Objective : To investigate the mechanisms of bromodomain-containing protein 4(BRD4) in TGF-β1-induced epithelial-mesenchymal transition in alveolar type II epithelial cells. Methods : MLE-12 cells were stimulated with different concentrations(5 ng/ml, 10 ng/ml) of TGF-β1 for 48 h to establish an EMT cell model. The cells were pretreated with 50 nmol/L BRD4 inhibitor JQ-1, 100 μmol/L high mobility group box 1 protein(HMGB1)inhibitor glycyrrhizin acid(GA), and 3 μg/ml rHMGB1. The experimental groups were divided as follows: control group, TGF-β1 group, JQ-1 group, JQ-1+TGF-β1 group, GA group, GA+TGF-β1 group, and JQ-1+TGF-β1+rHMGB1 group. The effect of JQ-1 on cell viability was examined using cell counting kit-8(CCK-8). The protein expression levels of CDH1, ZO-1, Vimentin, α-SMA, BRD4, HMGB1, TGF-β1, Smad2/3 and p-Smad2/3 were detected by Western blot. The cell migration ability was detected by a scratch test. Results : Compared with the control group, the levels of Vimentin and α-SMA in the TGF-β1 group increased, and the levels of CDH1 and ZO-1 protein decreased, suggesting that the EMT model was successfully established. In this model, the expression of BRD4 and HMGB1 significantly increased. Different concentrations of JQ-1 could inhibit the cell viability of MLE-12 in a concentration-dependent manner. Both JQ-1 and GA could effectively alleviate TGF-β1-induced EMT, and reduce the increase in HMGB1 expression and the activation of TGF-β1/Smad2/3 pathway caused by TGF-β1. Moreover, rHMGB1 treatment could reduce the effects of JQ-1 on EMT and the TGF-β1/Smad2/3 pathway. Additionally, both JQ-1 and glycyrrhizin could effectively decrease TGF-β1-induced cell migration, whereas rHMGB1 could alleviate the inhibitory effect of JQ-1 on the rate of cell migration. Conclusion BRD4 can regulate epithelial-mesenchymal transition in alveolar type II epithelial cells via HMGB1/TGF-β1/Smad2/3 signaling cascade, and BRD4 may be a potential target for inhibition of pulmonary fibrosis.

Objective To develop and validate an efficient and simple liquid chromatography with tandem mass spectrometry(LC-MS/MS)method for determination of polymyxin E in human plasma,and apply the established method in therapeutic drug monitoring(TDM)of polymyxin E.Methods The LC-MS/MS platform was based on AB SCIEX HPLC-4500MD system.Gradient elution was performed with 0.2％formic acid in water and 0.2％formic acid in acetonitrile.Phenomenex Kinetex XB-C18 column(100 mm × 2.1 mm,2.6 μm)were used.The analytes were detected by electrospray ionization(ESI)positive multiple reaction monitoring mode.The ion pairs for analytes(polymyxins E1,E2)and internal standard(polymyxins B1)were m/z 390.7→101.3,m/z 386.0→101.2,and m/z 402.3→101.2,respectively.Plasma samples were processed with protein precipitation method.Results Polymyxin E1 and E2 showed good linearity in the range of 0.031 2-6.24 mg/L and 0.006 15-1.23 mg/L,respectively.The within-run accuracy of polymyxin E1 and E2 in plasma ranged from 89.4％to 99.8％and 91.5％to 108.2％,respectively,while the between-run accuracy ranged from 91.8％to 104.7％and 95.6％to 105.2％,respectively.The within-run precision of polymyxin E1 and E2 in plasma ranged from 4.9％to 8.9％and 2.8％to 8.5％,respectively,while the between-run precision ranged from 4.1％to 7.6％and 4.2％to 9.8％,respectively.The average internal standard normalized matrix effect factors of polymyxins E1 and E2 were 96.9％-111.2％and 106.1％-112.8％in blank plasma samples from 6 different sources,102.5％-106.8％and 98.8％-105.2％in lipemic plasma,respectively,107.8％-108.9％and 106.9％-1 07.4％in hemolyzed plasma,respectively.The precision of matrix effects was less than 15.0％.The average recovery rate was 102.9％-107.5％for polymyxin E1 and E2,and 107.0％for internal standard polymyxin B1.The precision was less than 3.7％.Conclusions In this study,a simple and efficient LC-MS/MS method was established for determination of polymyxin E1 and E2 in human plasma,which is reliable in the therapeutic drug monitoring and pharmacokinetic study of polymyxin E.

