Objective:To investigate the effects of reduced nicotinamide adenine dinucleotide phosphooxidase 4 (NOX4) inhibitors GKT137831 and M2-type macrophages on oxidative stress markers NOX4, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the rat hepatic stellate cell line (HSC-T6).Methods:Rat bone marrow macrophages were extracted and induced using interleukin (IL)-4 to differentiate them into M2 phenotype macrophages. HSC-T6 activation was performed with 5 μg/L transforming growth factor β1 (TGF-β1). The proliferation condition of HSC-T6 cells stimulated by the NOX4 inhibitor GKT137831 at a concentration gradient of 5 to 80 μmol/L after 48 hours was detected using the Cell Counting Kit-8 (CCK-8) assay. The optimal drug concentration was chosen and divided into an HSC co-culture group (the control group) and five experimental groups: the TGF-β1 stimulation group, the TGF-β1 +GKT137831 stimulation group, the M2-type macrophage + HSC co-culture group, the M2-type macrophage +TGF-β1 stimulation group, and the M2-type + TGF-β1 + GKT137831 stimulation group. Reactive oxygen species (ROS) production level was detected in each cell using the DCFH-DA probe method. NOX4, α-smooth muscle actin (α-SMA), Nrf2, and HO-1 levels in each group of HSC cells were detected using the qRT-PCR method and the Western blot method. The t-test was used to compare the two groups. The one-way ANOVA method was used to compare multiple groups. Results:Intracellular ROS increased significantly following TGF-β1 stimulation. ROS relative levels in each cell group were 1.03±0.11, 3.88±0.07, 2.90±0.08, 0.99±0.06, 3.30±0.05, 2.21±0.11, F ?=?686.1, P ?=?0.001, respectively. The mRNA and protein expressions of NOX4, α-SMA, Nrf2, and HO-1 were significantly increased ( P ?

OBJECTIVE To investigate the metabolic changes in MEG-01 megakaryocytes after treatment with linezolid （LZD） from metabolomic perspective and its impact on the the proliferation of cells and generation of platelet precursors. METHODS MEG-01 cells were seeded in proliferation medium and divided into blank control group （untreated）， solvent control group （4‰DMSO）， and 100， 200， 400， 800 μg/mL LZD groups. The proliferation status of cells in each group was observed under the microscope； cell proliferation and viability were assessed. Cells were also seeded in differentiation medium and divided into blank control group （untreated）， solvent control group （4‰DMSO）， and 400 μg/mL LZD group； after 14 days of culture， platelet precursor generation was observed under the microscope； immunofluorescence staining was performed to count the proportion of cells producing pseudopodia， the relative length of pseudopodia was measured， and the expression levels of CD41 and CD42b mRNA were assessed. Cells from the solvent control group and the 400 μg/mL LZD group， cultured in differentiation medium for 14 days， were extracted and subjected to non-targeted metabolomics and targeted energy metabolomics analysis using liquid chromatography-tandem mass spectrometry. The relative content of pyruvate in cells， after being cultured for 14 days with the addition of pyruvate （10 mmol/L） or LZD （400 μg/mL）+pyruvate （10 mmol/L）， was measured and observed， as well as its effects on cell proliferation and platelet precursor generation. RESULTS 400 μg/mL LZD could significantly inhibit the proliferation of MEG-01 cells and the generation of platelet precursors （P＜0.01）. Non-targeted metabolomic analysis of MEG-01 cells after 400 μg/mL LZD treatment revealed significant changes in energy metabolism-related pathways such as mTOR signaling pathway， alanine， aspartate and glutamate metabolism， and central carbon metabolism in cancer. Targeted energy metabolomic analysis further showed that the relative contents of adenosine triphosphate， guanosine triphosphate， and pyruvate in MEG-01 cells were significantly reduced （P＜0.01）， while the relative contents of lactate were significantly increased （P＜0.01）. Compared with the LZD group， the relative content of pyruvate， cell count， the proportion of cells producing pseudopodia and the relative length of pseudopodia were significantly increased in the LZD+pyruvate group （P＜0.01）. CONCLUSIONS LZD may reduce pyruvate production by inhibiting mitochondrial energy metabolism， thereby suppressing megakaryocyte proliferation and platelet precursor production， ultimately leading to the occurrence of thrombocytopenia.

