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 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.

Cervi Cornu Pantotrichum is a precious animal-derived Chinese medicinal material, while there are often adulterants derived from animals not specified in the Chinese Pharmacopeia in the market, which disturbs the safety of medication. This study was conducted with the aim of strengthening the quality control of Cervi Cornu Pantotrichum and standardizing the medication. To achieve digital identification of Cervi Cornu Pantotrichum from different sources, a digital identification model was constructed based on ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UHPLC-QTOF-MS~E) combined with an adaptive boosting algorithm(Adaboost). The young furred antlers of sika deer, red deer, elk, and reindeer were processed and then subjected to polypeptide analysis by UHPLC-QTOF-MS~E. Then, the mass spectral data reflecting the polypeptide information were obtained by digital quantification. Next, the key data were obtained by feature screening based on Gini index, and the digital identification model was constructed by Adaboost. The model was evaluated based on the recall rate, F_1 composite score, and accuracy. Finally, the results of identification based on the constructed digital identification model were validated. The results showed that when the Gini index was used to screen the data of top 100 characteristic polypeptides, the digital identification model based on Adaboost had the best performance, with the recall rate, F_1 composite score, and accuracy not less than 0.953. The validation analysis showed that the accuracy of the identification of the 10 batches of samples was as high as 100.0%. Therefore, based on UHPLC-QTOF-MS~E and Adaboost algorithm, the digital identification of Cervi Cornu Pantotrichum can be realized efficiently and accurately, which can provide reference for the quality control and original animal identification of Cervi Cornu Pantotrichum.
Animals ; Algorithms ; Antlers/chemistry* ; Boosting Machine Learning Algorithms ; Chromatography, High Pressure Liquid/methods* ; Deer ; Drugs, Chinese Herbal/chemistry* ; Mass Spectrometry/methods* ; Quality Control ; Reindeer ; Tandem Mass Spectrometry/methods* ; Tissue Extracts/analysis*

Objective To investigate the expression and role of the DNA repair and recombination protein RAD54-like(RAD54L)in oral squamous cell carcinoma(OSCC).Methods Using OSCC-related data from The Cancer Genome Atlas(TCGA)database,the difference in RAD54L expression between OSCC and control samples was analysed using the Mann-Whitney rank sum test,and the potential value of RAD54L mRNA in OSCC diagnosis was assessed using the receiver operator characteristic curve.The correlation between RAD54L expression levels and clinicopathological data of OSCC patients was analysed using the chi-square test.Once OSCC samples were divided into two groups of high and low expression based on the median value of RAD54L mRNA expression,Cox regression analysis was used to compare the prognostic differences between the two groups.The differentially expressed genes between the groups were subse-quently screened using the DESeq2 package,and KEGG pathway enrichment analysis was performed using the cluster-Profiler package.The correlation between RAD54L mRNA and gene expression in the homologous recombination repair pathway was demonstrated by Spearman correlation analysis.After clarifying the bioinformatics significance of RAD54L,RAD54L knockdown experiments were performed in human oral squamous carcinoma cell line HSC-3,and the knock-down efficiency was verified through real-time quantitative polymerase chain reaction.After transfection,the changes in proliferation,migration,apoptosis,and cycle of HSC-3 cells were assessed by CCK-8,5-ethynyl-2'-deoxyuridine(EdU)staining,wound healing,apoptosis,and cell cycle assays.Results Bioinformatics analysis showed that the expression of RAD54L mRNA was higher in OSCC than in normal controls(P＜0.001)and had a high value in predicting poor prognosis(AUC=0.927).The high RAD54L expression group was associated with an increased proportion of male pa-tients(P=0.032),having a higher T-stage(P=0.040),clinical stage(P=0.027),and pathological grading(P=0.013).Once OSCC samples were divided into two groups of high and low expression using the median value of RAD54L mRNA expression,the prognosis of the group with high expression of RAD54L was poorer than that of the group with low expression(P=0.049).The differentially expressed genes between the high and low RAD54L expression groups two groups were mainly enriched in neuroactive ligand-receptor interactions,cytokine-cytokine receptor interactions,calci-um signaling pathway,cell cycle,gastric cancer,extracellular matrix receptor interactions,chemical carcinogenesis-DNA adducts,DNA replication,homologous recombination,and mismatch repair pathways(P＜0.05).In the homolo-gous recombination repair pathway,the expression of RAD54L was positively correlated with the expression of BRCA1,BLM,EME1,XRCC2,POLD1,TOPBP1,RAD51,BRIP1,RAD54B,BRCA2,and SYCP3(P＜0.05),and was strongly positively correlated with the expression of BRCA1,BLM,and EME1(R＞0.8,P＜0.05).The results of in vitro experi-ments showed that RAD54L expression was knocked down to approximately 25％in HSC-3 cells(P＜0.001).Compared with the control group,the RAD54L knockdown group showed a lower proliferation rate(P＜0.05),a lower proportion of EdU-positive cells(P＜0.001),a lower proportion of wound closure(P＜0.001),a higher proportion of G1-phase cells(P＜0.001),a lower proportion of S-phase cells(P＜0.001),and a higher proportion of apoptotic cells(P＜0.001).Conclusion RAD54L is highly expressed in OSCC and correlates with poor prognosis.Down-regulation of RAD54L expression inhibits the proliferation and migration of HSC-3 cells,promotes apoptosis,and impedes cell cycle progres-sion.

