Objective To investigate the effect of Didang Decoction-medicated serum on autophagy in high glucose(HG)-induced rat glomerular endothelial cells(RGECs)and explore the pathway that mediates its effect.Methods Primary RGECs were isolated and cultured using sequential sieving combined with collagenase digestion,followed by identification using immunofluorescence assay for factor Ⅷ.High glucose medium was used to induce RGECs to simulate a diabetic environment,and the effects of Didang Decoction-medicated serum and 3-MA(an autophagy inhibitor),either alone or in combination,on autophagy of HG-exposed cells were evaluated by observing autophagic vacuoles using monodansylcadaverine(MDC)staining.RT-qPCR and Western blotting were employed to measure mRNA and protein expression levels of Beclin-1,p62,LC3B,p-PI3K,p-Akt,and p-mTOR.Results Compared with the control cells,the HG-exposed RGECs showed significantly reduced autophagic fluorescence intensity,decreased Beclin-1 mRNA expression,increased p62 mRNA expression,downregulated Beclin-1 protein and LC3-Ⅱ/Ⅰ ratio,and upregulated p62,p-PI3K,p-Akt,and p-mTOR protein levels.Didang Decoction-medicated serum significantly enhanced autophagic fluorescence intensity in HG-exposed cells,increased Beclin-1 mRNA expression,decreased p62 mRNA expression,upregulated Beclin-1 protein,and downregulated p62,p-PI3K,p-Akt,and p-mTOR protein levels.Conclusion Didang Decoction-medicated serum enhances autophagy in HG-exposed RGECs by regulating the PI3K/Akt/mTOR signaling pathway,which sheds light on a new therapeutic strategy for diabetic nephropathy.

Objective To investigate the value of gut metabolites in predicting the development of severe acute pancreatitis(SAP),myocardial injury,and adverse outcomes in patients with acute pancreatitis(AP).Methods A total of 80 SAP patients admitted to Cangzhou Hospital of Integrated Chinese and Western Medicine from April 2023 to April 2024 were selected as the severe group,and 80 non-severe AP patients were selected as the non-severe group.The levels of serum amylase,lipase,acetic acid,propionic acid,butyric acid,and total short-chain fatty acids(SCFAs)in feces,serum bile acid(BA),trimethylamine n-oxide(TMAO),myocardial injury-related indicators[creatine kinase isoenzymes(CK-MB),cardiac troponin T(cTnT),N-terminal pro-b-type natriuretic peptide(NT-proBNP)],C-reactive protein(CRP),acute physiology and chronic health evaluation II(APACHE II)and bedside index for severity in acute pancreatitis(BISAP)were compared between the two groups at admission.Patients in the severe group were followed up for 30 days and divided into a survival subgroup(n=61)and a non-survival subgroup(n=19)based on their prognosis.The levels of total SCFAs,BA,and TMAO were compared between these two subgroups.Spearman correlation analysis was used to analyze the correlation of serum amylase,lipase,total SCFAs,BA,and TMAO levels with myocardial injury-related indicators and disease severity scores in all patients.Multivariate logistic regression analysis was used to identify the influencing factors for the occurrence of SAP.Receiver operating characteristic(ROC)curve analysis was used to evaluate the predictive value of total SCFAs,BA,TMAO,and their combination for the occurrence of SAP.Results Compared with the non-severe group,the severe group had significantly lower levels of acetic acid,propionic acid,butyric acid,and total SCFAs(P<0.01),and significantly higher levels of BA,TMAO,CK-MB,cTnT,NT-proBNP,CRP,and APACHE II and BISAP scores.Within the severe group,the non-survival subgroup had significantly lower levels of total SCFAs(P<0.05)and significantly higher levels of BA and TMAO(P<0.05)compared to the survival subgroup.Spearman analysis showed that the levels of CK-MB,cTnT,NT-proBNP,CRP,and the APACHE II and BISAP scores were negatively correlated with total SCFAs levels and positively correlated with BA and TMAO levels(P<0.001).Multivariate logistic regression analysis revealed that total SCFAs,BA,and TMAO were independent influencing factors for the occurrence of SAP(P<0.05).ROC curve analysis showed that the area under the curve(AUC)for total SCFAs,BA,TMAO,and their combination in predicting the occurrence of SAP were 0.951,0.797,0.790,and 0.974,respectively(P<0.001).The AUC for the combination of the three markers was larger than that of any single marker,indicating good predictive efficacy.Conclusion The levels of gut metabolites SCFAs,BA,and TMAO in SAP patients are independent factors associated with myocardial injury and prognosis.