Objective:To evaluate the efficacy and safety of first-line anti-tuberculosis (TB) drugs combined with linezolid in treatment of children with tuberculous meningitis (TBM).Methods:A retrospective cohort study design was performed . Eight-nine Children diagnosed as TBM during January 1 st 2016 and December 31 st 2023 in Department of Infectious Disease, Children′s Hospital of Chongqing Medical University were enrolled in the study. According to different treatment regimens, children were divided into a group of first-line anti-tuberculous drugs (isoniazid, rifampicin, pyrazinamide, ethambutol (HRZE)) and a group of HRZE and linezolid combination (HRZEL). The efficacy and safety of the 2 regimens were compared and the relationship between linezolid drug concentration and adverse reactions were analyzed. Comparisons between groups were performed using χ2 test and Mann-Whitney U test. Results:The 89 children with TBM included 53 males and 36 females with an onset age of 4.6 (1.4, 9.6) years. There were 27 cases in the HZREL group and 62 cases in the HRZE group. Before treatment, positive rate of interferon-gamma release assays (IGRA) in HRZEL group was lower than that in HRZE group (64% (16/25) vs.92% (55/60), χ2=9.82, P<0.05), but protein level of cerebrospinal fluid (CSF) was higher than that in HRZE group (1.2 (1.0, 2.0) vs.0.8 (0.4,1.4) g/L, Z=0.32, P<0.05). By the end of the intensive phase, there were no significant differences of rates of CSF improvement and etiology negativity between HRZEL group and HRZE group (both P>0.05).The 44 TBM children with high CSF protein (>1 g/L) included 25 males and 19 females with an onset age of 6.7 (3.0, 11.8) years. There were 21 cases in the HZREL group and 23 cases in the HRZE group accordingly. Before treatment, there were no significant differences of positive rate of IGRA test and CSF protein level between the 2 groups (62% (13/21) vs. 87% (20/23), 1.7 (1.1, 2.2) vs. 1.5 (1.2, 1.9) g/L, χ2=3.67, Z=0.23, both P>0.05). There were no significant differences in CSF indicators, etiology negativity or imaging remission between the two groups by the end of intensive phase (all P>0.05). Higher frequencies of granulocytopenia, gastrointestinal symptoms as well as withdrawal or change of drugs were found in HRZEL group when compared to those in HRZE group (44% (12/27) vs. 19% (12/62), 7% (2/27) vs. 0, 33% (9/27) vs. 3% (2/62), χ2=6.01, 4.70, 15.74, all P<0.05). Conclusions:The efficacy of HRZEL regimen is similar to conventional HRZE regimen in children with TBM, but with higher adverse effect. Prudentially evaluating the pros and cons of linezolid in the usage of drug-susceptible TB and carefully monitoring of linezolid associated adverse effects is suggested.

Objective To observe the distribution of atypical memory B cells in peripheral blood of children with frequently relapsing nephrotic syndrome(FRNS).Methods A total of 60 children with primary ne-phrotic syndrome(PNS)admitted to the hospital from October 2020 to March 2023 were selected as the re-search objects.According to the response to glucocorticoid(GC),they were divided into non-frequently relap-sing nephrotic syndrome(NFRNS)group(25 cases)and FRNS group(35 cases).A total of 20 age-and gen-der-matched healthy children were enrolled as the control group.The changes of atypical memory B cells in each group before and after GC treatment were compared,and the correlation between the changes and clinical data was analyzed.Results Before GC treatment,The percentages of total B cells(CD19+CD20+),total memory B cells(CD19+CD20+CD27+),resting memory B cells(CD19+CD20+CD21+CD27+)and atypical memory B cells(CD19+CD20+CD21-CD27-)in FRNS group and NFRNS group were significantly higher than those in control group.And the FRNS group was significantly higher than the NFRNS group(P＜0.05).After GC treatment,the percentages of total B cells,total memory B cells,resting memory B cells,acti-vated memory B cells(CD19+CD20+CD21-CD27+)and atypical memory B cells in FRNS group and NFRNS group were lower than those before GC treatment(P＜0.05).The FRNS group had a significantly higher pro-portion of atypical memory B cells than the NFRNS group and the control group(P＜0.05).Before GC treat-ment,the 24 h urinary protein in FRNS group and NFRNS group were higher than those in control group,and the levels of immunoglobulin G and albumin were lower than those in control group.The 24 h urinary protein in FRNS group was significantly higher than that in NFRNS group(P＜0.05).Before GC treatment,there was a positive correlation between 24 h urinary protein and the proportion of atypical memory B cells in FRNS group(P＜0.05).Conclusion There is abnormal distribution of atypical memory B cells in peripheral blood of FRNS children.The increase of atypical memory B cells can be used as a marker of recurrence of FRNS af-ter GC treatment.