RNA fluorescence aptamers are RNA sequences that can specifically bind to non-toxic,cell permeable,and self-fluorescent target molecules and activate their luminescent properties.These aptamers provide powerful tools for biosensing and imaging researches due to their simple structure,easy synthesis,and easy transfection.This article summarized the characteristics and development history of various RNA fluorescent aptamers,including Malachite Green,Spinach,Broccoli,Mango,Corn,and Pepper family,as well as their corresponding fluorescent groups.The applications of RNA fluorescent aptamers were also reviewed from two aspects:extracellular detection and cell imaging.This review might provide guidance for labeling,detection and interactions of molecules from proof of concept and clinical assessment to practical clinical and biomedical applications.

Professor ZHOU Peng has deeply discussed the pathological characteristics of psoriasis vulgaris,emphasizing that the disease is usually manifested deficiency interweaved with excess,leading to frequent recurrence and persistent refractory,which may lead to psychological and emotional problems of patients.This paper further expounds the effect of blood stasis on the pathogenesis,progression and prognosis of psoriasis,and puts forward a new method of combining Lingnan fire needling and filiform needling acupuncture technique to treat psoriasis vulgaris with blood stasis syndrome.Professor ZHOU Peng believes that the treatment principle of this disease is"regulating the mind first,rectifying blood as a base,syndrome differentiating and eliminating pathogenic factors",aiming at comprehensively considering the etiology and symptoms,in order to achieve more effective treatment results.Combined with the analysis of dermoscopic signs,it provides a possible improvement direction for the treatment of psoriasis vulgaris from a new perspective.

Objective To analyze the clinical efficacy and safety of glecaprevir/pibrentasvir in the treatment of pa-tients with hepatitis C virus(HCV)and human immunodeficiency virus(HIV)co-infection,and provide scientific basis for clinical treatment.Methods 89 initially treated non-cirrhotic patients with HCV/HIV co-infection in a hospital of Butuo County of Liangshan Prefecture from January 2021 to January 2022 were selected.All patients re-ceived glecaprevir/pibrentasvir treatment for 8 weeks and were followed up for 12 weeks.Virological response rate at the end-of-treatment and sustained virological response rate after 12 weeks(SVR12)of treatment as well as oc-currence of adverse reaction were recorded.Results Among 89 initially treated non-cirrhotic patients with HCV/HIV co-infection,most were middle-aged and young married men(n=79,88.8％).HIV was mainly transmitted through sexual contact(n=62,69.7％)and intravenous drug use(n=27,30.3％).The most common HCV geno-types were genotype 1b(n=33,37.1％)and genotype 3b(n=25,28.1％).All patients completed 8 weeks of treatment successfully and HCV RNA load at the end of treatment was below the detection limit(＜25 IU/mL).Eight patients failed to complete the follow-up,and the remaining 81(100％)patients achieved a sustained virologic re-sponse.There were no serious adverse reactions during the observation period,but 11 patients had mild adverse re-actions.Conclusion The 8-week treatment regimen of glecaprevir/pibrentasvir for non-cirrhotic patients with geno-type 1,3,and 6 HCV/HIV co-infection can achieve 100％SVR12,with high safety and tolerability,which can be used as a good choice for clinical treatment of these patients.