Objective To develop and validate an efficient and simple liquid chromatography with tandem mass spectrometry(LC-MS/MS)method for determination of polymyxin E in human plasma,and apply the established method in therapeutic drug monitoring(TDM)of polymyxin E.Methods The LC-MS/MS platform was based on AB SCIEX HPLC-4500MD system.Gradient elution was performed with 0.2％formic acid in water and 0.2％formic acid in acetonitrile.Phenomenex Kinetex XB-C18 column(100 mm × 2.1 mm,2.6 μm)were used.The analytes were detected by electrospray ionization(ESI)positive multiple reaction monitoring mode.The ion pairs for analytes(polymyxins E1,E2)and internal standard(polymyxins B1)were m/z 390.7→101.3,m/z 386.0→101.2,and m/z 402.3→101.2,respectively.Plasma samples were processed with protein precipitation method.Results Polymyxin E1 and E2 showed good linearity in the range of 0.031 2-6.24 mg/L and 0.006 15-1.23 mg/L,respectively.The within-run accuracy of polymyxin E1 and E2 in plasma ranged from 89.4％to 99.8％and 91.5％to 108.2％,respectively,while the between-run accuracy ranged from 91.8％to 104.7％and 95.6％to 105.2％,respectively.The within-run precision of polymyxin E1 and E2 in plasma ranged from 4.9％to 8.9％and 2.8％to 8.5％,respectively,while the between-run precision ranged from 4.1％to 7.6％and 4.2％to 9.8％,respectively.The average internal standard normalized matrix effect factors of polymyxins E1 and E2 were 96.9％-111.2％and 106.1％-112.8％in blank plasma samples from 6 different sources,102.5％-106.8％and 98.8％-105.2％in lipemic plasma,respectively,107.8％-108.9％and 106.9％-1 07.4％in hemolyzed plasma,respectively.The precision of matrix effects was less than 15.0％.The average recovery rate was 102.9％-107.5％for polymyxin E1 and E2,and 107.0％for internal standard polymyxin B1.The precision was less than 3.7％.Conclusions In this study,a simple and efficient LC-MS/MS method was established for determination of polymyxin E1 and E2 in human plasma,which is reliable in the therapeutic drug monitoring and pharmacokinetic study of polymyxin E.

Background Silicosis is an occupational disease mainly characterized by pulmonary progressive fibrosis induced by the accumulation of free silica (SiO₂) in the lungs due to long-term exposure to SiO₂ dust. It has been shown that neutrophil extracellular traps (NETs) are increased in the lung tissues of silicotic mice after 28 d SiO₂ exposure, but it is unclear how the levels of NETs change throughout entire progression of silicosis in mice. Objective To observe the levels of NETs and pathological changes in the lungs of silicotic mice after different duration of SiO₂ exposure, and to confirm the possible role and significance of NETsin the development of SiO₂-induced pulmonary fibrosis. Methods A total of 28 SPF male C57BL/6J mice were randomly divided into a control group, and a model group, and the model group was subdivided into, a 2 d model group, a 7 d model group, and a 28 d model group, with 7 mice in each group. The mice in the model groups were given intratracheal instillation with 10 mg SiO₂ suspension (50 μL), and the mice in the control group were received same volume of saline. Mice were sacrificed and samples were collected at designed time points. The pathological changes of lung tissues of mice were observed after hematoxylin-eosin (HE) and Van Gieson (VG) staining. Immunofluorescence was used to observe the NETs markers citrullination histone H3 (CitH3) and myeloperoxidase (MPO) in bronchoalveolar lavage fluid (BALF), and the percentage of NETs-positive cells was calculated. PicoGreen fluorescent dye kit was used to detect the content of extracelluar DNA (ex-DNA) in mouse BALF, and the expression levels of fibrosis-related proteins α-smooth muscle actin (α-SMA) and fibronectin (FN) and NETs marker CitH3 in lung tissues of mice were detected by Western blot (WB). Results Compared with the control group, inflammatory cells accumulation, alveolar wall thickening, and collagen deposition were obviously observed in the lungs of the silicosis model groups, and a large number of silicone nodules were recorded in the lung tissues in the 28 d group. Compared with the control group, the expressions of α-SMA and FN in the lung tissue of the 28 d group were significantly increased (P<0.05). The percentages of NETs in BALF increased significantly in the 2 d and the 7 d model group, then decreased in the 28 d model group (P<0.05). Compared with the control group (7.434±0.258) ng·mL⁻¹, the ex-DNA levels in BALF of mice in the 2 d [(35.110±6.331) ng·mL⁻¹], the 7 d [(39.491±6.948) ng·mL⁻¹], and the 28 d [(23.360±4.809) ng·mL⁻¹] model groups were increased (P<0.05), and the increase of ex-DNA in the 2 d and the 7 d model groups were statistically significant (P<0.05). In comparison with the control group, the protein level of CitH3 was significantly increased in the lung tissues of mice in the 7 d model group (P<0.05). Conclusion The content of NETs increases significantly and reaches a peak in the early inflammatory stage of silicosis, and decreases as the disease progresses to the fibrotic stage, suggesting that NETs may play a role in early stage of silicosis.