OBJECTIVES: To investigate the effect of Didang Decoction-medicated serum on autophagy in high glucose (HG)-induced rat glomerular endothelial cells (RGECs) and explore the pathway that mediates its effect. METHODS: Primary RGECs were isolated and cultured using sequential sieving combined with collagenase digestion, followed by identification using immunofluorescence assay for factor VIII. High glucose medium was used to induce RGECs to simulate a diabetic environment, and the effects of Didang Decoction-medicated serum and 3-MA (an autophagy inhibitor), either alone or in combination, on autophagy of HG-exposed cells were evaluated by observing autophagic vacuoles using monodansylcadaverine (MDC) staining. RT-qPCR and Western blotting were employed to measure mRNA and protein expression levels of Beclin-1, p62, LC3B, p-PI3K, p-Akt, and p-mTOR. RESULTS: Compared with the control cells, the HG-exposed RGECs showed significantly reduced autophagic fluorescence intensity, decreased Beclin-1 mRNA expression, increased p62 mRNA expression, downregulated Beclin-1 protein and LC3-II/I ratio, and upregulated p62, p-PI3K, p-Akt, and p-mTOR protein levels. Didang Decoction-medicated serum significantly enhanced autophagic fluorescence intensity in HG-exposed cells, increased Beclin-1 mRNA expression, decreased p62 mRNA expression, upregulated Beclin-1 protein, and downregulated p62, p-PI3K, p-Akt, and p-mTOR protein levels. CONCLUSIONS Didang Decoction-medicated serum enhances autophagy in HG-exposed RGECs by regulating the PI3K/Akt/mTOR signaling pathway, which sheds light on a new therapeutic strategy for diabetic nephropathy.
Animals ; Autophagy/drug effects* ; Signal Transduction/drug effects* ; Rats ; TOR Serine-Threonine Kinases/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Endothelial Cells/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Glucose ; Cells, Cultured ; Kidney Glomerulus/cytology* ; Rats, Sprague-Dawley

Objective To explore the effect of Echinetin(ECH)pretreatment on alleviating intestinal bar-rier dysfunction caused by severe acute pancreatitis(SAP).Methods Totally 36 rats were randomly divided into Sham group,SAP group and SAP+ECH group,with 12 rats in each group.Pancreatitis was induced by retrograde injection of 3％sodium taurocholate into pancreatic duct.Histological examination was performed on the pancreas of experimental rats to determine whether the pancreatitis model of rats was successfully con-structed.Intestinal barrier function was evaluated by intestinal pathological scores,serum diamine oxidase(DAO)activity and endotoxin levels,and bacterial translocation in mesenteric lymph nodes.The mRNA and protein expression levels of tight junction proteins ZO-1 and occludin were detected by quantitative fluorescent PCR(qPCR)and Western blot,and the protein expression levels of high mobility frame-1 protein(HMGB1),Toll-like receptor 4(TLR4)and protein kinase R(PKR)were detected by Western blot.Results ECH had no significant effect on the histological changes of pancreas,but could improve the intestinal mucosal barrier damage and membrane permeability associated with SAP.Although ECH does not affect the mRNA expres-sion levels of ZO-1 and occludin in ileum of SAP rats,it could significantly increase the expression levels of ZO-1 and occludin,and ECH treatment could significantly reduce the expression levels of HMGB1,TLR4 and PKR in ileum of SAP rats.Conclusion ECH can reduce the intestinal barrier dysfunction induced by SAP,and its effect on intestinal barrier function may be related to the inhibition of the HMGB1/TLR4/PKR pathway.

