Aim To investigate the pharmacological mechanism of Ebselenin(Ebselen,EbSe)in the treat-ment of Escherichia coli(E.coli)infection,which had no significant inhibitory effect on Gram-negative bacte-ria,based on previous studies.Methods After EbSe intervention in E.coli infected Raw264.7 cells,the via-bility of Raw264.7 cells was determined by CCK-8 method,the morphology and structure of Raw264.7 cells were observed by electron microscope,and the in-tracellular bacterial load of Raw264.7 cells was calcu-lated by coated plate method.Polarization status of peritoneal macrophages,Raw264.7 intracellular NO and ROS content and intracellular HO-1 expression in Raw264.7 and E.coli acutely infected mice after E.co-li infection by flow cytometry.qPCR was used to detect the expression of related mRNAs in Raw264.7 cells.qPCR was used to detect the intracellular GSH content in Raw264.7 cells by spectrophotometric assay,and the state of cytoskeletal proteins was observed by immuno-fluorescence.Western blot assay was performed to de-tect the intracellular Txnrd1 expression level.Results Microtiter method,CCK-8,and electron microscopy observations showed that EbSe had no effect on the growth of E.coli and Raw264.7 cells in vitro.The re-sults of smear plate counting showed that EbSe reduced the intracellular bacterial load of Raw264.7 in the in-fected group.Flow cytometry results showed that EbSe upregulated the number of M2-type macrophages.The EbSe-treated infected group had reduced intracellular NO and ROS levels and increased GSH levels.The qPCR results showed that the expression of IL-6,IL-1β,and iNOS was decreased,and the expression of HO-1,Txnrd1,and Glut1 was increased in DHB4-in-fected Raw264.7 cells after EbSe treatment.Cytoskel-etal staining showed that the morphology of the EbSe-treated infected cells was similar to that of oxPAPC-in-duced cells.Western blot results showed the expres-sion of Txnrd1 protein in EbSe-treated infected cells in-creased.Conclusion EbSe exerts anti-E.coli acute infection effect by regulating macrophage polarization and inhibiting macrophage excessive inflammatory state.

Aim To investigate the pharmacological mechanism of Ebselenin(Ebselen,EbSe)in the treat-ment of Escherichia coli(E.coli)infection,which had no significant inhibitory effect on Gram-negative bacte-ria,based on previous studies.Methods After EbSe intervention in E.coli infected Raw264.7 cells,the via-bility of Raw264.7 cells was determined by CCK-8 method,the morphology and structure of Raw264.7 cells were observed by electron microscope,and the in-tracellular bacterial load of Raw264.7 cells was calcu-lated by coated plate method.Polarization status of peritoneal macrophages,Raw264.7 intracellular NO and ROS content and intracellular HO-1 expression in Raw264.7 and E.coli acutely infected mice after E.co-li infection by flow cytometry.qPCR was used to detect the expression of related mRNAs in Raw264.7 cells.qPCR was used to detect the intracellular GSH content in Raw264.7 cells by spectrophotometric assay,and the state of cytoskeletal proteins was observed by immuno-fluorescence.Western blot assay was performed to de-tect the intracellular Txnrd1 expression level.Results Microtiter method,CCK-8,and electron microscopy observations showed that EbSe had no effect on the growth of E.coli and Raw264.7 cells in vitro.The re-sults of smear plate counting showed that EbSe reduced the intracellular bacterial load of Raw264.7 in the in-fected group.Flow cytometry results showed that EbSe upregulated the number of M2-type macrophages.The EbSe-treated infected group had reduced intracellular NO and ROS levels and increased GSH levels.The qPCR results showed that the expression of IL-6,IL-1β,and iNOS was decreased,and the expression of HO-1,Txnrd1,and Glut1 was increased in DHB4-in-fected Raw264.7 cells after EbSe treatment.Cytoskel-etal staining showed that the morphology of the EbSe-treated infected cells was similar to that of oxPAPC-in-duced cells.Western blot results showed the expres-sion of Txnrd1 protein in EbSe-treated infected cells in-creased.Conclusion EbSe exerts anti-E.coli acute infection effect by regulating macrophage polarization and inhibiting macrophage excessive inflammatory state.