Objective:To evaluate the efficacy and safety of θ short burst rapid pulse stimulation(TBS)in treating schizophrenia by meta-analysis.Methods:Randomized controlled trials(RCTS)on TBS in the treatment of schizophrenia were searched from CNKI,Wanfang,VIP,China Biomedicine,Web of science,PubMed,Embase and Cochrane Library databases to December 2022.The main study indicator was the Positive and Negative Symptoms Scale(PANSS).Risk quality assessment of the included literatures was performed by two reviewers and statistical analysis was performed using RevMan5.3.Results:A total of 13 RCTS with 641 patients were included.Meta-anal-ysis showed that TBS targeting the left dorsolateral prefrontal cortex(DLPFC)with intervention duration longer than 2 weeks decreased the PANSS total scores(WMD=-4.63,95％CI:-5.75--3.51,P＜0.001),positive symptom scores(WMD=-1.13,95％CI:-2.00--0.26,P＜0.05),negative symptom scores(WMD=-2.51,95％CI:-2.77--1.53,P＜0.001)and general psychopathological symptom scores(WMD=-1.20,95％CI:-1.80--0.60,P＜0.001).The adverse reactions of TBS included dizziness,and no serious adverse e-vents were reported.Conclusion:TBS has high safety,and stimulation of left dorsolateral prefrontal cortex targets for more than 2 weeks could effectively improve psychiatric symptoms in patients with schizophrenia.

Objective To establish an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for determination of tobramycin in human serum,and examine the utility of the method in a clinical pharmacokinetic study of tobramycin inhalation.Methods Serum samples were pretreated by solid phase extraction with tobramycin-D12 as internal standard.Chromatographic separation was performed on a TitankHilic(2.1 mm × 100 mm,3 μm)column.The mobile phase consisted of0.1％formic acid-acetonitrile and 0.1％formic acid aqueous solution at a flow rate of 0.4 mL/min.Electrospray ionization source and multiple reaction monitoring(MRM)scanning were used for monitoring the quantitative ion pairs with m/z 468.3→m/z 163.3(tobramycin)and m/z 480.6→m/z 166.2(tobramycin-D12).The established method was investigated in terms of selectivity,interaction,concomitant medication,standard curve and lower limit of quantitation,precision and accuracy,recovery,matrix effect,and stability of tobramycinin.Results The linear range of tobramycin was 0.050 0-10.0 mg/L(R2=0.999 5).The intra-and inter-batch precision was satisfactory(coefficient of variation[CV]≤3.6％).The accuracy ranged from-0.4％to 6.0％.The matrix effect factor(MF)in human serum samples(including hemolysis and lipemia)ranged from 92.2％to 94.9％(CV≤2.7％).The recovery of tobramycinin was 79.5％-81.9％in serum samples,while the recovery of internal standard was 78.9％.The analyte was stable in serum samples for 72 h at room temperature and for 274 days at-20℃/-70℃.The pharmacokinetic study of tobramycin inhalation in bronchiectasis patients showed that after continuous administration of tobramycin 300 mg twice a day to 3 patients,the mean Cmax of tobramycin was(0.72±0.61)mg/L on Day 1 and(0.76±0.73)mg/L on Day 28,respectively.The corresponding Tmax was(1.83±0.61)h and(1.50±0.50)h,respectively.Conclusions The UPLC-MS/MS method established in this study is sensitive,accurate and rapid.It is successfully applied to the clinical pharmacokinetic study of tobramycin inhalation.The method may be suitable for therapeutic drug monitoring of tobramycin in clinical practice.