Background and Aims:Triple-negative breast cancer(TNBC)is a clinically aggressive subtype of breast cancer with limited treatment options.Immune checkpoint blockade(ICB)combined with chemotherapy has emerged as a key neoadjuvant therapeutic strategy for TNBC.However,significant variability in ICB efficacy exists among patients,and the underlying mechanisms related to the tumor immune microenvironment(TME)remain unclear.Cancer-associated fibroblasts(CAFs),as major stromal components,regulate TME and influence immunotherapy responses.Fibroblast activation protein(FAP),a key marker of CAFs,has been associated with poor prognosis in multiple solid tumors,yet its immunological role in TNBC has not been systematically investigated.This study aims to elucidate the expression pattern of FAP in TNBC,its impact on the immune microenvironment and ICB efficacy,and to explore its potential immunosuppressive mechanisms and clinical implications.Methods:The data from TCGA and the I-SPY2 clinical trial were integrated to assess the association of FAP expression with prognosis,immune cell infiltration,and immune checkpoint molecule expression in TNBC.Immune landscape profiling was conducted using CIBERSORT,GSEA enrichment analysis,and differential gene expression analysis(DESeq2)to characterize the immune features associated with FAP expression and to identify downstream genes at the transcriptomic level.CAF models with FAP overexpression or knockdown were constructed and co-cultured with CD8? T cells to evaluate FAP's regulatory effects on CD8? T cell activity and apoptosis.The expression of COL1A1,a potential FAP-regulated gene identified from transcriptomic analysis,was validated using qPCR and Western blot.Finally,clinical data and multiplex immunofluorescence pathology samples from TNBC patients at Xiangya Hospital were analyzed alongside I-SPY2 data to determine the predictive value of FAP expression for pathological complete response(pCR)following neoadjuvant immunotherapy.Results:FAP was significantly upregulated in TNBC tumor tissues compared to normal tissues and associated with shorter overall survival.Multivariate Cox regression analysis identified FAP as an independent adverse prognostic factor.High FAP expression was correlated with reduced infiltration of CD8? T cells,NK cells,and Tfh cells,as well as upregulation of immune checkpoints including CD276,TIM-3,and PD-L2.In CAF models,FAP overexpression suppressed CD8? T cell activity and promoted apoptosis,while FAP knockdown had the opposite effect.Transcriptomic analysis showed that COL1A1 and other collagen-related genes were significantly upregulated in the FAP-high group and positively correlated with FAP expression;qPCR and Western blot confirmed that FAP positively regulates COL1A1 expression.Analysis of I-SPY2 data revealed that FAP-low patients receiving pembrolizumab plus chemotherapy had significantly higher pCR rates compared to FAP-high patients.Consistently,clinical data from the Xiangya cohort showed reduced CD8? T cell infiltration and lower pCR rates in FAP-high patients,with a ROC AUC of 0.857 for predicting treatment response.Conclusion:FAP-high CAFs contribute to the formation of an immunosuppressive TME in TNBC by promoting COL1A1 secretion,inhibiting CD8? T cell function,and upregulating immune checkpoint molecules.High FAP expression is associated with poor prognosis and reduced response to immunotherapy,highlighting FAP as both a prognostic biomarker and a potential therapeutic target for stratified and combination treatment strategies in TNBC.

