| BackgroundChronic Obstructive Pulmonary Disease(COPD)is a common,preventable and treatable disease that is characterized by persistent respiratory symptoms and airflow limitation that is due to airway and/or alveolar abnormalities usually caused by significant exposure to noxious particles or gases.Pathological changes not only restricted to the lung,but also with systemic and pulmonary adverse reactions.COPD is a leading cause of morbidity and mortality worldwide,that cause an economic and social burden.When patients suffering breathing difficulties,lung function damage is seriously,and the treatment cannot reverse lung function decline.The disability and mortality of COPD are predicted to increase over the coming decades due to continued exposure to environmental pollution and aging of the population.The pathogenesis of COPD is complex and unclear,the main pathogenesis include:airway and/or chronic inflammation of the lungs,oxidation/antioxidant imbalance,protease/antiprotease imbalance,airway and/or chronic inflammation of the lungs is the core pathogenesis.Cigarette smoking is the most important risk factor for COPD,other risk factors include environmental exposure,dust inhalation,genetic factors,physical and chemical factors,etc.Abnormal inflammatory characterized by the increasing numbers of inflammatory cells and activation,activated macrophages,neutrophils,T lymphocytes,eosinophils,secrete and release a large number of inflammatory mediators,such as tumor necrosis factor alpha(TNF-a);interleukin-6(IL-6),interleukin-8(EL-8),and transforming growth factor alpha(TGF-a),lead to oxidation/antioxidant imbalance,then the structure of lung tissue damaged.The inflammatory process is a key factor in the development of airflow limitation.Protease/antiprotease imbalance cause the lung tissue structure damages,and led to the extracellular matrix damages,causes airway reconstruction,and eventually lead to COPD.At the same time,oxidative stress can degrade the composition of the lung matrix,promote the occurrence and development of pulmonary inflammatory response,and cause the reconstruction of extracellular matrix.The combination of these factors leads to the reconstruction of airway,forming the dynamic pulmonary hyperinflation,which leads to airflow limitation and the clinical symptoms of dyspnea.High mobility group box-1 protein(HMGB1)is a kind of widespread highly conservative nucleoprotein,widely distributed in the nucleus and cytoplasm of prokaryotic and eukaryotic cells,participate in DNA transcription,replication,repair,and inflammation,etc.Intracellular HMGB binds to the minor groove of DNA,stabilizing nucleosome formation and regulating gene transcription and the activity of steroid hormone receptors.When cells received the external stimulation,HMGB1 can shift from immune cell nucleus to the cytoplasm,then release to outside of the cell.On the other hand,extracellular HMGB1 has been demonstrated to have a role in inflammatory processes,neural outgrowth,smooth muscle cell chemotaxis,migration and proliferation of mast cells,and cell proliferation in tumor and metastasis,and can mediate tumor cell transformation,growth,and drug resistance.Damage associated molecular pattern(DAMPs);including HMGB1,are released from damaged sort necrotic cells,and activated by the immune system.Studies found that HMGB1 is related to the inflammatory process,promote epithelial and mesenchymal cells(such as smooth muscle cell)proliferation.In the pathogenesis of COPD,HMGB1 also plays an important role in the disease process.Airway inflammatory cells such as neutrophils,macrophages and monocytes and airway structure such as epithelial cells,smooth muscle cells and alveolar cells can express HMGB1 at a high level,and HMGB1 can promote the release of other inflammatory cytokines,promote and maintain airway inflammation.Extracellular HMGB1 functions are mediated via binding to specific membrane receptors,leading to the activation of the nuclear factor-kapa B(NF-κB)pathway and also of mitogen activated protein kinase(MAPK).Wang et al.tried to target NF-κB with an inhibitor on mice with COPD obtaining a down regulation of HMGB 1 in lung tissue adding more elements in a blockage strategy of inflammation in an early phase of COPD.In another study,Ojo et al.inhibited RAGE and