OBJECTIVE: Patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection frequently develop central nervous system damage, yet the mechanisms driving this pathology remain unclear. This study investigated the primary pathways and key factors underlying brain tissue damage induced by the SARS-CoV-2 beta variant (lineage B.1.351). METHODS: K18-hACE2 and C57BL/6 mice were intranasally infected with the SARS-CoV-2 beta variant. Viral replication, pathological phenotypes, and brain transcriptomes were analyzed. Gene Ontology (GO) analysis was performed to identify altered pathways. Expression changes of host genes were verified using reverse transcription-quantitative polymerase chain reaction and Western blot. RESULTS: Pathological alterations were observed in the lungs of both mouse strains. However, only K18-hACE2 mice exhibited elevated viral RNA loads and infectious titers in the brain at 3 days post-infection, accompanied by neuropathological injury and weight loss. GO analysis of infected K18-hACE2 brain tissue revealed significant dysregulation of genes associated with innate immunity and antiviral defense responses, including type I interferons, pro-inflammatory cytokines, Toll-like receptor signaling components, and interferon-stimulated genes. Neuroinflammation was evident, alongside activation of apoptotic and pyroptotic pathways. Furthermore, altered neural cell marker expression suggested viral-induced neuroglial activation, resulting in caspase 4 and lipocalin 2 release and disruption of neuronal molecular networks. CONCLUSION These findings elucidate mechanisms of neuropathogenicity associated with the SARS-CoV-2 beta variant and highlight therapeutic targets to mitigate COVID-19-related neurological dysfunction.
Animals ; COVID-19/genetics* ; Mice ; Brain/metabolism* ; Apoptosis ; Mice, Inbred C57BL ; SARS-CoV-2/physiology* ; Pyroptosis ; Gene Expression Profiling ; Transcriptome ; Male ; Female

The chemical components of Carthami Flos were investigated by using macroporous resin, silica gel column chromatography, reversed-phase octadecylsilane(ODS) column chromatography, Sephadex LH-20, and semi-preparative high-performance liquid chromatography(HPLC). The planar structures of the compounds were established based on their physicochemical properties and ultraviolet-visible(UV-Vis), infrared(IR), high-resolution electrospray ionization mass spectrometry(HR-ESI-MS), and nuclear magnetic resonance(NMR) spectroscopic technology. The absolute configurations were determined by comparing the calculated and experimental electronic circular dichroism(ECD). Six flavonoid C-glycosides were isolated from the 30% ethanol elution fraction of macroporous resin obtained from the 95% ethanol extract of Carthami Flos, and identified as saffloquinoside F(1), 5-hydroxysaffloneoside(2), iso-5-hydroxysaffloneoside(3), isosafflomin C(4), safflomin C(5), and vicenin 2(6). Among these, the compounds 1 to 3 were new chalcone C-glycosides. The compounds 1, 2, 4, and 5 could significantly increase the viability of H9c2 cardiomyocytes damaged by oxygen-glucose deprivation/reoxygenation(OGD/R) at a concentration of 50 μmol·L~(-1), showing their good cardioprotective activity.
Glycosides/pharmacology* ; Flowers/chemistry* ; Drugs, Chinese Herbal/pharmacology* ; Carthamus tinctorius/chemistry* ; Chalcones/pharmacology* ; Animals

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.

Objective To develop and validate a fast Monte Carlo(MC)-based patient-specific quality assurance(PSQA)tool using the treatment log files that is suitable to be used in the online adaptive radiotherapy for pencil beam scanning proton therapy(PBSPT-ART).Methods The proposed tool first used the delivery log file of a PBSPT plan to reversely reconstruct the PBSPT(rPBSPT)plan,and then used an in-house developed graphic processing unit(GPU)-accelerated virtual particle MC(VPMC)dose engine to calculate the dose distribution of the rPBSPT plan.The rPBSPT dose calculated by VPMC was then compared to the rPBSPT dose calculated by another independent MC dose engine(MCsquare),using 3D gamma analysis to verify the accuracy of VPMC calculation.As a demonstration of the feasibility of developed log file-based PSQA,the VPMC calculated dose of the rPBSPT plan was compared to the pre-delivery second check dose of the corresponding PBSPT plan calculated by MCsquare,using 3D gamma analysis.3D gamma analysis employes a criterion of 2 mm/2%/10%.Twenty patients with different disease sites were representatively selected to validate the efficiency and accuracy of the tool.Results The average calculation time of a rPBSPT plan by VPMC was(5.88±4.00)s in the accuracy verification.Compared to MCsquare,the passing rate of the 3D gamma analysis was 99.47%±0.72%.In the proposed PSQA tool demonstration,the passing rate of comparing the VPMC calculated rPBSPT dose to MCsquare calculated second check dose of the corresponding PBSPT plan was 98.91%±0.92%.Conclusion The accuracy and efficiency of the tool can meet the requirements of PSQA in the online PBSPT-ART workflow.