Objective To develop dynamic prediction models based on multiple postnatal time points to support early diagnosis and individualized intervention for bronchopulmonary dysplasia(BPD)in preterm infants with gestational age<32 weeks.Methods Clinical data of 472 preterm infants with gestational age<32 weeks admitted to the Second Xiangya Hospital of Central South University between January 2016 and November 2020 were retrospectively analyzed.Multivariable logistic regression was applied to develop five independent prediction models at postnatal days 1,7,14,21,and 28.The performance of the models was assessed using the area under the receiver operating characteristic curve(AUC)and the Hosmer-Lemeshow test.Results Baseline characteristics such as gestational age and birth weight differed significantly between the BPD group(n=147)and the non-BPD group(n=325)(P<0.05).Predictors of BPD evolved across time points:on day 1,key predictors included gestational age,birth weight,Score for Neonatal Acute Physiology II(SNAP-II),invasive mechanical ventilation,and fraction of inspired oxygen>30%;by day 7,additional variables emerged,including fasting duration>2 days,mean feeding advancement rate<8.5 mL/(kg·d),neonatal respiratory distress syndrome,apnea of prematurity,and positive sputum culture;from day 14 onward,nutrition-and treatment-related indicators were incorporated additionally.The models demonstrated good discrimination at postnatal days 1,7,14,21,and 28,with AUCs of 0.917,0.927,0.939,0.944,and 0.968,respectively,and good calibration(Hosmer-Lemeshow P>0.05).Internal validation showed AUCs ranging from 0.899 to 0.958,indicating robust performance.Conclusions Dynamic postnatal prediction models incorporating indicators spanning perinatal factors,respiratory support,nutritional management,and therapeutic interventions demonstrate high predictive performance and facilitate dynamic risk assessment for BPD in preterm infants with gestational age<32 weeks.

Objective To develop dynamic prediction models based on multiple postnatal time points to support early diagnosis and individualized intervention for bronchopulmonary dysplasia(BPD)in preterm infants with gestational age<32 weeks.Methods Clinical data of 472 preterm infants with gestational age<32 weeks admitted to the Second Xiangya Hospital of Central South University between January 2016 and November 2020 were retrospectively analyzed.Multivariable logistic regression was applied to develop five independent prediction models at postnatal days 1,7,14,21,and 28.The performance of the models was assessed using the area under the receiver operating characteristic curve(AUC)and the Hosmer-Lemeshow test.Results Baseline characteristics such as gestational age and birth weight differed significantly between the BPD group(n=147)and the non-BPD group(n=325)(P<0.05).Predictors of BPD evolved across time points:on day 1,key predictors included gestational age,birth weight,Score for Neonatal Acute Physiology II(SNAP-II),invasive mechanical ventilation,and fraction of inspired oxygen>30%;by day 7,additional variables emerged,including fasting duration>2 days,mean feeding advancement rate<8.5 mL/(kg·d),neonatal respiratory distress syndrome,apnea of prematurity,and positive sputum culture;from day 14 onward,nutrition-and treatment-related indicators were incorporated additionally.The models demonstrated good discrimination at postnatal days 1,7,14,21,and 28,with AUCs of 0.917,0.927,0.939,0.944,and 0.968,respectively,and good calibration(Hosmer-Lemeshow P>0.05).Internal validation showed AUCs ranging from 0.899 to 0.958,indicating robust performance.Conclusions Dynamic postnatal prediction models incorporating indicators spanning perinatal factors,respiratory support,nutritional management,and therapeutic interventions demonstrate high predictive performance and facilitate dynamic risk assessment for BPD in preterm infants with gestational age<32 weeks.

Immobilized enzyme-based enzyme electrode biosensors, characterized by high sensitivity and efficiency, strong specificity, and compact size, demonstrate broad application prospects in life science research, disease diagnosis and monitoring, etc. Immobilization of enzyme is a critical step in determining the performance (stability, sensitivity, and reproducibility) of the biosensors. Random immobilization (physical adsorption, covalent cross-linking, etc.) can easily bring about problems, such as decreased enzyme activity and relatively unstable immobilization. Whereas, directional immobilization utilizing amino acid residue mutation, affinity peptide fusion, or nucleotide-specific binding to restrict the orientation of the enzymes provides new possibilities to solve the problems caused by random immobilization. In this paper, the principles, advantages and disadvantages and the application progress of enzyme electrode biosensors of different directional immobilization strategies for enzyme molecular sensing elements by specific amino acids (lysine, histidine, cysteine, unnatural amino acid) with functional groups introduced based on site-specific mutation, affinity peptides (gold binding peptides, carbon binding peptides, carbohydrate binding domains) fused through genetic engineering, and specific binding between nucleotides and target enzymes (proteins) were reviewed, and the application fields, advantages and limitations of various immobilized enzyme interface characterization techniques were discussed, hoping to provide theoretical and technical guidance for the creation of high-performance enzyme sensing elements and the manufacture of enzyme electrode sensors.