Background and Aims:Triple-negative breast cancer(TNBC)is a clinically aggressive subtype of breast cancer with limited treatment options.Immune checkpoint blockade(ICB)combined with chemotherapy has emerged as a key neoadjuvant therapeutic strategy for TNBC.However,significant variability in ICB efficacy exists among patients,and the underlying mechanisms related to the tumor immune microenvironment(TME)remain unclear.Cancer-associated fibroblasts(CAFs),as major stromal components,regulate TME and influence immunotherapy responses.Fibroblast activation protein(FAP),a key marker of CAFs,has been associated with poor prognosis in multiple solid tumors,yet its immunological role in TNBC has not been systematically investigated.This study aims to elucidate the expression pattern of FAP in TNBC,its impact on the immune microenvironment and ICB efficacy,and to explore its potential immunosuppressive mechanisms and clinical implications.Methods:The data from TCGA and the I-SPY2 clinical trial were integrated to assess the association of FAP expression with prognosis,immune cell infiltration,and immune checkpoint molecule expression in TNBC.Immune landscape profiling was conducted using CIBERSORT,GSEA enrichment analysis,and differential gene expression analysis(DESeq2)to characterize the immune features associated with FAP expression and to identify downstream genes at the transcriptomic level.CAF models with FAP overexpression or knockdown were constructed and co-cultured with CD8? T cells to evaluate FAP's regulatory effects on CD8? T cell activity and apoptosis.The expression of COL1A1,a potential FAP-regulated gene identified from transcriptomic analysis,was validated using qPCR and Western blot.Finally,clinical data and multiplex immunofluorescence pathology samples from TNBC patients at Xiangya Hospital were analyzed alongside I-SPY2 data to determine the predictive value of FAP expression for pathological complete response(pCR)following neoadjuvant immunotherapy.Results:FAP was significantly upregulated in TNBC tumor tissues compared to normal tissues and associated with shorter overall survival.Multivariate Cox regression analysis identified FAP as an independent adverse prognostic factor.High FAP expression was correlated with reduced infiltration of CD8? T cells,NK cells,and Tfh cells,as well as upregulation of immune checkpoints including CD276,TIM-3,and PD-L2.In CAF models,FAP overexpression suppressed CD8? T cell activity and promoted apoptosis,while FAP knockdown had the opposite effect.Transcriptomic analysis showed that COL1A1 and other collagen-related genes were significantly upregulated in the FAP-high group and positively correlated with FAP expression;qPCR and Western blot confirmed that FAP positively regulates COL1A1 expression.Analysis of I-SPY2 data revealed that FAP-low patients receiving pembrolizumab plus chemotherapy had significantly higher pCR rates compared to FAP-high patients.Consistently,clinical data from the Xiangya cohort showed reduced CD8? T cell infiltration and lower pCR rates in FAP-high patients,with a ROC AUC of 0.857 for predicting treatment response.Conclusion:FAP-high CAFs contribute to the formation of an immunosuppressive TME in TNBC by promoting COL1A1 secretion,inhibiting CD8? T cell function,and upregulating immune checkpoint molecules.High FAP expression is associated with poor prognosis and reduced response to immunotherapy,highlighting FAP as both a prognostic biomarker and a potential therapeutic target for stratified and combination treatment strategies in TNBC.

Objective:To build a systematic and standardized nutrition management plan for patients with chronic kidney disease.Methods:Based on the nutrition care procedure and model, a preliminary draft of a nutrition management plan for chronic kidney disease patients was developed through a literature search, quality evaluation, and group discussions. After two rounds of expert consultation and revision of the preliminary draft of the nutrition management plan, the final plan was formed.Results:A total of 32 experts were invited to complete two rounds of consultation. In two rounds of expert consultation, 32 questionnaires were distributed, and 32 and 31 valid questionnaires were collected, with valid response rates of 100.0% and 96.9%, respectively. The expert authority coefficients were 0.853 and 0.871, respectively. The final nutrition management plan for chronic kidney disease patients included six first-level items of nutrition management personnel: nutrition risk screening, nutrition assessment, nutrition treatment, nutrition monitoring, and nutrition health education, with 23 second-level and 52 third-level items.Conclusions:The constructed nutrition management plan for chronic kidney disease patients is scientific and can provide a reference for nutrition guidance.