TLR4 signalling,found that the blockage attenuated HMGB1 ability to increase epithelial cell reparation.The interaction of HMGB1 and its receptors exist outside and inside of cells,plays an important role in the process of cell growth,and the lung tissue damage may be avoided if these links is disturbed.Toll-like receptor 4(TLR-4)is one of the important members of pathogen-associated molecular patterns(PAMP)recognition receptor,via the identification and combination with pathogen associated molecular,prompting intracellular signal transduction pathway activation,promote the production and secretion of inflammation factors,and related closely to innate immunity and acquired immune,and the occurrence and development of various diseases.Its gene polymorphism is associated with COPD.HMGB1 as one of the DAMPs;combining with its corresponding receptors TLR-4;activate its downstream signal NF-κB,cause the release of inflammatory cytokines,including IL-6,IL-8 and TNF-a,promotes the inflammation development.The proinflammatory role of HMGB1 is closely related to signal transduction pathways of TLR-4.In the production and release process of cytokines,if the activity of TLR-4 was inhibited,the release of the cytokines induced by HMGB1 is reduced,so TLR-4 is thought to be one of the necessary receptors of HMGB1 proinflammatory response,and played an important role in the process of HMGB1 signaling transduction.Knockout the TLR-4 gene with siRNA can relieve HMGB1 related inflammatory mediators,such as IL-6,monocyte chemotactic protein 1,matrix metalloproteinases-2.Ulinastatin(UTI)also known as urinastatin,is one of the protease inhibitors,and mainly used in the treatment of disseminated intravascular coagulation,shock,acute pancreatitis,etc.,could restrain the generation of oxygen free radical and reduce the malondialdehyde levels.In vitro experiment results show that UTI can effectively suppress the release of TNF-a and IL-8 induced by lipopolysaccharide(LPS).In addition,UTI can effectively restrain ischemic reperfusion injury,protect the heart,liver and kidney function.Studies have shown that UTI can effectively inhibit a variety of hydrolytic enzyme activity,inhibit excessive release of a variety of inflammatory mediators,and play an important role as a protecting agent in tissue injury and inflammatory,it can also improve the body immunity,improving blood coagulation dysfunction etc.UTI can inhibit the release of inflammation mediators and prevent the occurrence and development of acute lung injury,so it is widely used in clinical treatment of acute lung injury.Whether used alone or combined with other drugs,UTI can effectively reduce multiple types of lung injury.UTI can inhibit the neutrophil infiltration in the lung tissue to reduce the expression of adhesion factor,thereby inhibit the release of a variety inflammatory mediators,effectively alleviate the inflammation in lungs,and reduce pulmonary congestion,edema and exudation,ultimately play an important role in improving pulmonary oxygenation and compliance.At present,the clinical commonly used drugs in the treatment of COPD are mainly bronchodilator(Long-acting beta 2 agonists,long-acting muscarinic antagonist,etc.),inhaled corticosteroids,phosphodiesterase inhibitors,antioxidant and immune modulators.UTI is a broad spectrum protease inhibitors,the application in pulmonary inflammatory diseases such as COPD may be a new treatment option.But it has not attracted wide attention,there is little research about the application of UTI in COPD.This study was undertaken to clarify the effects of UTI on inflammation caused by cigarette smoking stimulation,and COPD-induced lung injury and its mechanism from the perspective of in vitro cell experiments and in vivo animal experiments.The aim of this study was to provide a theoretical basis for the clinical application of UTI in the treatment of COPD.ObjectiveThis study was undertaken to clarify the effects of ulinastatin(UTI)on inflammation caused by cigarette smoking stimulation,and COPD-induced lung injury and its mechanism from the perspective of in vitro cell experiments and in vivo animal experiments.The aim of this study was to provide a theoretical basis for the clinical application of UTI in the treatment of COPD.Method1.The SD male rats was randomly assigned