Based on the regulation of mitochondrial fatty acid β-oxidation through the PGC1α/PPARα/CPT1A pathway, this study investigated the effect of Jianpi Qinghua Formula on the mitochondrial fatty acid β-oxidation pathway in the livers of mice with metabolic-associated fatty liver disease(MAFLD) induced by a high-fat diet. MAFLD mice were fed a high-fat diet to establish the model, and after successful modeling, the mice were divided into the model group, the Jianpi Qinghua Formula group, and the metformin group, with an additional control group. Each group was treated with the corresponding drug or an equivalent volume of saline via gavage. Body mass and food intake were measured regularly during the experiment. At the end of the experiment, blood lipid levels and liver function-related indices were measured, liver pathological changes were observed, and protein expression levels of PGC1α, PPARα, PPARγ, and CPT1A were detected by Western blot. The results showed that, with no difference in food intake, compared to the model group, the body mass of the Jianpi Qinghua Formula group and the metformin group was reduced, liver weight and liver index decreased, and levels of cholesterol, triglycerides, and low-density lipoprotein cholesterol(LDL-C) were lowered. Additionally, a decrease in alanine aminotransferase(ALT) and aspartate aminotransferase(AST) was observed. Hematoxylin and eosin(HE) staining revealed reduced pathological damage to hepatocytes, while oil red O staining showed improvement in fatty infiltration. The liver disease activity score decreased, and transmission electron microscopy revealed improvement in mitochondrial swelling and restoration of internal cristae. Western blot analysis indicated that Jianpi Qinghua Formula significantly increased the expression of PGC1α, PPARα, and CPT1A proteins in the liver and reduced the expression of PPARγ. These results suggest that the Jianpi Qinghua Formula improves mitochondrial function, promotes fatty acid oxidation, and alleviates the pathological changes of MAFLD. In conclusion, Jianpi Qinghua Formula can improve MAFLD by mediating mitochondrial fatty acid β-oxidation through the PGC1α/PPARα/CPT1A pathway.
Animals ; PPAR alpha/genetics* ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Carnitine O-Palmitoyltransferase/genetics* ; Male ; Liver/metabolism* ; Fatty Liver/genetics* ; Humans ; Mice, Inbred C57BL ; Diet, High-Fat/adverse effects*

Objective To investigate the effect of miR-185-5p-mediated targeted negative regulation of transmembrane 9 superfamily member 1(TM9SF1)on proliferation,migration and autophagy in lung adenocarcinoma cells.Methods The expression of miR-185-5p in lung adenocarcinoma tissues was analyzed using dataset GSE51853 downloaded from the Gene Expression Omnibus(GEO)database.Potential target proteins of miR-185-5p were predicted using online databases(miRTargetLink,miRTarbase,and DIANA-microT-CD),and autophagy-related proteins were obtained from HADb.The intersected results from these four databases was identified,and survival curves of vascular endothelial growth factor A(VEGFA)and TM9SF1 within the overlapping candidates were analyzed using the StarBase database.TM9SF1 3'UTR wild-type(WT)or TM9SF1 3'UTR mutant(MUT)reporter plasmids were separately co-transfected with miR-185-5p control plasmid(CON)or miR-185-5p overexpression plasmid(over-miR-185-5p)into HEK-293T cells.A dual-luciferase reporter gene assay was employed to assess the binding interaction between miR-185-5p and TM9SF1 and quantify the subsequent luciferase activity.Western blotting was used to assess TM9SF1 protein expression levels in A549 cells transfected with over-miR-185-5p.A549 cells were divided into three groups:(1)CON+NC group,co-transfected with miR-185-5p control plasmid and TM9SF1 control plasmid;(2)over-miR-185-5p+NC group,co-transfected with over-miR-185-5p and TM9SF1 control plasmid;(3)over-miR-185-5p+over-TM9SF1 group,co-transfected with both miR-185-5p and TM9SF1 overexpression plasmids.EdU cell proliferation assay,wound healing assay,and Transwell migration assay were performed to validate the effects of miR-185-5p targeted binding to TM9SF1 on proliferation and migration capacities in lung adenocarcinoma.Changes in autophagic flux and mitochondrial membrane potential(MMP)of lung adenocarcinoma cells were detected using stubRFP-sensGFP-LC3 lentivirus and JC-1 assays,respectively.Results In the GSE51853 dataset,miR-185-5p expression level was significantly lower in lung adenocarcinoma tissues compared with normal lung tissues(P<0.01).qRT-PCR analysis revealed that miR-185-5p expression was downregulated in lung adenocarcinoma cell lines NCI-H1299 and A549 compared with normal lung epithelial cells BEAS-2B(P<0.01).Bioinformatics predictions using miRTargetLink,miRTarbase,DIANA-microT-CD,and HADb databases indicated that miR-185-5p could target and regulate the autophagy-related protein TM9SF1.Dual-luciferase reporter assays and Western blotting demonstrated that miR-185-5p directly bound to the 3'UTR region of TM9SF1 mRNA,and overexpression of miR-185-5p significantly reduced the expression of target protein TM9SF1(P<0.05).EdU cell proliferation,wound healing,and Transwell migration assays demonstrated that miR-185-5p overexpression inhibited proliferation and migration capacities of lung adenocarcinoma cells,whereas TM9SF1 overexpression could attenuate this inhibition effect(P<0.05).Results of stubRFP-sensGFP-LC3 for autophagic flux analysis demonstrated that overexpression of miR-185-5p enhanced autophagic flux in A549 cells,whereas co-overexpression of miR-185-5p and TM9SF1 suppressed autophagic flux.JC-1 assays showed a decreased MMP level in A549 cells after miR-185-5p overexpression,with higher MMP level observed when miR-185-5p and TM9SF1 were co-overexpressed.Conclusion miR-185-5p may suppress proliferation,migration,and autophagy capacities in lung adenocarcinoma cells by targeting TM9SF1 through negative regulation.