Objective:To construct a novel dual-specific antibody targeting human CD123 (CD123 DuAb) and study its effects in acute myeloid leukemia (AML) .Methods:Based on the variable region of the CD123 monoclonal antibody independently developed at our institution, the CD123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein. Through a series of in vitro experiments, its activation and proliferation effect on T cells, as well as the effect of promoting T-cell killing of AML cells, were verified.Results:① A novel CD123 DuAb plasmid targeting CD123 was successfully constructed and expressed in the Expi-CHO eukaryotic system. ②The CD123 DuAb could bind both CD3 on T cells and CD123 on CD123 + tumor cells. ③When T cells were co-cultured with MV4-11 cells with addition of the CD123 DuAb at a concentration of 1 nmol/L, the positive expression rates of CD69 and CD25 on T cells were 68.0% and 44.3%, respectively, which were significantly higher than those of the control group ( P<0.05). ④Co-culture with CD123 DuAb at 1 nmol/L promoted T-cell proliferation, and the absolute T-cell count increased from 5×10 5/ml to 3.2×10 6/ml on day 9, and CFSE fluorescence intensity decreased significantly. ⑤ With the increase in CD123 DuAb concentration in the culture system, T-cell exhaustion and apoptosis increased. When the CD123 DuAb was added at a concentration of 1 nmol/L to the culture system, the proportion of CD8 + PD-1 + LAG-3 + T cells was 10.90%, and the proportion of propidium iodide (PI) - Annexin Ⅴ + T cells and PI + Annexin Ⅴ + T cells was 18.27% and 11.43%, respectively, which were significantly higher than those in the control group ( P<0.05). ⑥ The CD123 DuAb significantly activated T cells, and the activation intensity was positively correlated with its concentration. The expression rate of CD107a on T cells reached 16.05% with 1 nmol/L CD123 DuAb, which was significantly higher than that of the control group ( P<0.05). ⑦The CD123 DuAb promoted cytokine secretion by T cells at a concentration of 1 nmol/L, and the concentration of IFN-γ and TNF-α in the supernatant of the co-culture system reached 193.8 pg/ml and 169.8 pg/ml, respectively, which was significantly higher than that of the control group ( P<0.05). ⑧When CD123 DuAb was added at a concentration of 1 nmol/L to the co-culture system of T cells and CD123 + tumor cells, the killing intensity of T cells significantly increased, and the residual rates of CD123 + MV4-11 cells, CD123 + Molm13 cells, and CD123 + THP-1 cells were 7.4%, 6.7%, and 14.6% on day 3, respectively, which were significantly lower than those in the control group ( P<0.05) . Conclusion:In this study, a novel CD123 DuAb was constructed and expressed. In vitro experiments verified that the DuAb binds to CD123 + tumor cells and T cells simultaneously, promotes T-cell activation and proliferation, and facilitates their anti-leukemia effect, which provides a basis for further clinical research.

ObjectiveTo observe the protective effect of Didang Xianxiong decoction on the kidneys of diabetic rats， its regulation on the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway， and its influence on podocyte apoptosis and explore the mechanism of Didang Xianxiong decoction in improving diabetic nephropathy. MethodThe diabetic model was established by a single intraperitoneal injection of streptozotocin （STZ） solution of 55 mg·kg^-1. The successfully replicated model rats were randomly divided into the model group， Didang Xianxiong decoction group （8.10 g·kg^-1）， Xiao Xianxiongtang group （4.05 g·kg^-1）， Didangtang group （4.05 g·kg^-1）， and alagebrium （ALT-711） group （3 mg·kg^-1）， with six rats in each group. In addition， six rats were included in the blank group. After continuous administration for eight weeks， hematoxylin-eosin （HE） staining was used to observe the pathological changes in rats' kidney tissue. Masson staining was used to observe the degree of collagen deposition. Periodic acid-Schiff （PAS） staining was used to observe basement membrane lesions， and immunohistochemistry was used to detect the expression of phosphorylation （p）-PI3K and p-Akt proteins in rats' kidney tissue. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） method was used to detect podocyte apoptosis. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of PI3K and Akt in rats' kidney tissue. Western blot was used to detect the protein expression of PI3K， p-PI3K， Akt， p-Akt， B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， phosphorylation glycogen synthase kinase-3β （p-GSK-3β）， and Caspase-3 in the kidney tissue. ResultCompared with the normal group， the model group had compensatory expansion of glomeruli， proliferation of mesangial cells， a large amount of collagen deposition in the mesangial stroma， thickening of the basement membrane， decreased mRNA expression of PI3K and Akt， and inhibition of PI3K and Akt protein phosphorylation （P<0.01）. It also underwent enhanced apoptotic signaling， decreased expression of anti-apoptotic protein Bcl-2 （P<0.01）， and increased expression of Bax， p-GSK-3β， and Caspase-3 （P<0.01）. Compared with the model group， Didang Xianxiong decoction significantly improved kidney tissue pathology， increased mRNA expression of PI3K and Akt （P<0.01）， significantly up-regulated phosphorylation levels of PI3K and Akt proteins （P<0.01） and Bcl-2 expression （P<0.01）， downregulated the expression of Bax， p-GSK-3β， and Caspase-3 （P<0.01）， and weakened podocyte apoptotic signaling. ConclusionDidang Xianxiong decoction may promote the activation of the PI3K/Akt signaling pathway， inhibit podocyte apoptosis， and thus slow down the progression of diabetic nephropathy.

