， male rats were treated with anulus puncture for the modeling of IDD. Rats were randomly assigned into sham operation， model， Shentong Zhuyutang-containing serum （13.5 g·kg^-1）， and positive control （nimesulide dispersible tablets， 0.18 mg·kg^-1） groups. Rats in the drug intervention groups were administrated with corresponding agents at 1 mL·kg^-1， and those in the sham operation and model groups were administrated with equal volumes of normal saline， once daily for 28 consecutive days. At the end of the last administration， the histopathological changes in the intervertebral discs of rats were observed by hematoxylin-eosin staining and scored by the Masuda method. Western blot was employed to determine the protein levels of SIRT1， Nrf2， GPX4， and Collagen Ⅱ in the nucleus pulposus tissue. ResultsCompared with the control group， the IL-1β group of nucleus pulposus cells showed elevated levels of Fe²⁺， MDA， and ACSL4 （P<0.05）， decreased cell viability， lowered GSH level， and down-regulated protein levels of GPX4， Collagen Ⅱ， and Aggrecan （P<0.05）. Shentong Zhuyutang-containing serum and Fer-1 reversed the effects of IL-1β on the viability and ferroptosis of nucleus pulposus cells and up-regulated the protein levels of Collagen Ⅱ and Aggrecan in nucleus pulposus cells （P<0.05）. Compared with the control group， the IL-1β group showcased down-regulated expression of Sirt1 and Nrf2 in nucleus pulposus cells （P<0.05）. Compared with the IL-1β group， the high-dose Shentong Zhuyutang-containing serum+IL-1β group showed up-regulated expression of SIRT1 and Nrf2 in nucleus pulposus cells （P<0.05）. Compared with the high-dose Shentong Zhuyutang-containing serum+IL-1β group， the ML385 group showed down-regulated protein levels of Nrf2 and GPX4， lowered GSH level， and elevated Fe²⁺ and MDA levels （P<0.05）. In addition， the Ex 527 group showed down-regulated protein levels of SIRT1， Nrf2， and GPX4 （P<0.05）. The results of the animal experiment showed that compared with the sham operation group， the model group had severe degeneration of the intervertebral disc tissue with increased pathological score， up-regulated protein level of ACSL4 （P<0.05）， and down-regulated protein levels of SIRT1， Nrf2， GPX4， and Collagen Ⅱ （P<0.05）. Compared with the model group， the Shentong Zhuyutang group showed alleviated IDD with declined pathological score， down-regulated protein level of ACSL4 （P<0.05）， and up-regulated protein levels of SIRT1， Nrf2， GPX4， and Collagen Ⅱ （P<0.05）. ConclusionShentong Zhuyutang may activate the SIRT1/Nrf2 signaling pathway to inhibit the ferroptosis of nucleus pulposus cells， thereby delaying the process of IDD in rats.

As a new type of model organism， zebrafish is gradually gaining prominence in the field of scientific research. The unique biological characteristics and advantages of zebrafish make them play an increasingly important role in the quality evaluation of traditional Chinese medicine. Compared with other common experimental animals， zebrafish have a fast reproductive and growth speed and high embryo transparency， making them an ideal model for evaluating the quality of traditional Chinese medicine. This provides a new perspective and method for research on traditional Chinese medicine. With the growing global interest in traditional Chinese medicine， it has become crucial to find scientific and accurate methods to evaluate the quality and effectiveness of traditional Chinese medicine. The introduction of the zebrafish model has brought new breakthroughs in the quality evaluation of traditional Chinese medicine. To further promote the application of zebrafish in evaluating the quality of traditional Chinese medicine， this article systematically searched and sorted out the previous studies related to the application of zebrafish for this purpose since 2023. The commonly used disease models and indicators of zebrafish in evaluating the effectiveness of traditional Chinese medicine， as well as the mechanism of zebrafish in exploring the active ingredients of traditional Chinese medicine， were primarily reviewed. The application of zebrafish in evaluating the safety of traditional Chinese medicine and the typical examples in ensuring the quality of traditional Chinese medicine were demonstrated. The limitations encountered by zebrafish models in evaluating the quality of traditional Chinese medicine were highlighted. The resolution of these problems will help further improve the accuracy and reliability of zebrafish in evaluating the quality of traditional Chinese medicine. The article discussed the evaluation of effectiveness， safety， and quality control of zebrafish applied in traditional Chinese medicine， so as to provide a reference for establishing standards for traditional Chinese medicine and promoting its modernization in the future.

