The rapid screening of bioactive constituents within traditional Chinese medicine (TCM) presents a significant challenge to researchers. Prevailing strategies for the screening of active components in TCM often overlook trace components owing to their concealment by more abundant constituents. To address this limitation, a fishing strategy based on offline two-dimensional liquid chromatography (2D-LC) combined with surface plasmon resonance (SPR) was utilized to screen bioactive trace components targeting peroxiredoxin 3 (PRDX3), using Uncaria alkaloids (UAs) as a case study. Initially, an orthogonal preparative offline 2D-LC system combining a positively charged C₁₈ column and a conventional C₁₈ column under disparate mobile phase conditions was constructed. To fully reveal the trace alkaloids, 13 2D fractions of UAs were prepared, and their components were characterized using mass spectrometry (MS). Subsequently, employing PRDX3 as the targeting protein, a SPR-based screening approach was established and rigorously validated with geissoschizine methyl ether (GSM) serving as a positive control for binding. Employing this refined strategy, 29 candidate binding alkaloids were fished from the 13 2D fractions. Notably, combining offline 2D-LC with SPR increased the yield of candidate binding components from 10 to 29 when compared to SPR-based screening alone. Subsequent binding affinity assays confirmed that PRDX3 was a direct binding target for the 12 fished alkaloids, with isovallesiachotamine (IV), corynoxeine N-oxide (CO-N), and cadambine (CAD) demonstrating the highest affinity for PRDX3. Their interactions were further validated through molecular docking analysis. Subsequent intracellular H₂O₂ measurement assays and transfection experiments confirmed that these three trace alkaloids enhanced PRDX3-mediated H₂O₂ clearance. In conclusion, this study introduced an innovative strategy for the identification of active trace components in TCM. This approach holds promise for accelerating research on medicinal components within this field.

Objective:To evaluate the role of microRNA-149-5p (miR-149-5p) in resveratrol-induced reduction of lipopolysaccharide (LPS)-induced cardiomyocyte injury in rats.Methods:Rat cardiomyocyte cell line H9C2 was cultured and then divided into 5 groups ( n=27 each) using a random number method: control group (C group), LPS group, resveratrol group (RSV group), miR149-5p inhibitor negative control group (LRN group), and miR149-5p inhibitor group (LRI group). A cardiomyocyte injury model was prepared by incubating cells with culture medium containing 10 μg/ml LPS for 24 h. RSV group was incubated with resveratrol (final concentration of 10 μmol/L) for 24 h, followed by incubation with culture medium containing 10 μg/ml LPS for another 24 h. LRN group and LRI group were transfected with miR149-5p inhibitor negative control and miR149-5p inhibitor, respectively, and then the other treatments were similar to those previously described in RSV group. The cell viability was measured by CCK-8 assay, the apoptosis rate by flow cytometry, the concentration of lactate dehydrogenase (LDH) and content of glutathione (GSH) in the supernatant by microplate method, the content of malondialdehyde (MDA) by TBA reaction method, the activity of superoxide dismutase (SOD) by WST-1 method, the level of reactive oxygen species (ROS) by DCFH-DA fluorescent probe, the concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in the supernatant by enzyme-linked immunosorbent assay, and the expression of miR-149-5p by quantitative real-time polymerase chain reaction. Results:Compared with C group, the expression of miR-149-5p was significantly down-regulated, the cell viability was decreased, the concentrations of LDH, TNF-α and IL-6 in supernatant, apoptosis rate, ROS level and MDA content were increased, and the GSH content and SOD activity were decreased in LPS group ( P<0.05). Compared with LPS group, the expression of miR-149-5p was significantly up-regulated, the cell viability was increased, the concentrations of LDH, TNF-α and IL-6 in supernatant, apoptosis rate, ROS level and MDA content were decreased, and the GSH content and SOD activity were increased in RSV group ( P<0.05). Compared with RSV group or LRN group, the expression of miR-149-5p was significantly down-regulated, the cell viability was decreased, the concentrations of LDH, TNF-α and IL-6 in supernatant, apoptosis rate, ROS level and MDA content were increased, and the GSH content and SOD activity were decreased in LRI group ( P<0.05). Conclusions:The mechanism by which resveratrol alleviates LPS-induced cardiomyocyte injury is associated with the up-regulation of miR-149-5p expression and inhibition of cell apoptosis, oxidative stress and inflammatory responses in rats.