Objective To construct a risk prediction model for ventilator-associated pneumonia(VAP)in trauma patients and evaluate its efficacy.Methods A single-center retrospective study was conducted,trauma patients admitted to the department of emergency intensive care unit(EICU)of Hebei Cangzhou Hospital of Integrated Traditional Chinese Medicine and Western Medicine from January 1,2018 to January 1,2023 were selected as the study subjects,and the patients were divided into VAP group and non-VAP group.Differences between the two groups in variables including demographic characteristics,clinical data,and clinical scores.To prevent overfitting,differences between the groups were reduced using LASSO regression.Multifactor Logistic regression was used to identify risk factors for VAP in trauma patients and construct a risk prediction model.The model's discrimination was evaluated using the receiver operator characteristic curve(ROC curve)and area under the curve(AUC).The calibration curve was drawn and Hosmer-Lemeshow test were performed to evaluate the calibration degree of the model.Decision curve analysis(DCA)and clinical impact curve(CIC)were used to analyse the model's net benefit at different probability thresholds.Results A total of 888 trauma patients were included,among which 166 cases(18.7%)were diagnosed with VAP.Compared to the non-VAP group,the VAP group showed a significant increase in age,age-adjusted Charlson comorbidity index(aCCI)scores,white blood cell count(WBC),sequential organ failure assessment(SOFA)scores,length of ICU stay,and the proportion of patients with chest trauma,traumatic brain injury,and spinal cord injury.In contrast,hemoglobin(Hb),Glasgow coma scale(GCS)scores,and body mass index(BMI)were significantly lower in the VAP group(all P<0.05).Using LASSO regression,four variables were identified as important predictors for the occurrence of VAP in trauma patients:length of ICU stay,aCCI,WBC,and SOFA score.Multivariate Logistic regression showed that length of ICU stay[odds ratio(OR)and 95%confidence interval(95%CI)was 1.094(1.070-1.117)],aCCI[OR(95%CI)was 1.135(1.065-1.210)],WBC[OR(95%CI)was 1.139(1.104-1.176)],and SOFA score[OR(95%CI)was 1.137(1.080-1.197)]were independent risk factors for the occurrence of VAP in trauma patients(all P<0.05).Based on these influencing factors,a predictive model for VAP occurrence was constructed.ROC curve analysis showed that the AUC for predicting VAP occurrence in trauma patients was 0.876,with a 95%CI was 0.850-0.903,a sensitivity of 86.14%,and a specificity of 75.17%,indicating that the model has a high discriminative ability.Hosmer-Lemeshow test:χ2=7.7,P=0.2,Cox&Snell R2=0.236,Nagelkerke R2=0.387,the calibration curve was very close to the diagonal,and the mean absolute error(MAE)=0.03,indicating the model's predictions were highly consistent with actual clinical observations.The DCA and CIC curves indicate that within the threshold probability of<70%,using this model to identify high-risk groups for VAP in trauma patients and making clinical decisions can provide benefits in clinical practice.Conclusion The risk prediction model of VAP in trauma patients constructed in this study has high discrimination and calibration,which can provide reference for medical personnel to identify high-risk groups of VAP among trauma patients at an early stage and provide targeted intervention measures.