OBJECTIVE To analyze the influencing factors for the metabolism of voriconazole in adult patients， and to provide reference for the rational use of voriconazole in clinic. METHODS The clinical data of adult patients admitted in our hospital receiving voriconazole and therapeutic drug monitoring from April 2021 to March 2022 were collected. The trough concentration of voriconazole （c0） and plasma concentration of voriconazole-N-oxide concentration （cN） were determined， and voriconazole-to-voriconazole N-oxide concentration ratio （c0/cN） was calculated. Pearson’s correlation analysis was used to analyze the influencing factors for c0 and c0/cN of voriconazole. Multiple linear regression models were used to analyze the independent influencing factors for c0 and c0/cN of voriconazole. RESULTS The underlying diseases of the patients were mainly pneumonia， kidney disease and leukemia. The detected fungi were mainly Aspergillus， Candida and yeast-like fungi. Voriconazole was mainly administered by intravenous drip， especially in patients who used proton pump inhibitor in combination. The levels of C-reactive protein （CRP）， total bilirubin （TBIL）， direct bilirubin （DBIL） and indirect bilirubin （IBIL） were positively correlated with c0 of voriconazole， while platelet count and albumin levels were negatively correlated with voriconazole c0. The levels of CRP， TBIL and DBIL were positively correlated with c0/cN， while albumin levels were negatively correlated with c0/cN. Multiple linear regression analysis showed that the independent influencing factors of voriconazole c0 included the levels of CRP and IBIL， route of administration and dose of voriconazole， and the independent influencing factors of voriconazole c0/cN were the levels of CRP and DBIL and age. CONCLUSIONS The levels of CRP and IBIL， route of administration and dose of voriconazole are independent influencing factors of voriconazole c0； the levels of CRP and DBIL and age are independent influencing factors of voriconazole c0/cN. The influence of above indexes on the metabolism of voriconazole should be considered when using voriconazole clinically； and the route of administration and dose of voriconazole should be adjusted reasonably.

OBJECTIVE：To investigate the consistency and difference of f luorescence immunochromatographic and liquid chromatography-tandem mass spectrometry （LC-MS/MS）and enzyme multiplied immunoassay technique （EMIT）in the blood concentration monitoring of mycophenolic acid. METHODS ：Fluorescence immunochromatography ，LC-MS/MS and EMIT were used to detect the blood concentration of mycophenolic acid in 61 blood samples of children treated with mycophenolate mofetil ester orally at different time points. Kolmogorov-Smirnov method ，Wilcoxon pairing test ，Passing-Bablok regression ，Cusum method，Spearman correlation analysis ，Bland-Altman scatter diagram were adopted for statistical analysis. RESULTS ：Blood concentrations of mycophenolic acid ，which were determined by fluorescence immunochromatography ，LC-MS/MS and EMIT ， showed non-normal distribution. Passing-Bablok regression analysis showed that regression equation of fluorescence immunochromatography and LC-MS/MS ，fluorescence immunochromatographic method and EMIT were CFI=0.928 3CLC-MS/MS+0.961 7 and CFI＝0.880 7CEMIT－0.488 2（FI means fluorescence immunochromatographic ）. Spearman correlation analysis showed that the correlation coefficients between fluorescence immunochromatography and LC-MS/MS ，fluorescence immunochromatography and EMIT were 0.968 and 0.929， respectively （P＜0.000 1）. Bland Altman scatter plot analysis showed that 3.28％ of the 358341451@qq.com difference between fluorescence immunochromatography and LC-MS/MS was outside the consistency limit （±1.96SD）， and 1.64％ of the difference between fluorescence immuno- chromatography and EMIT was outside the consistency limit （± 1.96SD）. Wilcoxon pairing test showed that the results of fluorescence immunochromatography were higher than those of LC-MS/MS （Z＝3.76，P＝0.000 2）and lower than those of EMIT （Z＝－5.96，P＜0.000 1）. CONCLUSIONS ：Fluorescence immunochromatography shows good consistency and correlation with LC-MS/MS and EMIT ；the blood concentrations of mycophenolic acid detected by fluorescence immunochromatography were higher than those by LC-MS/MS and lower than those by EMIT . It can be used for bedside rapid detection. When using the test results of different methods for clinical medication ，the differences of test methods need to be considered.