ObjectiveBased on the zebrafish model， the hepatotoxicity and anti-osteoporotic activity of evodiamine （EVO） were studied. The mechanism of EVO in treating osteoporosis was explored by using network pharmacology and real-time polymerase chain reaction（Real-time PCR）. MethodsThree days after fertilization （3 dpf）， zebrafish were randomly selected and exposed to different concentrations of EVO solution for 96 hours. The mortality rate of zebrafish at different concentrations was calculated at the exposure endpoint， and a "dose-toxicity" curve was drawn. The 10% lethal concentration （LC₁₀） was calculated. Liver phenotype， acridine orange staining， and pathological tissue sections of liver-transgenic zebrafish ［CZ16 （gz15Tg.Tg （fabp 10a： ds Red； ela31： EGFP））］ were used to confirm hepatotoxicity of EVO. On this basis， prednisolone was used to create a model of osteoporosis in zebrafish. The skull development， area of the skull stained by alizarin red， and cumulative optical density were used as indicators to evaluate the anti-osteoporotic activity of EVO in a safe dose. Based on network pharmacology， the mechanism of action of EVO in the treatment of osteoporosis was predicted and verified through Real-time PCR. ResultsThe LC₁₀ of EVO on zebrafish （7 dpf） was determined to be 0.4 mg·L^-1. Compared with the control group， sublethal concentrations （10=0.36 mg·L^-1 and 1/2LC₁₀=0.18 mg·L^-1） significantly decreased the fluorescence area of zebrafish liver （P<0.05，P<0.01） and increased the number of liver cell apoptosis. The arrangement of liver tissue was disordered and loose， and the vacuole was obvious， especially in the 0.36 mg·L^-1 group. Compared with the control group， under safe dose conditions， 0.08 and 0.02 mg·L^-1 of EVO had no significant effect on the liver. Prednisolone administration significantly reduced the mineralization area and cumulative optical density of zebrafish skulls （P<0.01） compared with the control group. The mineralization area and cumulative optical density of zebrafish skulls in the 0.08 and 0.02 mg·L^-1 groups of EVO were significantly increased compared with the model group （P<0.01）. Network pharmacology prediction suggested that EVO may regulate key targets such as avian sarcoma viral oncogene homolog （SRC）， signal transducer and activator of transcription 3 （STAT3）， interleukin-8 （CXCL8） and tyrosine kinase receptor 2 （ERBB2） to regulate signaling pathways related to lipid and atherosclerosis in diabetic complications， as well as the regulation of inflammatory mediators of tryptophan （TRP） channels. Real-time PCR experiment results indicated that EVO could regulate the above-mentioned targets， as well as genes related to osteoblasts and osteoclasts. ConclusionExcessive doses of EVO can lead to hepatotoxicity in zebrafish. Under safe dosage conditions， EVO can regulate relevant targets to exert anti-osteoporotic activity， providing a reference for the clinical safe use of EVO and ideas for the development of new drugs for osteoporosis.