Objective To develop and validate a fast Monte Carlo(MC)-based patient-specific quality assurance(PSQA)tool using the treatment log files that is suitable to be used in the online adaptive radiotherapy for pencil beam scanning proton therapy(PBSPT-ART).Methods The proposed tool first used the delivery log file of a PBSPT plan to reversely reconstruct the PBSPT(rPBSPT)plan,and then used an in-house developed graphic processing unit(GPU)-accelerated virtual particle MC(VPMC)dose engine to calculate the dose distribution of the rPBSPT plan.The rPBSPT dose calculated by VPMC was then compared to the rPBSPT dose calculated by another independent MC dose engine(MCsquare),using 3D gamma analysis to verify the accuracy of VPMC calculation.As a demonstration of the feasibility of developed log file-based PSQA,the VPMC calculated dose of the rPBSPT plan was compared to the pre-delivery second check dose of the corresponding PBSPT plan calculated by MCsquare,using 3D gamma analysis.3D gamma analysis employes a criterion of 2 mm/2%/10%.Twenty patients with different disease sites were representatively selected to validate the efficiency and accuracy of the tool.Results The average calculation time of a rPBSPT plan by VPMC was(5.88±4.00)s in the accuracy verification.Compared to MCsquare,the passing rate of the 3D gamma analysis was 99.47%±0.72%.In the proposed PSQA tool demonstration,the passing rate of comparing the VPMC calculated rPBSPT dose to MCsquare calculated second check dose of the corresponding PBSPT plan was 98.91%±0.92%.Conclusion The accuracy and efficiency of the tool can meet the requirements of PSQA in the online PBSPT-ART workflow.

Objective:To explore the correlation between ASXL1 gene mutation characteristics and clinical manifestations and prognosis in patients with myelodysplastic syndrome(MDS).Methods:The clinical date of 264 patients with MDS in Xuzhou Central Hospital,Southeast University from August 2010 to April 2024 was retrospectively analyzed.The patients were divided into ASXL1wt group and ASXL1mut group according to the presence of ASXL1 gene mutation,and the correlation between gene mutation characteristics and clinical manifestations and prognosis was analyzed.Results:Compared with ASXL1wt group,the ASXL1mut group had a higher age of onset(P＜0.05),a higher proportion of males(P＜0.05),while the incidence of del(5q)was lower(P＜0.01).The mutation frequency of ASXL1 in MDS patients was 21.97％,and most of them were frameshift mutations.The p.Gly646fs was the most common amino acid variant,with a mutation frequency of 20.69％.The median overall survival(OS)and leukemia-free survival of patients with this sequence variant was 18.1 and 23.8 months,respectively,while in those without this sequence variant was 30 months and not reached,and the differences were statistically significant(P＜0.05).The results of multivariate analysis showed that the mutation of NRAS,WT1,KIT gene and the p.Gly646fs sequence mutation of ASXL1 gene were independent prognostic factors for OS in ASXL1mut patients.The median OS of ASXL1wt and ASXL1mut patients was 27.9(21.3-40.4)and 23.7(18.6-NA)months,respectively(P＞0.05).Among 58 ASXL1mut patients,5 cases(8.6％)transformed to acute leukemia,including 3 cases with RUNX1 mutation and 3 cases with TET2 mutation.Among 206 ASXL1wt patients,28 cases(13.6％)transformed to acute leukemia.The difference in leukemia transformation rate between the two groups was not statistically significant(P＞0.05).The efficacy of different treatment regimens was similar in the ASXL1mut group,while in the ASXL1wt group,patients receiving allogeneic hematopoietic stem cell transplantation had a significantly better prognosis than those receiving other treatment regimens(P＜0.001).The overall response rate to demethylation therapy was 68.7％and 67.6％in ASXL1mut and ASXL1wt group,respectively,and the difference between the two groups was not significant(P＞0.05).Conclusion:The overall survival of MDS patients with ASXL1mut is poor.The patients with p.Gly646fs sequence mutation have a higher proportion of bone marrow blasts and a worse prognosis.There are no statistical differences in efficacy of different treatment strategies in ASXL1mut group.ASXL1 mutation shows no significant effect on the response of MDS to hypomethylating agent therapy.

