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AIM:To investigate the influence of matrine (MA) on the phenotype switching of mouse mono-cytes and alveolar macrophages induced by bleomycin ( BLM) .METHODS:All mice were randomly divided into normal saline (NS) group, BLM group, BLM+NS group and BLM +MA group.The mice were administered with BLM at 2.5 mg/kg via oropharyngeal instillation .The mice in BLM+MA group were treated with MA (15 mg· kg-1 · d-1 ) by oral gavage following BLM administration .The mice were sacrificed on days 3, 7, 14, and 21.The lungs were removed for pathological analysis .The circulating monocyte subsets and polarization state of bronchoalveolar lavage fluid ( BALF)-de-rived alveolar macrophages were analyzed by flow cytometry .RESULTS:The results of HE and Masson trichrome staining in BLM and BLM+NS groups exhibited classical pathological stages of lung fibrosis , including acute inflammation phase and later fibrosis phase .Compared with BLM +NS group, MA treatment alleviated the inflammatory response and the de-gree of fibrosis induced by BLM (P<0.05).There was a rapid change of circulating Ly6Chi monocytes and its magnitude was positively associated with the pulmonary inflammatory response .An expansion of M2-like alveolar macrophages was positively correlated with the magnitude of lung fibrosis .Moreover , MA treatment partially normalized the phenotype switc-hing of monocytes and alveolar macrophages .CONCLUSION:Matrine treatment attenuates BLM-induced pulmonary injury partially via modulating the phenotype switching of monocytes and alveolar mocrophages .
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Objective Pneumosilicosis is characterized by pulmonary fibrosis and cannot be effectively treated at present. This study was to explore the changes of monocyte subsets in the mouse model of silicon dioxide-induced experimental pneumosilicosis and the correlation of the changes with lung inflammatory injury and pulmonary fibrosis. Methods A total of 100 male C57BL/6J mice weighing 18-22 g were equally randomized into a normal saline (NS) group and a silicon dioxide (quartz) group.The model of experimental pneumosilicosis was established by oropharyngeal aspiration of quartz suspension.At 1, 3, 7, 14, and 28 days after treat-ment, the mice were sacrificed and the proportions of different circulating monocyte subpopulations determined by flow cytometry.Dif-ferent types of inflammatory cells in the bronchoalveolar lavage fluid ( BALF) were routinely counted.The inflammation score and col-lagen volume fraction ( CVF) of the lung tissue were obtained by HE and picrosirius red staining. Results At 7 days after quartz treatment, silicotic nodules were observed in the lung tissue.Compared with the NS controls, the model mice showed significantly in-creased inflammation score and CVF at 7 days (0.920 ±0.049 vs 1.400 ±0.089, P0.05).Compared with the NS controls, the quartz-treated mice showed markedly increased proportion of Ly6Chimonocytes at all time points, which peaked at 7 days (58.750 ±2.386 vs 78.300 ±2.517, P<0.01), with a positive corre-lation with the inflammation score (P<0.01) and CVF of the lung tissue (P<0.01) at 7 and 28 day. Conclusion The propor-tions of circulating Ly6Chi and Ly6Clo monocytes changed dynamically in the murine model of quartz-induced experimental pneumosilico-sis.The increased proportion of the Ly6Chi monocyte subpopulation might be closely related with lung inflammatory injury and pulmona-ry fibrosis in pneumosilicosis.
