The Expression And Clinic Significance Of Proinflammatory Cytokine HMGB1 In Asthma And COPD

BACKGROUND AND AIM:Bronchial asthma (asthma) is a chronic respiratory disease characterized by chronic airway inflammation, mucus hypersecretion, airway hyperresponsiveness and airway remolding, which has a great harmful impact on health. Chronic Obstructive Pulmonary Disease (COPD) also a major public health problem. It is the fourth leading cause of chronic morbidity and mortality in the world.Chronic inflammation is involved in the pathogenesis of asthma and COPD. Cytokines play a key role in orchestrating the chronic inflammation and structural changes of the respiratory tract in both asthma and COPD. Proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, are found in increased amounts in the sputum and BAL fluid in individuals with asthma and COPD and amplify inflammation. Although the clinical symptoms of both diseases are caused by airway narrowing as a result of inflammation in the airways, there are marked differences in the patterns of underlying inflammation including the different expression of cytokines.High mobility group box 1 (HMGB1) is a new proinflammatory cytokines to amply and maintain inflammation.HMGB1, nuclear and cytosolic ubiquitous protein, is a DNA-binding protein which participates in maintaining nucleosome structure, regulation of gene transcription and so on. Recently, however, this protein has been identified as acting as a pro-inflammatory mediator when found extracellularly in animal models and human disease. HMGB-1 is actively secreted by innate immune cells such as macrophages and monocytes. HMGB1 is passively released by injured and necrotic cells and has been shown to stimulate necrosis-induced inflammation. Moreover, HMGB1 induces other cytokines such as TNF-α, IL-1, IL-6, and IL-8, and is also an activator of endothelial cells (HUVEC) leading to the upregulation of adhesion molecules. HMGB-1 has been shown to interact with toll-like receptor (TLR) 2, TLR 4, and the receptor for advanced glycation end products (RAGE) in established cell lines and animal models. This leads to a downstream translocation of NF-κB inducing immunostimulatory and chemotactic responses. Anti-HMGB-1 antibodies have been demonstrated to confer protection in animal models of sepsis, endotoxemia, and arthritis. Elevated HMGB-1 levels in serum have been found in clinical inflammatory conditions such as sepsis and rheumatoid arthritis.Because many cytokines like TNF-α,IL-1βwhich were found in increased amounts in asthma and COPD patients, can induce HMGB1 to release from immune cells such as macrophages and monocytes, We suppose that HMGB1 may highly expresses in asthma and COPD patients,but now litter research is made about the expression and effect on asthma and COPD.So the purposes of this research are to investigate the different expression of HMGB1 in induced sputum and plasma of asthma and COPD patients, to investigate the correlation between the expression of HMGB1 and the severity of disease and inflammation phenotype,subsequently to investige the expression of HMGB1 in a OVA sensitized murine model of allergic asthma, in order to determine the effect on asthma and COPD. METHODSClinical trial1,Firstly we made up the inclusion criteria and exclusion criteria of this experiment.2,Then we collected datas of patients and the patients underwent flow-volume spirometry.3,Then we collected induced sputums and plasma of patients.4,At last we examined the HMGB1 level of induced sputum and plasma using ELISA assay.Animal experimentsthe expression of HMGB1 in lung tissue and BALF in a OVA sensitized murine model of allergic asthma.18 female BALB/c mice were randomly by table of random numbers divided into 3 groups:control group, OVA group (asthma group), OVA/DM group(dexamethasone group).Airway hyperresponsiveness to MeCh (Sigma; 0,3.125,6.25,12.5,25 and 50 mg/ml in isotonic saline) was assessed by whole body plethysmography (Buxco), Penh value of different groups were recorded and compared.BAL fluid was isolated, total cells recovered were counted, and the cellular composition of BALF was determined using Hematoxylin-eosin staining. Cytokines (IL-4,IFN-γ,HMGB1) in BAL of mice were examined by ELISA assay.Pulmonary histopathology:mice lungs were excised and fixed. These tissues were then embedded in paraffin, cut sections and stained with hematoxylin and eosin (H&E).The expression of HMGB1 was examined by western blotting, Quantity One software was used to analyze the comparative production of HMGB1 protein. Statistical analysisStatistical analysis was performed using SPSS 13.0 software. All data were expressed as mean±SD. One-way analysis of variance (one-way ANOVA) or Welkch test and LSD or Dunnett's T3 methods were used to determine differences between experimental groups according to test of homogeneity of variance. Chi-square test was