Objective : To investigate the expression of class A scavenger receptor 1(MSR1) in the lungs of silico⁃ sis mice and its role in inflammation and lipid metabolism mediated by mouse mononuclear macrophages (RAW264. 7) . Methods : 24 C57BL/6 male mice were randomly divided into control group , exposed 7 d group , exposed 14 d group , exposed 28 d group , with 6 mice in each group. RAW264. 7 cells were divided into control group , siRNA⁃MSR1 group , SiO2 group and siRNA⁃MSR1 + SiO2 stimulation group. The pathological changes of lung tissue in mice were observed by HE and VG staining. Lipid accumulation was observed under oil red O staining microscope. Immunohistochemical staining (IHC) was used to detect the expression and localization of MSR1 . The expression of MSR1 , tumor necrosis factor (TNF) Ⅳα , interleukin (IL) Ⅳ6 and IL⁃1β were detected by Western blot. Results : Compared with the control group , HE and VG staining results showed that inflammatory cells gathered and collagen distribution increased in the lung tissue of silicosis mice. Oil red O staining showed that a large number of orange⁃red lipid droplets appeared in the lung tissue of mice. IHC results showed that the expression of MSR1 was up⁃regulated in silicosis inflammation stage. Western blot results showed that the expression of MSR1, TNF⁃α , IL⁃6 and IL⁃1β was up⁃regulated in silicosis inflammation stage (P < 0. 05) . The expression of MSR1 in the SiO2 cell stimulation group was up⁃regulated ( P < 0. 05 ) , and the expression of MSR1 in the siRNA⁃MSR1 group decreased (P < 0. 05) , and lipid droplets also appeared in the SiO2 cell stimulation group. The accumulation of lipid droplets in siRNA⁃MSR1 + SiO2 stimulation group was lower than that in SiO2 group (P < 0. 01) . ELISA results showed that the expression of TNF⁃α , IL⁃6 and IL⁃1β in SiO2 cell stimulation group was up⁃regulated ( P <0. 05) . Compared with SiO2 group , the expression of TNF⁃α , IL⁃6 and IL⁃1β in siRNA⁃MSR1 + SiO2 stimulation group was down⁃regulated (P < 0. 05) . Conclusion MSR1 is involved in the regulation of lipid components and the release of inflammatory factors in lung tissue and cells of silicosis mice. Inhibition of MSR1 expression can an⁃ tagonize the inflammatory response and abnormal lipid accumulation in macrophages. MSR1 may be a potential therapeutic target for future intervention in the progression of silicosis.

Objective : To explore the regulatory role of microRNA⁃455 ⁃3p ( miR⁃455 ⁃3p) in lymphangiogenesis of rat silicosis model , and to investigate the effect of miR⁃455 ⁃3p targeted regulation of vascular endothelial growth factor C (VEGF⁃C) on the tubular structure formation of human lymphatic endothelial cells ( HLECs) . Methods: The rats were randomly divided into the silicosis model group and the normal control group. The silicosis model group were injected with silicon dioxide (SiO2 )dust suspension , and the control group was injected with the same amount of normal saline. HE , Masson and immunohistochemistry staining were used to observe the pathological changes and lymphangiogenesis of lung tissue. The expression levels of miR⁃455 ⁃3p and VEGF⁃C in lung tissues of rats were detected by Quantitative real⁃time PCR ( RT⁃qPCR) and Western blot; The miR⁃455 ⁃3p inhibitors and negative controls ( NC) were transfected into HLECs , and the expression levels of miR⁃455 ⁃3p and VEGF⁃C in cells were detected by RT⁃qPCR and Western blot. The migration ability of HLECs was detected by scratch test , the ability of tubular structure formation was detected by matrigel tube formation test , and dual luciferase experiments were used to verify the targeting relationship between miR⁃455 ⁃3p and VEGF⁃C. Results : Compared with the normal control group , in the silicosis model group , a large number of inflammatory cells gathered and collagen gradually deposited in the pulmonary interstitium , and there was lymphatic hyperplasia in the lung. The expression of miR⁃455 ⁃3p in the lung tissue was lower than that in the control group , and the expression of VEGF⁃C was higher than that in the control group ; After transfection with HLECs , compared with the NC group , the expression of miR⁃455 ⁃3p in the cells of the Inhibitors group decreased , the expression of VEGF⁃C increased , and the ability of cell migration and tubular structure formation increased(P < 0. 05) ; VEGF⁃C was confirmed as a target gene of miR⁃455 ⁃3p by the dual luciferase experiments. Conclusion miR⁃455 ⁃3p can affect the tubular structure formation ability of HLECs and regulate lymphangiogenesis by targeting the expression of VEGF⁃C.