to control group(n=10),COPD group(n=10)and UTI group(n=10).The rats in the control group were perfused by tracheal with physiological saline(100μ/per)without smoking exposure.And COPD model was established with smoking exposure and perfused by tracheal with lipopolysaccharide(LPS,100μl/per)in the COPD group.In UTI group,COPD model was established with the same method in COPD group,then,from 29d,the rats were treated with caudal vein infusion of 100000U/day of UTI,twice a day,for 7d.The pulmonary function of SD rats in each group was detected with small animal lung function instrument,the appearance and pathologiwere observed.The cell apoptosis in lung tissue was detected with TUNEL staining cal changes of lung tissue protocol in each group.The expression of toll-like receptor 4(TLR-4),myeloid differentiation factor 88(MyD88),tumor necrosis factor receptor associated factor-6(TRAF-6),lecktin-like oxidized low density lipoprotein receptor-1(LOX-1),high mobility group box-1 protein(HMGB1)mRNA and protein were detected by fluorescence quantitative PCR and western blot in lung tissue of rats in each group.The correlation between TLR-4 and HMGB1 protein,and the correlation between TLR-4 and HMGB1 protein expression and lung function in rats was analyzed.2.SD rats alveolar type Ⅱ epithelial cells(AT-Ⅱ cells)were isolated and cultured,then identified by HE staining,electron microscopy and modified papanicolaou stain.AT-Ⅱ cells were stimulated by cigarette smoke extract(CSE)to simulate the changes of epithelial cells at the onset of COPD,followed by the intervention of UTI.The CCK-8 test was used to detect the cell proliferation rate in control group(AT-Ⅱ cells regular cultured),CSE group(100μL CSE solution with the concentration of 100μg/mL was added to AT-Ⅱ cells)and UTI group(UTI solution with concentration of 10000U/mL was added to AT-Ⅱ cells,cultured for 24h,and then 100μL CSE solution with the concentration of 100μg/mL was added).The concentration of IL-6,IL-8 and TNF-a in cell culture supernatant were detected by enzyme-linked immuno sorbent assay(ELISA).3.For statistical analyses,SPSS 19.0(SPSS Inc,Chicago,IL)was used.Results1.The small animal lung function instrument was used to measure the lung function of SD rats in control group,COPD group,UTI group.The results showed that the FEV0.3 in the three groups were 5.56±0.31,3.89±0.32,5.07±0.41ml,respectively;the FVC were 6.68 ± 0.43;4.71 ± 0.39,6.11 ± 0.45ml,respectively;FEV0.3/FVC ratio were 90.10±2.78%,74.24±2.31%,83.26±2.56%,respectively,and the PEF were 36.78±3.89,26.16±3.12,32.48±3.26ml/s,respectively.Compared with the control group,the lung function indexs of COPD group were significantly lower(P<0.05,respectively).Compared with the COPD group,lung function indexs in UTI group were increased significantly(P<0.05,respectively).2.Compared with the control group,the lung tissue of the COPD group was damaged,a large number of inflammatory cells were infiltrated,and a large number of alveoli were destroyed.The lung tissue structure of the UTI group was more integrallty,the number of damaged alveoli was decreased and the infiltration of inflammatory cells was relieved.The results of TUNEL showed that a small number of apoptotic cells were observed in lung tissue of the control group,and the number of TUNEL positive cells were much more in COPD group than control group.Compared with the COPD group,the number of TUNEL positive cells in UTI group was significantly reduced,close to the control group.3.Compared with the control group,the relatively expression of TLR-4 mRNA in COPD group,UTI group were 2.78±0.31,1.67±0.26,respectively;the relatively expression of MyD88 mRNA in the two group were 2.67±0.28,1.98±0.25,respectively;the relatively expression of TRAF-6 mRNA in the two group were 2.34±0.41,1.85±0.36,respectively;the relatively expression of LOX-1 mRNA in the two group were 1.89±0.29,1.38±0.37,respectively;the relatively expression of HMGB1 mRNA in the two group were 3.31±0.35,2.01±0.29,respectively.The TLR-4,MyD88,TRAF-6,LOX-1,HMGB1 mRNA in lung tissue of COPD group and UTI group were significantly higher than that in control group(P<0.05,respectively).Compared with the COPD group,the levels of TLR-4,MyD88,TRAF-6,LOX-1 and HMGB1 mRNA were significantly decreased in UTI group(P<0.05,respectively).4.Western