Objective:To investigate the relationship between the BCL2 family protein BIM and ibrutinib resistance in chronic lymphocytic leukemia (CLL) and to analyze its regulatory mechanisms on apoptosis and autophagy.Methods:RNA sequencing (RNA-seq) was used to examine changes in the expression of BCL2 family proteins in samples from patients with CLL, MEC1 cell lines, and ibrutinib-resistant cell lines (MR). Western blot was used to analyze changes in BIM protein expression during apoptosis in MR. shRNA knockdown was used to assess the effects of BIM on cell proliferation and apoptosis. RNA-seq and the autophagy inhibitor chloroquine treatment were used to study autophagy-related changes in MR.Results:BIM expression was significantly downregulated before and after drug resistance in CLL primary cells and MEC1 cell lines ( P<0.0001). Knockdown of BIM in CLL cells inhibited ibrutinib-induced apoptosis and promoted cell proliferation ( P<0.05 for both). In addition, protective autophagy was increased in MR and apoptosis was increased after administration of chloroquine and small interfering RNA. The increased expression of LC3-Ⅱ protein in BIM-knockdown cell lines ( P<0.01) suggested that reduction of BIM may mediate autophagy activation. Conclusion:Downregulation of BIM may be a key factor in promoting ibrutinib resistance in CLL by activating protective autophagy. These findings provided a potential target for improving CLL treatment.

Objective To explore the effect and preliminary mechanism of the plant-derived immunostimulatory molecule,ophiopogonin,on enhancing the immune response of a subunit vaccine with the receptor-binding domain(RBD)of coronavirus spike protein as the antigen.Methods CCK-8 assay was used to determine the cytotoxicity of ophiopogonin D'(OPD')on bone marrow-derived dendritic cells(BMDCs).Female Balb/c mice were randomly divided into RBD,RBD/OPD',RBD/Alum,and control groups.The immunization dose was 5 μg of antigen per mouse and 100 μg of adjuvant per mouse,and immunization was carried out according to the intramuscular injection immunization procedure on days 0,21,and 42.The titers of specific IgG and its subtype antibodies were detected by ELISA.The cytokine levels in the supernatant of splenocytes were detected using ELISA.The number of splenocytes secreting IFN-γ was detected by ELISpot.Laser confocal microscopy was employed to observe the uptake of antigen by BMDCs.The phagocytic ability of BMDCs for antigen was quantitatively analyzed by flow cytometry.The mechanism of its enhanced immune effect was preliminarily explored using transcriptomics technology combined with bioinformatics research.Results When the concentration of OPD'was less than 5 μg/mL,the survival rate of BMDCs was 100%.After a single intramuscular injection in mice,except for a slight decrease in body weight,the other biochemical indicators were within corresponding normal ranges.After intramuscular injection immunization of the vaccine,the titers of serum-specific IgG,IgG1,and IgG2a in the RBD/OPD'group were significantly higher than those in the RBD group(P<0.05).Compared with the RBD group,the RBD/OPD'group induced a high-level Th1 cell immune response of IL-1β,TNF-α,and IFN-γ(P<0.01)and had more lymphocytes secreting IFN-γ(P<0.001).Laser confocal microscopy displayed that BMDCs took up more antigens after OPD'treatment,which was further confirmed with flow cytometry in quantitative analysis on antigen uptake rate(P<0.01).Transcriptomics results indicated that there was more significant enrichment of the PPAR signaling pathway in the RBD/OPD'group than the RBD group,suggesting that OPD'may activate the PPAR signaling pathway to exert its adjuvant effect.Conclusion OPD'effectively enhances the immune response of the RBD subunit vaccine,and its action mechanism may be related to the activation of the PPAR signaling pathway.

Objective To establish a mouse model of infection with the minimum lethal dose of Vibrio vulnificus(V.vulnificus)and to evaluate the protective efficacy of inactivated whole-cell(IWC)vaccine using this model.Methods A mouse model of lethal-dose infection was established by intraperitoneal injection of different doses of V.vulnificus.Bacterial colonization in the organs was detected with tissue homogenate plating,and pathological changes in the organs were observed after tissue section staining.Flow cytometry was used to detect immune cell responses after liver tissues were digested into single-cell suspension.IWC vaccine of V.vulnificus was prepared,and the mice were immunized through different routes to observe the protective efficacy of the vaccine.Results A mouse model of infection with the minimum lethal dose at 1×106 CFU of V.vulnificus was successfully established.After infection,the bacteria were mainly colonized in the liver of mice and caused severe pathological damages.Compared with the uninfected mice,the proportion of neutrophils in the liver was significantly increased in the infected mice,whereas the proportions of B cells and T cells were correspondingly decreased(P<0.05).A single intramuscular or intraperitoneal injection of the IWC vaccine could protect the mice effectively against lethal infection of V.vulnificus in 7 d later(P<0.01),although the level of serum IgG having no significant increase.Conclusion A mouse model of lethal-dose infection with V.vulnificus is successfully established,with histopathological characteristics.The IWC vaccine of V.vulnificus rapidly mediates immune protection in this model probably independent of IgG.