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Objective To compare the strengths and limitations of the old and revised guidelines for the diagnosis in patients with idiopathic pulmonary fibrosis(IPF).Methods Patients who were admitted and diagnosed as interstitial lung diseases (ILDs) in our hospital from 2009 to 2014 were enrolled in our study.Eachpatient was reevaluated respectively according to the old and revised guidelines of IPF.Results A total of 553 cases were initially reviewed,among whom 355 cases were excluded for pulmonary fibrosis secondary to definite underlying diseases,28 excluded due to high resolution computed tomography(HRCT) not done,26 excluded because serum immunology examination was not available.The remaining 144 cases were finally enrolled in this study including 92 males and 52 females with median age 21-92 (68 ± 11) years old.Twenty five patients (17.4％,25/144) met the diagnostic criteria of IPF by the old guideline.While by the revised guideline,53 patients (36.8％,53/144) were diagnosed as classical IPF,29 patients (20.1％,29/144) as probable cases,another 69 non-IPF patients accounting for 43.1％ (62/144).The result revealed that there's a significant difference between the two guidelines in the diagnosis of IPF.Conclusions The revised guideline favors an early diagnosis of IPF and simplifies the diagnostic process.However the possibility of over diagnosis or missed diagnosis by the revised guideline does exist.On the other hand,despite of the delayed diagnosis by the old guideline,it may reduce the misdiagnosis of IPF in some circumstance.
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AIM: To establish a cell line of stable silencing of P2X7 receptor (P2X7R) expression through short hairpin RNA ( shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line.METHODS:Stable silencing of P2X7 R gene in the RAW264.7 cells was achieved by re-combinant shRNA plasmid targeting murine P2X7 R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X7 R silencing in G418 resistant cells was verified by immunofluorescent micros-copy and real-time PCR, respectively.The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation as-say.The cell cycle distribution and apoptosis were evaluated by flow cytometry.RESULTS:The expression of P2X7 R at mRNA and protein levels was down-regulated by 80% in shP2X7 R group compared with negative control ( NC) plasmid transfection.In addition, P2X7 R-silencing cells exhibited higher proliferative activity compared with NC and wild-type RAW264.7 cells (P<0.05).Compared with NC cells, P2X7R silencing resulted in an increase in the phagocytosis of the cells ( P<0.05) .CONCLUSION:A cell line RAW264.7 of stable silencing of P2X7 R expression was successfully es-tablished.P2X7 R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macro-phages.
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Objective:To screen differentially expressed proteins in serum proteomic patterns in patients with Rheumatoid Arthritis associated Interstitial Lung Disease by Surface enhanced laser desorption /ionization mass spectrometry with Protein Chip ,and evaluate the value of a preliminary study.Methods: WCX2 ( Weak cation exchange chip ) and Surface enhanced laser desorption/ionization mass spectrometry with ProteinChip were performed to test serum proteins in 19 Rheumatoid Arthritis associated Interstitial Lung Disease,15 Rheumatoid Arthritis patients and 13 health adults,Biomarker Wizard ,Biomarker Pattern and Spss13.0 software were used in combination to analyze the data.Results:The mass/charge ratio ( M/Z) value of 142 protein peaks were detected in serum , there were 4 proteins differentially expressed with statistical significance among them ( P<0.05;Student′s t-test ) , which were the proteins with M/Z 3382.59 ,3453.39 ,11886.0 ,5825.48;the proteins with M/Z 11689.4 ,2266.49 ,1020.22 ,4392.28 ,5074.38 and 2764.69 were selected to establish the optimal diagnostic model.The sensitivity was of was 90%(9/10),the specificity was of 90%(18/20).Conclusion: Surface enhanced laser desorption/ionization time of flight mass spectrum can screen out the significantly different proteins in serum of patients with Rheumatoid Arthritis associated Interstitial Lung Disease.As bomarkers , both of their sensitivity and specificity were high and determination have good prospects for clinical application .