Blot was used to measure the TLR-4,MyD88,TRAF-6,LOX-1 and HMGB1 protein in control group,COPD group,UTI group.The results showed that TLR-4 protein in the three groups were 0.82±0.11,1.21 ±0.12,0.93±0.10,respectively;the MyD88 protein were 0.56±0.12,0.73±0.11,0.61 ±0.10,respectively;the TRAF-6 protein were 0.58±0.14,0.76±0.13,0.62±0.10,respectively;the LOX-1 protein were 0.51±0.09,0.95±0.10,0.72±0.11,respectively;the HMGB1 protein were 0.79±0.14,1.36±0.15,1.04±0.13,respectively.The TLR-4,MyD88,TRAF-6,LOX-1,HMGB1 protein in lung tissue of COPD group and UTI group were significantly higher than that in control group(P<0.05,respectively).Compared with COPD group,the levels of TLR-4,MyD88,TRAF-6,LOX-1 and HMGB1 protein were significantly decreased in UTI group(P<0.05,respectively).5.The expression levels of TLR-4 and HMGB1 were positively correlated in the control group,COPD group,UTI group(r = 0.764,P = 0.010;r = 0.814,P =0.004;r = 0.805,P = 0.005,respectively).There was a significant negative correlation between FEV0.3 and TLR-4 and HMGB1 protein expression(r =-0.845,P<0.001;r =-0.820,P<0.001,respectively).6.Alveolar AT-Ⅱ cells HE staining results show that the cell’s cytoplasm contains a lot of dark particles,a white halo around the dark particles,these characteristics are in conformity with the characteristics of the AT-Ⅱ cells.Electron microscopy observation showed that a number of brown lamellar bodies were observed around the nucleus in the field of view,and the lamellar structures were different in size and in concentric circles or parallel arrangement,in which part of the lamellar bodies had been released.The structural integrity of cell membrane and the nucleus was observed,a variety of organelles could be founded in the cytoplasm,and mitochondrial content was rich.The cytoplasm contains organelles such as rough endoplasmic reticulum and Golgi complex.Modified papanicolaou stain results show that there were a large number of dark particles in cytoplasm,there were white halo around the dark particles Of the 400 cells counted,364 AT-Ⅱ cells were counted,and the proportion of AT-Ⅱ cells in the isolated cells was 90%.7.The results of CCK-8 showed that the effect of UTI to cell proliferation activity of AT-Ⅱ cells was highest at 24h at the concentration of 10000U/mL,and the effect of CSE to cell proliferation activity of AT-Ⅱ cells was lowest at 2h at the concentration of 1000μg/mL.Therefore,the study took these concentration and action time as experimental conditions.The proliferation activity of the cells was detected by CCK-8 method.The OD450 of control group,CSE group,UTI group was 1.24±0.73,0.56±0.12,0.91 ±0.25,respectively.The results showed that the proliferation activity of CSE group and UTI group was significantly lower than that of control group(P<0.05,respectively),the proliferation activity of UTI group was significantly higher than that of CSE group(P<0.05).8.The levels of IL-6,IL-8 and TNF-a in the supernatant of the three groups were detected by ELISA.The results showed that the levels of IL-6 in control group,CSE group,UTI group was 0.7 ±0.12,2.65±0.77,1.42±0.56 pg/mL,respectively.The levels of IL-8 in the three group was 0.3910.08,1.51±0.65,0.68±0.32 pg/mL,respectively.The levels of TNF-a in the three group was 0.73±0.11,2.79±0.72,1.28±0.35 pg/mL,respectively.Compared with the control group,the IL-6,IL-8 and TNF-α levels in CSE group and UTI group were significantly higher than those in control group(P<0.05,respectively).And the IL-6,IL-8 and TNF-a levels in UTI group were significantly lower than that in CSE group(P<0.05,respectively).Conclusion1.UTI plays a protective role on lung function in SD rats COPD model.2.UTI plays a protective role on the inflammation of lung tissue,the mechanism may be related to the inhibition of TLR-4,MyD88,TRAF-6,LOX-1,HMGB1 expression.3.TLR-4 and HMGB1 protein expression levels were positively correlated,FEV0.3 and TLR-4,HMGB1 protein expression level were negative correlated,suggesting that UTI may protect the lung tissue injury caused by COPD through the inhibition the TLR-4/HMGB1 signaling pathways.4.CSE stimulation to AT-Ⅱ cells in vitro can increase IL-6,IL-8 and TNF-αlevel significantly;However,after UTI intervention,IL-6,IL-8 and TNF-α is decreased obviously. |
PDF Full Text Request