Objective To construct an artificial intelligence assisted diagnosis model based on deep learning for dynamically recognizing gastric lesions and their locations under gastroscopy in real time,and to evaluate its ability to detect and recognize gastric lesions and their locations.Methods The gastroscopy videos of 104 patients in our hospital was retrospectively analyzed,and the video frames were manually annotated.The annotated picture frames of lesion category were divided into the training set and the validation set according to the ratio of 8∶2,and the annotated picture frames of location category were divided into the training set and the validation set according to the patient sources at the ratio of 8∶2.These sets were utilized for training and validating the respective models.YoloV4 model was used for the training of lesion recognition,and ResNet152 model was used for the training of location recognition.The accuracy,sensitivity,specificity,positive predictive value,negative predictive value and location recognition accuracy of the auxiliary diagnostic model were evaluated.Results A total of 68 351 image frames were annotated,with 54 872 frames used as the training set,including 41 692 frames for lesion categories and 13 180 frames for location categories.The validation set consisted of 13 479 frames,comprising 10 422 frames for lesion categories and 3 057 frames for location categories.The lesion recognition model achieved an overall accuracy of 98.8%,with a sensitivity of 96.6%,specificity of 99.3%,positive predictive value of 96.3%,and negative predictive value of 99.3% in validation set.Meanwhile,the location recognition model demonstrated an top-5 accuracy of 87.1% .Conclusion The artificial intelligence assisted diagnosis model based on deep learning for real-time dynamic recognition of gastric lesions and their locations under gastroscopy has good ability in lesion detection and location recognition,and has great clinical application prospects.

OBJECTIVE: To observe the effect of Shugan Tiaoshen acupuncture (acupuncture for soothing the liver and regulating the mentality) combined with western medication on depression and sleep quality in the patients with depression-insomnia comorbidity due to COVID-19 quarantine, and investigate the potential mechanism from the perspective of cortical excitability. METHODS: Sixty patients with depression-insomnia comorbidity due to COVID-19 quarantine were randomly divided into an acupuncture group and a sham-acupuncture group, 30 cases in each one. The patients of both groups were treated with oral administration of sertraline hydrochloride tablets. In the acupuncture group, Shugan Tiaoshen acupuncture was supplemented. Body acupuncture was applied to Yintang (GV 24⁺), Baihui (GV 20), Hegu (LI 4), Zhaohai (KI 6), Qihai (CV 6), etc. The intradermal needling was used at Xin (CO₁₅), Gan (CO₁₂) and Shen (CO₁₀). In the sham-acupuncture group, the sham-acupuncture was given at the same points as the acupuncture group. The compensatory treatment was provided at the end of follow-up for the patients in the sham-acupuncture group. In both groups, the treatment was given once every two days, 3 times a week, for consecutive 8 weeks. The self-rating depression scale (SDS) and insomnia severity index (ISI) scores were compared between the two groups before and after treatment and 1 month after the end of treatment (follow-up) separately. The cortical excitability indexes (resting motor threshold [rMT], motor evoked potential amplitude [MEP-A], cortical resting period [CSP]) and the level of serum 5-hydroxytryptamine (5-HT) were measured before and after treatment in the two groups. RESULTS: After treatment and in follow-up, SDS and ISI scores were decreased in both groups compared with those before treatment (P<0.05), and the scores in the acupuncture group were lower than those in the sham-acupuncture group (P<0.05), and the decrease range in the acupuncture group after treatment was larger than that in the sham-acupuncture group (P<0.05). After treatment, rMT was reduced (P<0.05), while MEP-A and CSP were increased (P<0.05) in the acupuncture group compared with that before treatment. The levels of serum 5-HT in both groups were increased compared with those before treatment (P<0.05). The rMT in the acupuncture group was lower than that in the sham-acupuncture group, while MEP-A and CSP, as well as the level of serum 5-HT were higher in the acupuncture group in comparison with the sham-acupuncture group (P<0.05). CONCLUSION Shugan Tiaoshen acupuncture combined with western medication can relieve depression and improve sleep quality in the patients with depression-insomnia comorbidity due to COVID-19 quarantine, which is probably related to rectifying the imbalanced excitatory and inhibitory neuronal functions.
Humans ; Depression ; Quarantine ; Serotonin ; Sleep Initiation and Maintenance Disorders ; COVID-19 ; Acupuncture Therapy ; Comorbidity