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Objective The unbalanced phenotype of pe-ripheral blood monocyte is closely related to the pathological progres-sion of pulmonary fibrosis .The present study was designed to address the dynamic changes of circulating monocyte subsets in the experimen-tal mouse model of pulmonary fibrosis , and explore the relationship of circulating monocyte subsets with pulmonary inflammation and fibro-sis. Methods A total of 100male C 57BL/6J mice were random-ized as control group and a bleomycin A 5 group to be treated with sterile saline and bleomycin A5 at 2 mg/kg, respectively.The mice were sacrificed on day 1, 3, 7, 14, and 21 after treatment.The inflammation score and collagen volume fraction ( CVF) of the lung tissue were obtained by HE and Masson staining .The total number and different types of cells in the bronchoalveolar lavage fluid ( BALF) were counted using the routine method .The mRNA expressions of collagens ⅠandⅢwere determined by real-time PCR, the content of hydroxyproline (HYP) assayed by the chloramine-T method, and the proportions of different monocyte subsets measured by flow cytometry . Results Compared with the saline control , the bleo-mycin A5 group showed significantly increases in the inflammation score at 3 and 7 days ( P<0 .01 ) , CVF at 14 and 21 days ( P<0.01), and the numbers of total cells and macrophages in BALF at 3-21 days, the count of neutrophils granulocytes at 1-3 days (P<0.01), The numbers of neutrophile granulocyles were significant higer than that in control groups on the 1st(9.086 ±1.268 vs 1.108 ±0.229), 3rd(5.551 ±0.511 vs 0.315 ±0.100) and 7th(8.093 ±0.922 vs 0.249 ±0.074)day.The mRNA expressions of collagens ⅠandⅢat 14 and 21 days (P<0.05), the content of HYP at 7-21 days (P<0.01), and the proportion of Ly6Chi mon-ocytes on day 1, which peaked on day 3 (P<0.01) and then decreased from day 14 to 21.The proportion of Ly6Chi monocytes was positively correlated with the inflammation score (P<0.000 1) and CVF of the lung tissue (P=0.001 3). Conclusion In the mouse model of bleomycin A5-induced pulmonary fibrosis, dynamic changes of circulating Ly6Chi and Ly6Clo monocyte subsets occurred in different pathophysiological stages .Compared with the pathological process of inflammatory infiltration , Ly6Chi circulating monocytes displayed a rapid response to tissue injury and inflammation .The increased proportion of Ly6Chi monocyte subsets might be closely re-lated with pulmonary inflammation and fibrosis .
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Objective To identify whether the helper T lymphocyte 1 (Th1)/helper T lymphocyte 2 (Th2) of patients' serum and bronchoalveolar lavage fluid (BALF) at admission could represent the severity of idiopathic pulmonary fibrosis (IPF) and whether its change at six months could predict the progression of the disease.Methods Eighty-three patients with IPF were subjected to pulmonary function tests (PFFs),dyspnea scores,arterial blood gas analysis,six-minute walk test (6MWT) and high-resolution computed tomography (HRCT).The serum and BALF specimen of these patients were obtained as well as 20 control serum and 10 control BALF specimen.A total of 55 patients were followed up,and their BALF and serum levels of interferon γ (IFNγ) and IL-4 were detected by enzyme-linked immunoadsorbent assay (ELISA).The correlation between the IFNγ/IL-4 levels (at admission and the change of that at six months follow-up) and the clinical,physiological and image features in the IPF patients were analyzed.Results The baseline serum and BALF level of IFNγ/IL-4 (0.8 ± 0.3) in the IPF patients was lower than that in the control group (1.4 ± 0.2),which showed significant correlation with the course of disease,dyspnea scores,FEV1 ％,FVC％,TLC％,maximum desaturation,6MWD and CT-fib (all P values ＜ 0.05).The serum level of IFNγ/IL-4 showed positive correlation with CT-alv (r =0.340,P ＜ 0.01).During follow-up,no statistic difference was found in the serum levels of IFNγ,IL-4 and IFNγ/IL-4 between the IPF patients with or without glucocorticoids treatment.There were significant aggravation in the dyspnea scores,FEV1％,FVC％,CT-alv,CT-fib,IFNγ and IL-4 at six months follow-up.Significant correlation had been showed between the change of the serum IFNγ/IL-4 level with the changes of the dyspnea scores,FVC％,TLC％,DLCO％,6MWD and CT-fib in the IPF patients (P ＜0.05).Conclusions There are disequilibrium of the Th1/Th2 in the serum and the BALF of the IPF patients.The Th1/Th2 level could represent severity of the disease,and the serum level change of Th1/Th2 in the follow-up could predict the progression of the diseases in the IPF patients.