Objective To collect and analyze outpatient prescription data for exogenous cough treatment from a hospital in Shenzhen,and to identify the characteristics and medication patterns of traditional Chinese medicine(TCM).Methods This study collected prescriptions from a hospital in Shenzhen for exogenous cough treatment in January 2024.R language for data mining were used to analyze the medication frequency,clustering patterns,and association rules in the treatment of exogenous cough by TCM and to explore the medication patterns and the usage of classic formulas in TCM for this condition.Results A total of 451 outpatient prescriptions for exogenous cough were include,the top ten most frequently used herbs were Licorice,Bitter almond,Bellflower,Ephedra,Tangerine peel,Stemonae radix,Tuckahoe,Pinellia ternata,Nepeta,Bulb of thunberg fritillary.Cluster analysis and association rules revealed that San'ao decoction,Kikyodon soup,and Zhisou powder were commonly prescribed,targeting lung function,dispersing wind,and resolving phlegm to effectively alleviate cough symptoms.Significant differences in medication usage were observed across different syndrome types.For the wind cold attacking lung pattern,the core herbs were warm in nature and focused on dispersing with acrid-warm properties.Conversely,the treatment of wind heat attacking the lung pattern typically involved cold-natured herbs,with a primary focus on clearing and draining lung heat.Stratification by age revealed that the pediatric group often used drugs with mild properties,such as Stemona and Shegan.The young adult group tended to be prescribed cold-natured drugs like Forsythia and Hogfonnel Root.The middle-aged and elderly group preferred warming and tonifying drugs such as Japanese Catnip and Perilla frutescens.Conclusion The TCM treatment of exogenous cough primarily focuses on releasing the exterior and dispersing the lung.Due to the region's subtropical monsoon climate and temperature fluctuations physicians flexibly modified classical formulas such as San'ao decoction and Zhisou powder according to individual constitutions and symptom patterns.This targeted,syndrome-based approach effectively disperses the lung qi,stops cough and transforms phlegm,and alleviates cough.

Objective To collect and analyze outpatient prescription data for exogenous cough treatment from a hospital in Shenzhen,and to identify the characteristics and medication patterns of traditional Chinese medicine(TCM).Methods This study collected prescriptions from a hospital in Shenzhen for exogenous cough treatment in January 2024.R language for data mining were used to analyze the medication frequency,clustering patterns,and association rules in the treatment of exogenous cough by TCM and to explore the medication patterns and the usage of classic formulas in TCM for this condition.Results A total of 451 outpatient prescriptions for exogenous cough were include,the top ten most frequently used herbs were Licorice,Bitter almond,Bellflower,Ephedra,Tangerine peel,Stemonae radix,Tuckahoe,Pinellia ternata,Nepeta,Bulb of thunberg fritillary.Cluster analysis and association rules revealed that San'ao decoction,Kikyodon soup,and Zhisou powder were commonly prescribed,targeting lung function,dispersing wind,and resolving phlegm to effectively alleviate cough symptoms.Significant differences in medication usage were observed across different syndrome types.For the wind cold attacking lung pattern,the core herbs were warm in nature and focused on dispersing with acrid-warm properties.Conversely,the treatment of wind heat attacking the lung pattern typically involved cold-natured herbs,with a primary focus on clearing and draining lung heat.Stratification by age revealed that the pediatric group often used drugs with mild properties,such as Stemona and Shegan.The young adult group tended to be prescribed cold-natured drugs like Forsythia and Hogfonnel Root.The middle-aged and elderly group preferred warming and tonifying drugs such as Japanese Catnip and Perilla frutescens.Conclusion The TCM treatment of exogenous cough primarily focuses on releasing the exterior and dispersing the lung.Due to the region's subtropical monsoon climate and temperature fluctuations physicians flexibly modified classical formulas such as San'ao decoction and Zhisou powder according to individual constitutions and symptom patterns.This targeted,syndrome-based approach effectively disperses the lung qi,stops cough and transforms phlegm,and alleviates cough.

Objective:To investigate the relationship between the BCL2 family protein BIM and ibrutinib resistance in chronic lymphocytic leukemia (CLL) and to analyze its regulatory mechanisms on apoptosis and autophagy.Methods:RNA sequencing (RNA-seq) was used to examine changes in the expression of BCL2 family proteins in samples from patients with CLL, MEC1 cell lines, and ibrutinib-resistant cell lines (MR). Western blot was used to analyze changes in BIM protein expression during apoptosis in MR. shRNA knockdown was used to assess the effects of BIM on cell proliferation and apoptosis. RNA-seq and the autophagy inhibitor chloroquine treatment were used to study autophagy-related changes in MR.Results:BIM expression was significantly downregulated before and after drug resistance in CLL primary cells and MEC1 cell lines ( P<0.0001). Knockdown of BIM in CLL cells inhibited ibrutinib-induced apoptosis and promoted cell proliferation ( P<0.05 for both). In addition, protective autophagy was increased in MR and apoptosis was increased after administration of chloroquine and small interfering RNA. The increased expression of LC3-Ⅱ protein in BIM-knockdown cell lines ( P<0.01) suggested that reduction of BIM may mediate autophagy activation. Conclusion:Downregulation of BIM may be a key factor in promoting ibrutinib resistance in CLL by activating protective autophagy. These findings provided a potential target for improving CLL treatment.