OBJECTIVE: To evaluate the dynamic changes of glucocorticoid (GC) dose and the feasibility of GC discontinuation in rheumatoid arthritis (RA) patients under the background of Chinese medicine (CM). METHODS: This multicenter retrospective cohort study included 1,196 RA patients enrolled in the China Rheumatoid Arthritis Registry of Patients with Chinese Medicine (CERTAIN) from September 1, 2019 to December 4, 2023, who initiated GC therapy. Participants were divided into the Western medicine (WM) and integrative medicine (IM, combination of CM and WM) groups based on medication regimen. Follow-up was performed at least every 3 months to assess dynamic changes in GC dose. Changes in GC dose were analyzed by generalized estimator equation, the probability of GC discontinuation was assessed using Kaplan-Meier curve, and predictors of GC discontinuation were analyzed by Cox regression. Patients with <12 months of follow-up were excluded for the sensitivity analysis. RESULTS: Among 1,196 patients (85.4% female; median age 56.4 years), 880 (73.6%) received IM. Over a median 12-month follow-up, 34.3% (410 cases) discontinued GC, with significantly higher rates in the IM group (40.8% vs. 16.1% in WM; P<0.05). GC dose declined progressively, with IM patients demonstrating faster reductions (median 3.75 mg vs. 5.00 mg in WM at 12 months; P<0.05). Multivariate Cox analysis identified age <60 years [P<0.001, hazard ratios (HR)=2.142, 95% confidence interval (CI): 1.523-3.012], IM therapy (P=0.001, HR=2.175, 95% CI: 1.369-3.456), baseline GC dose ⩽7.5 mg (P=0.003, HR=1.637, 95% CI: 1.177-2.275), and absence of non-steroidal anti-inflammatory drugs use (P=0.001, HR=2.546, 95% CI: 1.432-4.527) as significant predictors of GC discontinuation. Sensitivity analysis (545 cases) confirmed these findings. CONCLUSIONS RA patients receiving CM face difficulties in following guideline-recommended GC discontinuation protocols. IM can promote GC discontinuation and is a promising strategy to reduce GC dependency in RA management. (Trial registration: ClinicalTrials.gov, No. NCT05219214).
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Arthritis, Rheumatoid/drug therapy* ; Glucocorticoids/therapeutic use* ; Medicine, Chinese Traditional ; Retrospective Studies

