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 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 explore host factors interacting with influenza virus nucleoprotein （NP） and study their effects on influenza virus replication， as well as the mechanism of gardenia jasminoides iridoid glycoside （IGE） in inhibiting influenza virus. MethodA yeast two-hybrid system was utilized to screen host factors that interacted with influenza virus NP. Heterogeneous nuclear ribonucleoprotein D0 （HNRNPD）， glucosamine-6-phosphate deaminase 1 （GNPDA1）， poly（rC）-binding protein 1 （PCBP1）， and protein inhibitor of activated signal transducer and activator of transcription （STAT） protein 1 （PIAS1） were validated by immunoprecipitation assay. The effects of PIAS1 and HNRNPD on influenza virus replication were compared by a dual luciferase assay， and the effects of IGE on influenza virus replication were examined in the presence of transfected ribonucleoprotein （RNP） and knockdown of PIAS1. ICR mice were randomly divided into a normal group， model group， oseltamivir phosphate group， and high， medium， and low dose IGE groups， with 10 mice in each group. In addition to the normal group， each group was infected with the influenza A virus FM1 strain by nasal drip to establish a viral pneumonia model. The high， medium， and low dose IGE groups were given drugs of 50， 25， and 12.5 mg∙kg^-1 by gavage， and the oseltamivir phosphate group was given the drug of 27.5 mg∙kg^-1 by gavage. Equal amounts of distilled water were instilled in the normal and model groups for four consecutive days. Later， protein expression of PIAS1， NP， phosphorylated （p）-STAT3， STAT3， p-STAT1， and STAT1 were detected in the lung tissue by Western blot. ResultIn yeast two-hybrid assays， 16 potential host targets interacting with influenza virus NP were identified. Immunoprecipitation experiments revealed that HNRNPD and PIAS1 could interact with influenza virus NP. The dual luciferase reporter assays found that both PIAS1 knockdown and overexpression significantly affected IAV RNP activity （P<0.05， P<0.01）， and the effect of HNRNPD on IAV RNP was not significant. Both high and low dose IGE groups reduced influenza virus replication （P<0.05） and reversed the increase in influenza virus replication caused by the knockdown of PIAS1（P<0.05， P<0.01）. The expressions of PIAS1， NP， p-STAT3， p-STAT1， and STAT1 in the lung tissue of infected mice were reduced to different degrees in each IGE group （P<0.05， P<0.01）. ConclusionPIAS1 interacts with influenza virus NP and is able to inhibit influenza virus replication. IGE may exert antiviral effects by inhibiting the activity of IAV RNP through the PIAS1/STAT1 pathway.

In this study,nine hybridoma cells secreting monoclonal antibodies against deltamethrin were prepared,and the monoclonal antibody 4D4E11 with best sensitivity was selected to develop indirect enzyme-linked immunosorbent assay (ic-ELISA) and gold nanoparticle-based lateral flow immunoassay (LFIA) for detection of deltamethrin. The optimal working buffer for ic-ELISA was 0.01 mol/L phosphate buffer (pH 7.4) containing 0.2 mol/L NaCl and 20％ methanol,while 0.01 mol/L phosphate buffer (pH 7.4) containing 1 mol/L NaCl,5‰Tween-20 and 10％methanol for LFIA. Under the optimal conditions,the half inhibition concentration (IC50) and limit of detection (IC10) of ic-ELISA were 10.60 ng/mL and 1.43 ng/mL respectively,and the limit of detection of the developed LFIA was 0.5μg/mL. The developed ic-ELISA and LFIA showed no cross-reactivities (CRs) with eight kinds of analogues of deltamethrin,which indicated the excellent specificity of proposed immunoassays. The average recoveries of the ic-ELISA in spiked tomato,cabbage and lettuce samples were 79.8％-92.6％with relative standard deviations of 0.8％-5.5％. The detection results of LFIA were consistent with the spiked concentrations in the range of 1-5 mg/kg. Meanwhile,the results of ic-ELISA and LFIA showed close correlation with high performance liquid chromatography (HPLC) in the test of blind lettuce samples. The experimental results demonstrated that the two immunoassays proposed here were suitable for rapid detection of deltamethrin with high sensitivity and high accuracy.