Objective:To understand the status quo of emotional intelligence, emotional labor, and humanistic caring ability of medical staff, and to clarify their internal relationship.Methods:Convenience sampling was used to select 713 medical staff from a grade A tertiary hospital in Chengdu, Sichuan Province, China. Emotional Intelligence Scale, Humanistic Caring Scale, and Emotional Labor Scale were used to measure the emotional intelligence, humanistic caring ability, and emotional labor of medical staff. SPSS 22.0 software was used to establish a database for statistical description and analysis. Process 3.2 software was used to analyze the mediating effect.Results:In humanistic caring ability, the average score of comprehension dimension was the highest (75.62±8.20) and the average score of patience dimension was the lowest (58.53±5.01). In emotional labor, the average score of the deep action dimension was the highest (23.39±3.85) and the average score of the surface action dimension was the lowest (17.73±3.18). In emotional intelligence, the average score of self-emotion evaluation dimension was the highest (21.76±3.30) and the average score of other-emotion evaluation dimension was the lowest (20.07±3.71). Positive correlations were found between humanistic caring ability and emotional intelligence, between humanistic caring ability and emotional labor, and between emotional intelligence and emotional labor ( P<0.01). Humanistic caring ability had a partial mediating effect between emotional intelligence and emotional labor. Humanistic caring ability had direct and indirect effects on emotional labor, and the effect sizes were 0.279 and 0.029, respectively. Conclusion:Emotional intelligence has a direct positive predictive effect on emotional labor, humanistic caring ability as an intermediary variable indirectly and positively predicts emotional labor. Humanistic caring ability plays a partial mediating role between emotional intelligence and emotional labor. Attention should be paid to the emotional labor of medical staff, and the emotional labor of medical staff should be improved through targeted training on emotional intelligence and humanistic caring ability. These efforts will improve the current situation and establish a harmonious doctor-patient relationship.

MicroRNAs (miRNAs) play critical roles in the development and progression in various cancers.Dysfunctional miR-9 expression remains ambiguous,and no consensus on the metastatic progression of ovarian cancer has been reached.In this study,results from the bioinformatics analysis show that the 3'-UTR of the E-cadherin mRNA was directly regulated by miR-9.Luciferase reporter assay results confirmed that miR-9 could directly target this 3'-UTR.miR-9 and E-cadherin expression in ovarian cancer tissue was quantified by qRTPCR.Migration and invasion were detected by wound healing and Transwell system assay in SKOV3 and A2780.qRT-PCR and Western blot were performed to detect the epithelial-mesenchymal transition-associated mRNA and proteins.Immunofluorescence technique was used to analyze the expression and subcellular localization of Ecadherin,N-cadherin,and vimentin.The results showed that miR-9 was frequently upregulated in metastatic serous ovarian cancer tissue compared with paired primary ones.Upregulation of miR-9 could downregulate the expression of E-cadherin but upregulate the expression of mesenchymal markers (N-cadherin and vimentin).Overexpression of miR-9 could promote the cell migration and invasion in ovarian cancer,and these processes could be effectively inhibited via miR-9 inhibitor.Thus,our study demonstrates that miR-9 may promote ovarian cancer metastasis via targeting E-cadherin and a novel potential therapeutic approach to control metastasis of ovarian cancer.