used to compare the mean percentage and constituent ratio. Spearman's rank correlation test was used for variables.Significance was accepted when p< 0.05.RESULTS:The result of Clinical trial1,Induced sputums and peripheral blood were collected from 30 healthy control subject,51 asthmatic patients and 49 COPD patients.2,The plasma HMGB1 level (11.86±6.81 ng/ml) in COPD group was higher compared with healthy control group (4.14±1.65 ng/ml) and asthma group (6.22±4.04 ng/ml) (P=0.000);compared with healthy control group,it elevated in asthma group(p<0.005). Induced sputum HMGB1 level (15.51±10.81 ng/ml) in COPD group was higher compared to healthy control group (1.01±1.61 ng/ml) and asthma group (4.45±3.44 ng/ml) (P=0.000);compared to healthy control group,it elevated in asthma group(p=0.000).3,there are no different plasma HMGB1 levels in COPD patients between stable period and exacerbation period(p=0.974). HMGB1 significantly elevated in sputum of patients during acute COPD exacerbation compared with when they were clinically stable(p=0.029).4,there are no different plasma and induced sputum HMGB1 levels between asthma patients of different Severity. HMGB1 were significantly elevated in the sputum of severe and very severe COPD patients compared with moderate COPD patients (p=0.029) 5,The plasma HMGB1 levels showed significantly negative correlations with FVC(r=-0.486, p=0.000);PEF(r=-0.545, p=0.000); FEV1(r=-0.514, p=0.000); FEV1%(r=-0.447,p=0.000); FEV1/FVC (r=-0.437, p=0.000). The induced sputum HMGB1 levels showed significant negative correlations with FVC (r=-0.604,p=0.000);PEF (r=-0.684, p=0.000); FEV1 (r=-0.685, p=0.000) FEV1%(r=-0.658,p=0.000); FEV1/FVC (r=-0.601,p=0.000). The plasma HMGB1 level showed significantly positive correlations with peripheral neutrophilic counts (r=0.324, p=0.001) and percentange of peripheral neutrophilic granulocyte (r=0.374, p=0.000).The induced sputum HMGB1 level showed significantly positive correlations with percentange of induced sputum neutrophilic counts (r=0.504, p=0.000)and induced sputum neutrophilic counts(r=0.593, p=0.000).Results of animal experiments1. Airway responsiveness to MeCh increased along with its concentration in different groups. Mice in the OVA group have the most high%baseline Penh value compared to mice in other groups.A significantly increased%baseline Penh value in OVA group (419.94±72.57) and DM group (285.48±118.43) compared with control group (286.16±96.49) from 6.25 mg/ml MeCh (P<0.05). A significantly decreased %baseline Penh value in mice of DM group compared with OVA group (P<0.05)2. Level of IL-4 in BAL were elevated in OVA group (66.25±18.07 pg/ml), higher than those of control group (38.30±9.60pg/ml) (P<0.05), decreased in OVA/DM group (P<0.01). There were no significantly different IFN-γlevel in BALF between control group (14.41±7.65 pg/ml) and OVA group (6.31±2.17 pg/ml)(P=0.124), Level of IFN-γin BALF decreased in OVA/DM group(9.86±1.77 pg/ml) compared with OVA group (P<0.05)3. The numbers of total cells, eosinophils in OVA group (7.92±1.02)×105ml were higher compared with control group (3.48±0.51)×105ml (P<0.01). The HMGB1 level of OVA group in BALF(6.31±4.05 ng/ml)were significantly higher than those of control group (2.59±0.73ng/ml)(P=0.017), but the level of HMGB1 in OVA/DM group (3.39±0.50 ng/ml) don't significantly decrease compared with OVA group. (P=0.052)4. H&E staining showed that epithelial cells hypertrophy/hyperplasia, mucus hypersecretion, tremendous eosinophil and lymphocytes infiltration were observed in OVA group. Dexamethasone treatment inhibited the airway inflammation to some degree.5. HMGB1 protein in lung tissue of mice was examined by Western blotting, the comparative production of HMGB1 protein in OVA group (2.08±0.87) was higher compared with control group (0.85±0.30) (P=0.032), but the expression of HMGB1 protein (1.15±0.48) in OVA/DM group don't significantly decrease compared with OVA group. (P=0.133)CONCLUSIONS:1,HMGB1 highly expresses both in plasma and induced sputum of asthma and COPD patients,We propose that the HMGB1 might is involved in the pathogenesis of asthma and COPD.2,HMGB1 play a important role in exacerbation period of COPD.The HMGB1 level showed significantly negative correlations with lung function parameters,Maybe HMGB1 is a new marker of the COPD severity.3,The plasma HMGB1 level showed significantly positive correlations with peripheral neutrophilic counts, the induced sputum HMGB1 level showed significantly positive correlations with induced sputum neutrophilic counts,we suppose that HMGB1 is mainly secreted from stimulated neutrophilic granulocyte in Peripheral blood and induced sputum. 4,The expression of HMGB1 in lung tissue and the level of HMGB1 in BAL significantly increases in a OVA sensitized murine model of allergic asthma, it implies that HMGB1 is involved in the chronic inflammatory process of asthma.