Objective:To explore the correlation between ASXL1 gene mutation characteristics and clinical manifestations and prognosis in patients with myelodysplastic syndrome(MDS).Methods:The clinical date of 264 patients with MDS in Xuzhou Central Hospital,Southeast University from August 2010 to April 2024 was retrospectively analyzed.The patients were divided into ASXL1wt group and ASXL1mut group according to the presence of ASXL1 gene mutation,and the correlation between gene mutation characteristics and clinical manifestations and prognosis was analyzed.Results:Compared with ASXL1wt group,the ASXL1mut group had a higher age of onset(P＜0.05),a higher proportion of males(P＜0.05),while the incidence of del(5q)was lower(P＜0.01).The mutation frequency of ASXL1 in MDS patients was 21.97％,and most of them were frameshift mutations.The p.Gly646fs was the most common amino acid variant,with a mutation frequency of 20.69％.The median overall survival(OS)and leukemia-free survival of patients with this sequence variant was 18.1 and 23.8 months,respectively,while in those without this sequence variant was 30 months and not reached,and the differences were statistically significant(P＜0.05).The results of multivariate analysis showed that the mutation of NRAS,WT1,KIT gene and the p.Gly646fs sequence mutation of ASXL1 gene were independent prognostic factors for OS in ASXL1mut patients.The median OS of ASXL1wt and ASXL1mut patients was 27.9(21.3-40.4)and 23.7(18.6-NA)months,respectively(P＞0.05).Among 58 ASXL1mut patients,5 cases(8.6％)transformed to acute leukemia,including 3 cases with RUNX1 mutation and 3 cases with TET2 mutation.Among 206 ASXL1wt patients,28 cases(13.6％)transformed to acute leukemia.The difference in leukemia transformation rate between the two groups was not statistically significant(P＞0.05).The efficacy of different treatment regimens was similar in the ASXL1mut group,while in the ASXL1wt group,patients receiving allogeneic hematopoietic stem cell transplantation had a significantly better prognosis than those receiving other treatment regimens(P＜0.001).The overall response rate to demethylation therapy was 68.7％and 67.6％in ASXL1mut and ASXL1wt group,respectively,and the difference between the two groups was not significant(P＞0.05).Conclusion:The overall survival of MDS patients with ASXL1mut is poor.The patients with p.Gly646fs sequence mutation have a higher proportion of bone marrow blasts and a worse prognosis.There are no statistical differences in efficacy of different treatment strategies in ASXL1mut group.ASXL1 mutation shows no significant effect on the response of MDS to hypomethylating agent therapy.

Objective To investigate the population characteristics,distribution of traditional Chinese medicine(TCM)syndromes and influencing factors of long-term prognosis of diarrhea-predominant irritable bowel syndrome(IBS-D),and to provide evidence for the formulation of intervention program for IBS-D patients.Methods A total of 124 patients with IBS-D admitted to the medical institutions of the project team members from July 2020 to August 2022 were selected.According to the scoring results of IBS Quality of Life Measure(IBS-QOL),the patients were divided into the good prognosis group(81 cases)and the poor prognosis group(43 cases).The distribution of TCM syndromes in patients with IBS-D was explored,and the difference of IBS-QOL scores of the patients between good prognosis group and poor prognosis group was compared.Univariate logistic regression analysis and multivariate logistic regression analysis were used to determine the main risk factors for poor prognosis in patients with IBS-D.Results(1)The analysis of population characteristics showed that there was no significant difference in the proportion of male and female patients with IBS-D.The patients with IBS-D were usually middle-aged,and had a large interval span of the course of disease.The severity of their symptoms was mostly moderate.All of the patients with IBS-D had various degrees of anxiety and depression,and had nutritional imbalance.(2)The distribution of TCM syndromes in the patients with IBS-D were shown as the following:78 cases were identified as liver depression and spleen deficiency type,accounting for 62.90％;26 cases were identified as spleen-qi deficiency type,accounting for 20.97％;20 cases were identified as spleen and kidney yang deficiency type,accounting for 16.13％.(3)Analysis of IBS-QOL score showed that compared with the good prognosis group,the items scores of negative emotion,physical function,behavioral disorder,health status,being fastidious about food,social function,sexual behavior and interpersonal relationship of IBS-QOL in the poor prognosis group were significantly lowered(P＜0.01).(4)The univariate analysis showed that the risk of poor prognosis in patients with IBS-D would be increased by the factors of age,education level,course of disease,severity of symptoms,anxiety state,depression state,TCM syndrome types,Acute Physiology and Chronic Health Evaluation scoring system Ⅱ(APACHE 11)score,complication of neurological diseases,hemoglobin level,albumin level and total protein level(P＜0.01).(5)The multivariate Logistic regression analysis showed that the risk factors for poor prognosis of IBS-D patients involved age,education level below junior high school,the severity of symptoms being severe,Self-Rating Anxiety Scale(SAS)score,Self-Rating Depression Scale(SDS)score,TCM syndrome being liver depression and spleen deficiency type,hemoglobin level,albumin level and total protein level(P＜0.01).Conclusion Most of IBS-D patients exert long-term poor prognosis,and their long-term prognosis is affected by the factors of age,education level,severity of symptoms,anxiety and depression state,nutritional imbalance and TCM syndrome being liver depression and spleen deficiency type.The identification of the risk factors of poor prognosis will provide evidence for the formulation and adjustment of clinical intervention programs.