Objective To investigate the risk factors for bronchopulmonary dysplasia(BPD)in twin preterm infants with a gestational age of<34 weeks,and to provide a basis for early identification of BPD in twin preterm infants in clinical practice.Methods A retrospective analysis was performed for the twin preterm infants with a gestational age of<34 weeks who were admitted to 22 hospitals nationwide from January 2018 to December 2020.According to their conditions,they were divided into group A(both twins had BPD),group B(only one twin had BPD),and group C(neither twin had BPD).The risk factors for BPD in twin preterm infants were analyzed.Further analysis was conducted on group B to investigate the postnatal risk factors for BPD within twins.Results A total of 904 pairs of twins with a gestational age of<34 weeks were included in this study.The multivariate logistic regression analysis showed that compared with group C,birth weight discordance of>25%between the twins was an independent risk factor for BPD in one of the twins(OR=3.370,95%CI:1.500-7.568,P<0.05),and high gestational age at birth was a protective factor against BPD(P<0.05).The conditional logistic regression analysis of group B showed that small-for-gestational-age(SGA)birth was an independent risk factor for BPD in individual twins(OR=5.017,95%CI:1.040-24.190,P<0.05).Conclusions The development of BPD in twin preterm infants is associated with gestational age,birth weight discordance between the twins,and SGA birth.

AIM:To evaluate the efficacy,safety,and economy of camrelizumab(CAM)combined with platinum-containing chemotherapy(CT)for the first-line treatment of locally advanced/meta-static non-small cell lung cancer(NSCLC).METH-ODS:Chinese and English databases such as Pubmed,the Cochrane Library,China Knowledge Network,Wanfang Data,and other related web-sites were systematically searched.After literature screening,quality assessment,and data extraction of the literature according to the inclusion and ex-clusion criteria,two researchers conducted a rapid health technology assessment(HTA).RESULTS:A total of 7 systematic evaluations/Meta-analyses and 17 economics evaluations were included.In terms of effectiveness,compared to docetaxel che-motherapy,CAM+CT significantly prolonged the overall survival(OS),progression-free survival(PFS),and improved the objective remission rate(ORR)of mutation-negative patients with locally ad-vanced/metastatic NSCLC.Compared with CT and pembrolizumab(PEM),CAM+CT significantly pro-longed the PFS,and improved the ORR of mutation-negative patients with locally advanced/metastatic NSCLC.Subgroup analysis showed that CAM+CT significantly prolonged PFS in patients with PD-L1 ≥1％and PD-L1 ≥ 50％compared with CT.Compared with CT,CAM+CT significantly prolonged the OS and PFS of mutation-negative patients with locally advanced/metastatic squamous NSCLC.Compared with sintilimab(SIN),CAM+CT significantly pro-longed the PFS of mutation-negative patients with locally advanced/metastatic squamous NSCLC.Sub-group analysis showed that CAM+CT significantly prolonged OS in patients with PD-L1＜1％com-pared with CT.In terms of safety,CAM+CT was comparable in terms of the occurrence of all grades of adverse events,but the incidence of grade 3 or higher treatment-related adverse events was significantly increased compared with CT and PEM for mutation-negative locally advanced/meta-static NSCLC patients.CAM+CT was significantly in-creased the occurrence of all grades of adverse events compared with CT,but was comparable in terms of the occurrence of grade 3 or higher treat-ment-related adverse events.In terms of economy,CAM+CT has a cost-effectiveness advantage over CT for patients with mutation-negative advanced/metastatic squamous NSCLC.CAM+CT has a cost-effectiveness advantage over CT and PEM+CT;and CAM+CT does not have a cost-effectiveness ad-vantage over SIN+CT for patients with mutation-negative locally advanced/metastatic non-squa-mous NSCLC.CONCLUSION:CAM+CT has good ef-ficacy and cost-effectiveness for the first-line treat-ment of locally advanced/metastatic NSCLC,and the safety aspect is compared with CT,PEM or slightly worse.