Objective To monitor the susceptibility of clinical isolates to antimicrobial agents in healthcare facilities in major regions of China in 2023.Methods Clinical isolates collected from 73 hospitals across China were tested for antimicrobial susceptibility using a unified protocol based on disc diffusion method or automated testing systems.Results were interpreted using the 2023 Clinical & Laboratory Standards Institute (CLSI) breakpoints.Results A total of 445199 clinical isolates were collected in 2023,of which 29.0％ were gram-positive and 71.0％ were gram-negative.The prevalence of methicillin-resistant strains in Staphylococcus aureus,Staphylococcus epidermidis and other coagulase-negative Staphylococcus species (excluding Staphylococcus pseudintermedius and Staphylococcus schleiferi) (MRSA,MRSE and MRCNS) was 29.6％,81.9％ and 78.5％,respectively.Methicillin-resistant strains showed significantly higher resistance rates to most antimicrobial agents than methicillin-susceptible strains (MSSA,MSSE and MSCNS).Overall,92.9％ of MRSA strains were susceptible to trimethoprim-sulfamethoxazole and 91.4％ of MRSE strains were susceptible to rifampicin.No vancomycin-resistant strains were found.Enterococcus faecalis had significantly lower resistance rates to most antimicrobial agents tested than Enterococcus faecium.A few vancomycin-resistant strains were identified in both E.faecalis and E.faecium.The prevalence of penicillin-susceptible Streptococcus pneumoniae was 93.1％ in the isolates from children and and 95.9％ in the isolates from adults.The resistance rate to carbapenems was lower than 15.0％ for most Enterobacterales species except for Klebsiella,22.5％ and 23.6％ of which were resistant to imipenem and meropenem,respectively .Most Enterobacterales isolates were highly susceptible to tigecycline,colistin and polymyxin B,with resistance rates ranging from 0.6％ to 10.0％.The resistance rate to imipenem and meropenem was 21.9％ and 17.4％ for Pseudomonas aeruginosa,respectively,and 67.5％ and 68.1％ for Acinetobacter baumannii,respectively.Conclusions Increasing resistance to the commonly used antimicrobial agents is still observed in clinical bacterial isolates.However,the prevalence of important crabapenem-resistant organisms such as crabapenem-resistant K.pneumoniae,P.aeruginosa,and A.baumannii showed a slightly decreasing trend.This finding suggests that strengthening bacterial resistance surveillance and multidisciplinary linkage are important for preventing the occurrence and development of bacterial resistance.

Objective To investigate the changing distribution and antibiotic resistance profiles of clinical isolates of Staphylococcus in hospitals across China from 2015 to 2021.Methods Antimicrobial susceptibility testing was conducted for the clinical isolates of Staphylococcus according to the unified protocol of CHINET(China Antimicrobial Surveillance Network)using disk diffusion method and commercial automated systems.The CHINET antimicrobial resistance surveillance data from 2015 to 2021 were interpreted according to the 2021 CLSI breakpoints and analyzed using WHONET 5.6.Results During the period from 2015 to 2021,a total of 204,771 nonduplicate strains of Staphylococcus were isolated,including 136,731(66.8％)strains of Staphylococcus aureus and 68,040(33.2％)strains of coagulase-negative Staphylococcus(CNS).The proportions of S.aureus isolates and CNS isolates did not show significant change.S.aureus strains were mainly isolated from respiratory specimens(38.9±5.1)％,wound,pus and secretions(33.6±4.2)％,and blood(11.9±1.5)％.The CNS strains were predominantly isolated from blood(73.6±4.2)％,cerebrospinal fluid(12.1±2.5)％,and pleural effusion and ascites(8.4±2.1)％.S.aureus strains were mainly isolated from the patients in ICU(17.0±7.3)％,outpatient and emergency(11.6±1.7)％,and department of surgery(11.2±0.9)％,whereas CNS strains were primarily isolated from the patients in ICU(32.2±9.7)％,outpatient and emergency(12.8±4.7)％,and department of internal medicine(11.2±1.9)％.The prevalence of methicillin-resistant strains was 32.9％in S.aureus(MRSA)and 74.1％in CNS(MRCNS).Over the 7-year period,the prevalence of MRSA decreased from 42.1％to 29.2％,and the prevalence of MRCNS decreased from 82.1％to 68.2％.MRSA showed higher resistance rates to all the antimicrobial agents tested except trimethoprim-sulfamethoxazole than methicillin-susceptible S.aureus(MSSA).Over the 7-year period,MRSA strains showed decreasing resistance rates to gentamicin,rifampicin,and levofloxacin,MRCNS showed decreasing resistance rates to gentamicin,erythromycin,rifampicin,and trimethoprim-sulfamethoxazole,but increasing resistance rate to levofloxacin.No vancomycin-resistant strains were detected.The prevalence of linezolid-resistant MRCNS increased from 0.2％to 2.3％over the 7-year period.Conclusions Staphylococcus remains the major pathogen among gram-positive bacteria.MRSA and MRCNS were still the principal antibiotic-resistant gram-positive bacteria.No S.aureus isolates were found resistant to vancomycin or linezolid,but linezolid-resistant strains have been detected in MRCNS isolates,which is an issue of concern.