ObjectiveTo investigate the effects of Pinelliae Rhizoma Praeparatum （PRP） on lung tissue， gut microbiota， and short-chain fatty acid （SCFA） metabolism in a model of mice with cold fluid retention in the lung and explore its mechanism of action in resolving dampness and phlegm based on the interconnection between the lung and large intestine. MethodsFifty female ICR mice were randomly divided into a normal group， model group， positive control group （Xiaoqinglong granules， 6.5 g·kg^-1）， and high-dose and low-dose PRP decoction groups （3.0， 1.5 g·kg^-1）， with 10 mice in each group. A model of mice with cold fluid retention in the lung was established using ovalbumin （OVA） sensitization combined with cold-water immersion. Drug interventions were conducted from day 18 to day 33 for 15 consecutive days. The airway resistance value of the mice was measured using a non-invasive pulmonary function analyzer. Phlegm-resolving effects were evaluated via a microplate reader. Eosinophil and neutrophil counts in bronchoalveolar lavage fluid （BALF） were analyzed using an automated hematology analyzer. Serum levels of total immunoglobulin E （IgE）， interferon-γ （IFN-γ）， interleukin-4 （IL-4）， and BALF levels of interleukin-6 （IL-6） and interleukin-8 （IL-8） were quantified by enzyme-linked immunosorbent assay （ELISA）. Lung histopathology was assessed using hematoxylin-eosin （HE） staining. Immunohistochemistry （IHC） was employed to detect mucin 5AC （MUC5AC） and aquaporin 5 （AQP5） protein expression in lung tissue. Gut microbiota composition was analyzed via agarose gel electrophoresis， and fecal SCFA levels were measured by gas chromatography-mass spectrometry （GC-MS）. ResultsCompared with the normal group， the model group exhibited significantly increased airway resistance value （RI） （P<0.05）， elevated eosinophil and neutrophil counts and IL-6 and IL-8 levels in BALF （P<0.05）， increased serum IgE and IL-4 levels （P<0.05）， with reduced IFN-γ levels （P<0.05）. It also showed thickened bronchial walls， widened alveolar septa， narrowed lumens， and mucus plugs in lung tissue， upregulated MUC5AC protein expression and downregulated AQP5 protein expression （P<0.05）， decreased relative abundance of beneficial gut microbiota （Firmicutes， Clostridia， Clostridiales， Lactobacillaceae， and Lactobacillus）， and increased abundance of harmful microbiota （Bacteroidetes， Bacteroidia， Bacteroidales， Muribaculaceae， and Muribaculum）. In addition， the model group presented reduced fecal SCFA levels （acetate， propionate， and butyrate） （P<0.05）. After the intervention of PRP decoction， compared to the model group， all drug administration groups showed decreased RI （P<0.05）， increased phenol red excretion， declined eosinophil and neutrophil counts and IL-6， IL-8， IgE， and IL-4 levels （P<0.05）， and improved IFN-γ levels （P<0.05） and lung pathology improved. The MUC5AC protein expression decreased （P<0.05）， and the AQP5 protein expression increased （P<0.05）. The disorder of gut microbiota was improved， and the diversity of gut microbiota was restored， with a significantly increased relative abundance ratio of beneficial microbiota （P<0.05） and a significantly reduced relative abundance ratio of harmful microbiota （P<0.05）. The SCFA levels （acetate， propionate， and butyrate） increased （P<0.05）. The efficacy indicators of serum inflammatory factors （IgE， IL-4， and IFN-γ）， phlegm-resolving effect， airway resistance， total pathological score， and the protein expression of MUC5AC and AQP5 were correlated with gut microbiota and SCFAs. ConclusionPRP decoction alleviates cold-phlegm syndrome by modulating the gut-lung axis， promoting beneficial gut microbiota， enhancing SCFA production， restoring the balance of gut microbiota， and suppressing respiratory inflammation. This study provides novel insights into the TCM theory of interconnection between the lung and large intestine.

ObjectiveTo investigate the effects of Pinelliae Rhizoma Praeparatum （PRP） on lung tissue， gut microbiota， and short-chain fatty acid （SCFA） metabolism in a model of mice with cold fluid retention in the lung and explore its mechanism of action in resolving dampness and phlegm based on the interconnection between the lung and large intestine. MethodsFifty female ICR mice were randomly divided into a normal group， model group， positive control group （Xiaoqinglong granules， 6.5 g·kg^-1）， and high-dose and low-dose PRP decoction groups （3.0， 1.5 g·kg^-1）， with 10 mice in each group. A model of mice with cold fluid retention in the lung was established using ovalbumin （OVA） sensitization combined with cold-water immersion. Drug interventions were conducted from day 18 to day 33 for 15 consecutive days. The airway resistance value of the mice was measured using a non-invasive pulmonary function analyzer. Phlegm-resolving effects were evaluated via a microplate reader. Eosinophil and neutrophil counts in bronchoalveolar lavage fluid （BALF） were analyzed using an automated hematology analyzer. Serum levels of total immunoglobulin E （IgE）， interferon-γ （IFN-γ）， interleukin-4 （IL-4）， and BALF levels of interleukin-6 （IL-6） and interleukin-8 （IL-8） were quantified by enzyme-linked immunosorbent assay （ELISA）. Lung histopathology was assessed using hematoxylin-eosin （HE） staining. Immunohistochemistry （IHC） was employed to detect mucin 5AC （MUC5AC） and aquaporin 5 （AQP5） protein expression in lung tissue. Gut microbiota composition was analyzed via agarose gel electrophoresis， and fecal