Objective To construct SpyCatcher-mi3 nanoparticle vaccine delivery vectors,evaluate their role in enhancing the immunogenicity of the ovalbumin CD8+T-cell epitope peptide,OVA257-264,and determine its protective effect in a model which mice were immunized and subcutaneously challenged with E.G7-OVA tumor cells.Methods SpyCatcher-mi3 proteins were expressed by E.coli and purified by affinity chromatography and anion exchange chromatography sequentially.OVA257-264-SpyTag peptide was obtained by synthesis.The OVA257-264-mi3 nanoparticles were produced by the SpyTag/SpyCatcher system.The toxicity of OVA257-264-mi3 was evaluated using hemolysis assay,CCK-8 assay and mouse experiment.A total of 42 female SPF-grade C57BL/6 mice(6～8 weeks old,18～20 g)were randomly divided into OVA257-264-mi3,OVA257-264,and control groups,with 14 mice in each group.Then the mice in each group were immunized on days 0,14 and 28.In 14 d after the last immunization,the amounts of spot-forming cells(SFCs,indicating IFN-γ secreting cells in splenic lymphocytes)were determined using ELISpot assay to evaluate their immunogenicity.After the immunized mice were subcutaneously implanted with E.G7-OVA tumor cells,the antitumor effect of the vaccine in prophylactic xenograft tumor model was evaluate by observing tumor volumes with a caliper and tumor growth with MRI.Results Both SpyCatcher-mi3 and OVA257-264-mi3 could be self-assembled to form homogeneous and stable nanoparticles,with an average particle size of about 43.8 and 91.3 nm,respectively.The OVA257-264-mi3 was safe for in vitro and in vivo toxicity evaluation.The number of IFN--y secreting cells per 1 × 106 splenic lymphocytes reached 253 in the OVA257-264-mi3 group of mice,significantly higher than that in the OVA257-264 group and the Control group(P＜0.05).The tumor volume of mice in the OVA257-264-mi3 group was about 151.1 mm3 on day 22,which was significantly smaller than that of the OVA257-264 group and the Control group(P＜0.05),and the survival rate during the observation period reached 60％,which was significantly higher than that of the OVA257-264 groups(P＜0.05).Conclusion Nanoparticle vaccine OVA257-264-mi3 is successfully constructed,and it shows enhancing effect on the immunogenicity of the antigen epitope peptide,and exerts protective effect on prophylactic xenograft tumor model,providing a theoretical basis for the research of tumor neoantigen vaccines.

Objective To develop a matrix metalloproteinase(MMP)-responsive hyaluronic acid(HA)-based controlled-release material for brain-derived neurotrophic factor(BDNF)to provide a novel therapeutic strategy for intervention and repair of traumatic brain injury(TBI).Methods HA was modified with amination,followed by condensation with Suflo-SMCC carboxyl group to form amide,and then linked with glutathione(GSH)to synthesize HA-GSH.The recombinant glutathione S-transferase(GST)-tissue inhibitor of metalloproteinase(TIMP)-BDNF(GST-TIMP-BDNF)expression plasmid was constructed using molecular cloning technique with double enzyme digestion by Bam H Ⅰ and Eco R Ⅰ.The recombinant GST-TIMP-BDNF protein was expressed in the Escherichia coli prokaryotic expression system,and purified by ion exchange chromatography,confirmed by Western blotting.MMP diluents were supplemented with PBS,MMP inhibitor marimastat,and varing concentrations(0.4,0.6,0.8 mg/ml)of GST-TIMP-BDNF or GST-BDNF.MMP-2 activity was analyzed using an MMP activity detection kit to evaluate the inhibitory effect of the recombinant protein on MMP.Primary rat neurons were extracted and cultured to establish an iron death model induced by RSL3.The effect of recombinant protein GST-TIMP-BDNF on neuronal injury was detected by immunofluorescence staining.Results MRI hydrogen spectrum identification confirmed the successful synthesis of HA-GSH.Western blotting results showed the successful expression of the recombinant protein GST-TIMP-BDNF containing the GST tag using the E.coli prokaryotic expression system.MMP activity detection results indicated that the recombinant protein GST-TIMP-BDNF had a superior inhibitory effect on MMP-2 activity compared to GST-BDNF(P<0.05).Immunofluorescence staining results showed a significant increase in fluorescence intensity in rat neurons treated with GST-TIMP-BDNF after RSL3 induction(P<0.05).Conclusion A MMP-responsive HA-based BDNF controlled-release material has been successfully developed,exhibiting a protective effect on neuron damage.