【Objective】 To investigate the profiles of RhC, c, E, and e antigens and phenotypes in 4 704 inpatients from multiple regions, i. e. Nanning, Guangzhou and Shenzhen, and provide data information for compatibility blood transfusion of Rh blood group. 【Methods】 The Rh blood group antigens were detected by microcolumn gel cards from three manufactures. If the test and the control results are inconsistent, a third-party reagent would be used, and traditional tube method for confirmation if needed. The Chi-square test and Fisher's exact test were used to analyze antigen frequency and Rh phenotypes in each region. 【Results】 Among the 4 704 inpatients, the frequency of C, c, E, and e antigen was e(91.77%)>C(85.64%)>c(49.62%)>E(41.60%), and Rh phenotypes distribution was CCee(49.40%)>CcEe(27.53%)>Ccee(8.16%)>ccEE(7.74%)>ccEe(4.89%)>CCEe(0.96%)>ccee(0.83%)>CcEE(0.47%)>CCEE(0.02%). There were significant differences in Rh blood type distribution among Nanning, Guangzhou and Shenzhen(P<0.05). Differences in Rh phenotype distribution between male and female were noticed in Shenzhen(P< 0.05), but not in Nanning or Guangzhou. 【Conclusion】 The distribution of Rh blood group in Shenzhen, Nanning and Guangzhou were significantly different from each other, therefore regional characteristics should be considered when carrying out Rh-compatible blood transfusion, so as to guarantee the security of transfusion and reduce the incidence of unexpected antibodies.

Objective:To explore the etiology, clinical diagnosis and treatment strategy of Lesch Nyhan syndrome.Methods:We retrospectively analyzed 2 patients with severe dyskinesia, mental retardation and complicated renal calculi who were admitted to the first people's Hospital of Zhengzhou in August 2019. Case 1, male, 9 years old, had multiple urinary calculi for 1 year. The patient came to the local hospital because double multiple kidney stones and bladder stonesa year ago. The patient had been treated with transurethral holmium laser lithotripsy for bladder stones. The results of infrared spectrum showed that the bladder stone was anhydrous uric acid stone. A week ago, color Doppler ultrasound showed multiple kidney stones and bladder stones. The patient was underdeveloped, mentally retarded and had a full-term cesarean section. There was no history of hypoxia, asphyxia and rescue of the patient. He had the following clinical manifestations: In the waking state, he was no language response to any stimulation. The nasolabial fold on the right was shallow and the corner of the mouth was oblique to the left. He lost the large movements such as lifting head, sitting alone, standing. The trunk showed torsion spasticity, limb muscle strength 2-3, limbs showing spastic hypertonia, limb joints stiff, hands showing fist-like, no involuntary movement and muscle fasciculation. The biceps reflex and knee tendon reflex were not elicited, and the pathological reflex was positive. Serum uric acid was 517 μmol/L. The Case 2 came from the same family, male, 6 years old, had the similar symptoms to his elder brother case 1. The family members complained on behalf of the child about intermittent fever for more than 2 years. The imaging examination of case 2 revealed kidney stones. Serum uric acid was 373 μmol/L. Whole Exome Sequencing and Sanger Sequencing were used to find the genetic causes of the two siblings. The NCBI-Homologene database was used to find the homologous sequence of the human HPRT1 gene, and the human HPRT1 gene sequence was compared with other species to analyze the protein conservation. The online website PredictProtein (http: //www.predactprotein) was used to predict the two-dimensional structure of the HPRT1 gene. The reported cases were summarized and same with the treatment plan.Results:A De novo mutation [c.571T>G(p.Tyr191Asp)] was found in the HPRT1 gene of the child, which was inherited from the mother. Lesch Nyhan syndrome can be diagnosed by the results of gene examination combined with clinical manifestations. The amino acid Tyr at the 191 position and the amino acids before and after it were highly conserved. Amino acid 191 was involved in the β-strand of the protein. We treated the patients with the lowest dose of allopurinol and children's conventional dose of potassium sodium bicitrate granules, and low purine diet. After 3 months of treatment, the serum uric acid was decreased, and the urinary calculi did not increase significantly.Conclusions:Combining with the clinical manifestations of children, HPRT1 gene might be the cause of pediatric disease and the two siblings could be diagnosed as Lesch-Nyhan syndrome. For such patients, the lowest dose of allopurinol and children's conventional dose of potassium sodium hydrogen citrate granule combined with diet could be more effective.