OBJECTIVE： To establish the method for simultaneous determination of clopidogrel （CLP）， its intermediate metabolite （2-O-CLP）， inactive metabolite （CLPCA） and active metabolite （CLPTM） in human plasma. METHODS： Totally 90 patients diagnosed as stroke were selected from the First Affiliated Hospital of Army Medical University. They were given one CLP tablet （75 mg/tablet） orally on an empty stomach in the morning. Blood samples were collected 2 h after taking the tablet. CLPTM- D was formed by derivation of CLPTM with 2-bromo-3’-methoxyacetophenone and extracted by precipitation of acetonitrile protein together with the other three substances to be measured. LC-MS/MS method was adopted. The determination was performed on Agilent poroshell 120 EC-C18 column with mobile phase consisted of acetonitrile （0.1％ formic acid） and water （0.1％ formic acid） （90 ∶ 10， V/V）. The quantitation analysis was performed using multiple reaction monitoring at the specific ion transitions of m/z 308.1→198.1 （CLPCA）， 322.3→212.0 （CLP）， 338.3→155.0 （2-O-CLP）， 504.4→354.1 （CLPTM-D） and 264.0→154.1 （ticlopidine， internal standard）， respectively. RESULTS： The retention time of CLPCA， CLP， 2-O-CLP， CLPTM-D and internal standard were 2.01， 3.32， 2.83， 2.68， 1.87 min， respectively. The linear range of CLPCA， CLP， 2-O-CLP and CLPTM-D were 100-10 000， 0.2-20， 0.3-30， 0.5-50 ng/mL （all r≥0.999 5）. The intra-day and inter-day RSD were all less than 9.5％ （n＝5）. Accuracy ranged from 93.5％-98.9％ （n＝5）， and extraction recovery was from 85.4％ to 95.9％ （n＝5）. The matrix effect ranged from 2.7％-6.2％ （n＝5）. In stability tests （storing at －80 ℃ for 3 months， 3 freeze-thaw cycles， storing at 4 ℃ for 8 h）， RE of CLP， CLPCA and CLPTM-D were all lower than 10.0％ （n＝5）. CONCLUSIONS： Established LC-MS/MS method has the advantages of high specificity， accuracy and reliability， and can be used to detect the concentration of CLP and its three metabolites in human plasma.

Theory of treatment from spleen is being widely applied in in the process of Chinese medical therapies and had already shown its clinical efficacy. The purpose of this paper was to explain this theory in the fields of the theoretical contents, physiology, pathology and clinical practices.

Objective To investigate the roles of SENP1 in regulation of biological characteristics of NK cells.Methods Lentivirus-mediated-Senp1-small-hairpinRNA (shRNA) transduction was applied to NK92 cells.The expression of SENP1 in NK92 cells was determined by real-time PCR and Western blot.The proliferation of NK92 cells was detected by CCK-8 assay.The apoptosis of NK92 cells was determined by Annexin Ⅴ and PI labeling.The cytotoxicity of NK92 cells against K562 cells was evaluated by luciferase reporter assay.Results Treatment of NK92 cells with IL-21 resulted in SENP1 upregulation.Lentivirus mediated SENP1 knockdown reduced proliferation and increased apoptosis in NK-92 cells,but SENP1 inhibition had slight impact on the cytotoxic ability of NK92 cells to kill K562 cells.Conclusion SENP1 mediates the regulatory effect of IL-21 on the proliferation and survival of NK92 cells.

Objective To study the biofilm formation ability and related gene distribution of Staphylococcus (S .) aureus iso‐lated from urinary tract infections to provide the theoretical basis for the prevention and treatment of clinical infection .Methods The minimal inhibitory concentration was detected using the agar double dilution method .The bacterial adhesion ability was deter‐mined by flat colony counting method .The biofilm formation ability was analyzed by the 96‐well crystal violet staining method .The biofilm‐associated genes were detected by PCR amplification .Results Eleven clinical strains of S .aureus were high resistant to pen‐icillin and erythromycin ,whereas were all sensitive to vancomycin and nitrofurantoin .All the isolates had a strong ability of adhe‐sion ,but the biofilm formation ability was weak .Among them ,the icaAD and icaBC genes were amplified in 10 S .aureus isolates . Conclusion The adhesion ability and biofilm formation ability of S .aureus isolated from urinary tract infections have the strain differences ,and ica is an important gene of S .aureus biofilm formation .