SCFA levels were measured by gas chromatography-mass spectrometry （GC-MS）. ResultsCompared with the normal group， the model group exhibited significantly increased airway resistance value （RI） （P<0.05）， elevated eosinophil and neutrophil counts and IL-6 and IL-8 levels in BALF （P<0.05）， increased serum IgE and IL-4 levels （P<0.05）， with reduced IFN-γ levels （P<0.05）. It also showed thickened bronchial walls， widened alveolar septa， narrowed lumens， and mucus plugs in lung tissue， upregulated MUC5AC protein expression and downregulated AQP5 protein expression （P<0.05）， decreased relative abundance of beneficial gut microbiota （Firmicutes， Clostridia， Clostridiales， Lactobacillaceae， and Lactobacillus）， and increased abundance of harmful microbiota （Bacteroidetes， Bacteroidia， Bacteroidales， Muribaculaceae， and Muribaculum）. In addition， the model group presented reduced fecal SCFA levels （acetate， propionate， and butyrate） （P<0.05）. After the intervention of PRP decoction， compared to the model group， all drug administration groups showed decreased RI （P<0.05）， increased phenol red excretion， declined eosinophil and neutrophil counts and IL-6， IL-8， IgE， and IL-4 levels （P<0.05）， and improved IFN-γ levels （P<0.05） and lung pathology improved. The MUC5AC protein expression decreased （P<0.05）， and the AQP5 protein expression increased （P<0.05）. The disorder of gut microbiota was improved， and the diversity of gut microbiota was restored， with a significantly increased relative abundance ratio of beneficial microbiota （P<0.05） and a significantly reduced relative abundance ratio of harmful microbiota （P<0.05）. The SCFA levels （acetate， propionate， and butyrate） increased （P<0.05）. The efficacy indicators of serum inflammatory factors （IgE， IL-4， and IFN-γ）， phlegm-resolving effect， airway resistance， total pathological score， and the protein expression of MUC5AC and AQP5 were correlated with gut microbiota and SCFAs. ConclusionPRP decoction alleviates cold-phlegm syndrome by modulating the gut-lung axis， promoting beneficial gut microbiota， enhancing SCFA production， restoring the balance of gut microbiota， and suppressing respiratory inflammation. This study provides novel insights into the TCM theory of interconnection between the lung and large intestine.

ObjectiveTo investigate the potential machanism of Xiaozhong Zhitong Mixture （消肿止痛合剂， XZM） in the treatment of diabetes foot ulcer （DFU）. MethodsFifty SD rats were randomly divided into blank group， model group， XZM group， inhibitor group， XZM plus inhibitor group （combination group）， with 10 rats in each group. Except for the blank group， rats were fed with high-sugar， high-fat， high-cholesterol diet， intraperitoneally injected with streptozotocin， and subjected to skin defect to establish DFU model. After successful modeling， the XZM group and the combination group were given 1 ml/（100 g·d）of XZM by gavage， while the blank group， model group， and inhibitor group were all given an equal volume of 0.9% sodium chloride injection by gavage. Thirty minutes later， the inhibitor group and the combination group were intraperitoneally injected with 5 mg/（kg·d） of Notch1 inhibitor DAPT. All groups were treated once a day. After 14 days of administration， the skin tissue from the dorsal foot of the blank group rats and wound tissue from the other groups were collected. The pathological changes of granulation tissue in the wound were detected using hematoxylin eosin （HE） staining. The microvascular density （MVD） in wounds was detected through immunohistochemical staining. Real time fluorescence quantitative polymerase chain reaction （RT-PCR） and western blotting were used to detect the mRNA and protein levels of Notch1 homolog （Notch1）， Delta-like ligand 4 （Dll4）， Delta-like ligand 4 （VEGF）， and angiopoietin 2 （Ang-2）， respectively. ResultsHistological results showed that the epidermal structure in the dorsal foot skin tissue of the rats in the blank group was intact. In the wound tissue of the model group， the epidermis exhibited excessive keratinization， vacuolar cytoplasm， and a large number of inflammatory cells infiltrating the tissue， while in the XZM group， a large amount of scab formation was observed in the epidermis， with no significant inflammatory cell infiltration and a noticeable increase in fibroblasts. In the combination group and the inhibitor group， partial epidermal scab formation was observed in the wound tissue with a small amount of inflammatory cell infiltration. Compared to those in the blank group， the MVD in the wound tissue increased in the model group， as well as the mRNA expression and protein levels of Notch1 and Dll4， while VEGFA and Ang-2 mRNA expression and protein levels significantly decreased （P＜0.05 or P＜0.01）. Compared to those in the model group， the MVD in the wound tissue of all medication groups significantly increased， and the mRNA and protein levels of Notch1 and Dll4 decreased， while VEGFA and Ang-2 mRNA expression and protein levels increased （P＜0.05 or P＜0.01）. Compared to the XZM group， the inhibitor group and the combination group showed decreased MVD in wound tissue， increased Notch1 and Dll4 mRNA and protein levels， and decreased expression of VEGFA and Ang-2 mRNA and proteins （P＜0.05 or P＜0.01）. ConclusionXZM can effectively promote wound healing in DFU rats， and its mechanism of action may be related to the inhibition of Dll4/Notch1 signaling pathway in the wound tissue， therey promoting angiogenesis.