Objective To explore the effect of several cytokines, including interferon-γ, interleukin-10 and interlekin-4, on promoter activity of human BAFF (B-cell activating factor belonging to tumor necrosis factor family) gene. Methods A construct of phBAFF 1.02 containing sequence form -1349 bp to -329 bp of human BAFF gene, linking with chloramphenicol acetyltransferase (CAT) as reporter gene, was transiently transfected into human HL-60 cells, a kind of myeloid tumor cell lines. The cells were subsequently treated with IFN-γ, IL-10 and IL-4, and the CAT activity was assessed 24 hours after stimulation with each cytokines. Results IFN-γ of 5 ng/ml, IL-10 of 100 ng/ml could increase the CAT activity of phBAFF 1.02 to 4.18 and 2.13 folds respectively compared to the control. IL-4 at 100 ng/ml had no effect on promoter activity of human BAFF gene. Combination of IFN-γ, IL-10 and IL-4 could increase the CAT activity of phBAFF 1.02 to 3.41 and 1.58 folds respectively compared with controls. Conclusion IFN-γ and IL-10 can increase the promoter activity of human BAFF gene. IL-4 treatment can not affect the CAT activity driven by BAFF promoter. However, IL-4 can decrease the upregulating effect of IFN-γ and IL-10 on phBAFF1.02. These provide essential evidence for future study on the interaction mechanism of cytokines and BAFF in autoimmune diseases.

Objective To explore the effect of IFN-γ, IL-10 and IL-4 on B cell activating factor (BAFF) expression in human HL-60 cells, a kind of myeloid tumor cell lines, and its possible regulation mechanism. Methods Cultured human HL-60 cells were treated with IFN-γ, IL-10 and IL-4 for 1-3 days. The expression of membrane-bound BAFF on HL-60 cells was examined by flow cytometry, the amount of soluble BAFF was detected by ELISA assay, and the level of BAFF mRNA was tested by real-time PCR method. A functional 1021 bp fragment of the 5'-tlanking region of the human BAFF gene (-1349 to -329 bp) was cloned and investigated with serial 5'-deletion. The 5'-deleted promoters were recombinated with chloramphenicol acetyltransferase (CAT) as reporter gene. These five recombinant plasmids were transiently transfected to HL-60 cells with liposomal transfectian method. Promoters activities were determined by CAT reporter gene assay(CAT-ELISA) in those transfected cells treated with different cytokines. Results The results showed that the expression of membrane-bound BAFF, soluble BAFF and BAFF mRNA in human HL-60 cells were significantly elevated (P ＜ 0. 05) after incubated with IFN-γ and IL-10. In addition, IFN-γ and IL-10 showed significantly (P ＜ 0. 05) increased effects on promoter activity in human BAFF gane. And the cytokines-responsive sequences were located between -929 and -719 bp of the BAFF promoter region. Conclusion The enhancement of IFN-γ and IL-10 on BAFF expression and synthesis were regnla-ted by promoter activation. Our in vitro studies also raise the possibility to investigate the mechanisms regula-ting BAFF expression in other tumor cells of myeloid origin under pathological circumstances.

Objective To explore the differential expression of Toll-like receptor 4(TLR4)mRNA in peripheral blood mononuclear cells(PBMCs)between the patients with ankylosing spondylitis(AS),rheumatoid arthritis(RA)and the healthy individuals as well as its clinical significance.Methods The expression level of TLR4 mRNA in PBMCs was determined from 60 AS patients,30 RA patients and 30 healthy controls by real-time fluorescence quantitative RT-PCR.Erythrocyte sedimentation rate(ESR)and plasma C-reactive protein(CRP)were detected automatically respectively by ESR automatic analyzer and specific protein electrophoresis analyzer.Results The expression level of TLR4 mRNA in PBMC was significantly higher in patients with AS or RA than in the controls,while no significant difference was observed between the two disease groups.Furthermore,both HLA-B27-positive and -negative AS patients showed higher TLR4 mRNA level than healthy controls,and HLA-B27-positive AS patients higher than RA patients.However,there was no significant difference between HLA-B27-positive,-negative AS and RA patients.In HLA-B27-positive AS patients,close correlations between TLR4 mRNA and ESR or CRP were observed,but no correlation observed in HLA-B27 negative patients.In RA patients,the level of TLR4 mRNA correlated with CRP.Conclusion The expression level of TLR4 mRNA is elevated in PBMC from either AS or RA patients.Although the upregulation of TLR4 mRNA is not associated with whether HLA-B27 is positive or not in AS patients,it is more significant in HLA-B27-positive AS than in RA.Furthermore,the correlations between the expression of TLR4 mRNA and ESR or CRP are influenced by HLA-B27 antigen in AS patients.In conclusion,the present results indicate that the abnormal expression of TLR4 might play a role in the development and progression of AS and RA.