Objective To understand the current situation of human brain donation in Hebei Province by analyzing the basic information of Human Brain Bank samples of Hebei Medical University in order to provide basic data support for subsequent scientific research.Methods The samples collected from the Human Brain Bank of Hebei Medical University were analyzed(from December 2019 to February 2024),including gender,age,cause of death,as well as quality control data such as postmortem delay time,pH value of cerebrospinal fluid and and RNA integrity number and result of neuropathological diagnosis.Results Until February 2024,30 human brain samples were collected and stored in the Human Brain Bank of Hebei Medical University,with a male to female ratio of 9∶1.Donors over 70 years old accounted for 53％.Cardiovascular and cerebrovascular diseases(36.67％)and nervous system diseases(23.33％)accounted for a high proportion of the death causes.The location of brain tissue donors in Shijiazhuang accounted for 90％donations,and the others were from outside the city.The postmortem delay time was relatively short,90％within 12 hours and 10％more than 12 hours.69.23％of the brain samples had RNA integrity values greater than 6.Cerebrospinal fluid pH values ranged from 5.8 to 7.5,with an average value of 6.60±0.45.Brain weights ranged from 906-1496 g,with an average value of(1210.78±197.84)g.Three apolipoprotein E(APOE)alleles were detected including five genotypes(ε2/ε3,ε2/ε4,ε3/ε3,ε3/ε4,ε4/ε4).Eleven staining methods related to neuropathological diagnosis had been established and used.A total of 12 cases were diagnosed as neurodegenerative diseases(including Alzheimer's disease,Parkinson's disease,multiple system atrophy,corticobasal degeneration and progressive supranuclear palsy,etc.),accounting for 40％donated brains.The comorbidity rate of samples over 80 years old was 100％.Conclusion The summary and analyses of the data of brain donors in the Human Brain Bank of Hebei Medical University can reflect the current situation of the construction and operation of the brain bank in Hebei Province,and it can also be more targeted to understand and identify potential donors.Our information can provide reference for the construction of brain bank and provides more reliable materials and data support for scientific research.

UHPLC-Q-Exactive Orbitrap MS/MS was used to systematically analyze and compare the alkaloids in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Praeparata. After the samples were pretreated in the solid-phase extraction cartridges, 0.1% ammonium hydroxide(A)-acetonitrile(B) was used for gradient elution. The LC-MS method for characterization of alkaloids in the three herbal medicines was established in ESI positive ion mode to collect high resolution MS data of reference substances and samples. On the basis of the information of reference substance cracking behavior, retention time, accurate molecular mass, and related literature, a total of 155 alkaloids were identified in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Prae-parata. Specifically, 130, 127, and 92 alkaloids were identified in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Praeparata, respectively. Monoester alkaloids and amino-alcohol alkaloids were dominant in the three herbal medicines, and the alkaloids in Aconiti Kusnezoffii Radix and Aconiti Radix were similar. This paper can provide a reference for elucidating the pharmacological effects and clinical application differences of the three herbal medicines produced from plants of Aconitum.
Tandem Mass Spectrometry ; Aconitum ; Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal ; Alkaloids ; Plants, Medicinal