Objective To investigate the correlation between levels of intercellular adhesion molecule-1 (ICAM-1), platelet surface P-selectin (CD62P), and inflammatory factors and cerebral artery stenosis in patients with acute cerebral infarction (ACI). Methods A total of 305 patients with ACI complicated with cerebral artery stenosis admitted to Zhongwu Hospital of Suqian City and Xinyi People's Hospital from January 2021 to December 2023 were selected as the research subjects. According to the degree of cerebral artery stenosis, they were divided into grade I group (stenosis degree<50%, n=85), grade II group (stenosis degree of 50%-75%, n=128), and grade III group (stenosis degree>75%, n=92). Sixty-eight ACI patients without cerebral artery stenosis during the same period were included in the reference group. The levels of serum inflammatory factors [C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8)], ICAM-1 and CD62P were compared among the four groups. Spearman analysis was used to analyze the correlation between each factor and degree of cerebral artery stenosis. Results The levels of CRP, TNF-α, IL-6, IL-8, ICAM-1 and CD62P in the grade I, II and III groups were higher than those in the reference group. The levels of these factors were higher in the grade II and III groups than those in the grade I group, while the levels of various factors were higher in the grade III group than those in the grade II group (P<0.05). Spearman analysis showed that CRP, TNF-α, IL-6, IL-8, ICAM-1, and CD62P were positively correlated with the degree of cerebral artery stenosis in patients with ACI complicated with cerebral artery stenosis (P<0.05). Conclusion The levels of serum inflammatory factors, ICAM-1 and CD62P are significantly correlated with cerebral artery stenosis degree in patients with ACI.

Objective To investigate the correlation between levels of intercellular adhesion molecule-1 (ICAM-1), platelet surface P-selectin (CD62P), and inflammatory factors and cerebral artery stenosis in patients with acute cerebral infarction (ACI). Methods A total of 305 patients with ACI complicated with cerebral artery stenosis admitted to Zhongwu Hospital of Suqian City and Xinyi People's Hospital from January 2021 to December 2023 were selected as the research subjects. According to the degree of cerebral artery stenosis, they were divided into grade I group (stenosis degree<50%, n=85), grade II group (stenosis degree of 50%-75%, n=128), and grade III group (stenosis degree>75%, n=92). Sixty-eight ACI patients without cerebral artery stenosis during the same period were included in the reference group. The levels of serum inflammatory factors [C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8)], ICAM-1 and CD62P were compared among the four groups. Spearman analysis was used to analyze the correlation between each factor and degree of cerebral artery stenosis. Results The levels of CRP, TNF-α, IL-6, IL-8, ICAM-1 and CD62P in the grade I, II and III groups were higher than those in the reference group. The levels of these factors were higher in the grade II and III groups than those in the grade I group, while the levels of various factors were higher in the grade III group than those in the grade II group (P<0.05). Spearman analysis showed that CRP, TNF-α, IL-6, IL-8, ICAM-1, and CD62P were positively correlated with the degree of cerebral artery stenosis in patients with ACI complicated with cerebral artery stenosis (P<0.05). Conclusion The levels of serum inflammatory factors, ICAM-1 and CD62P are significantly correlated with cerebral artery stenosis degree in patients with ACI.