The Research Of Relationship Of Thrombin Activity And Airway Remodeling In Asthmatic Rats

Bronchial asthma referred to as asthma,it is a common respiratory disease,characterized by the presence of airway inflammatory cells and the change of airway structure.It is also called "airway remodeling".It can lead to irreversible airway obstruction, airway hyper-reaction, decreased lung function, etc.irway remodeling is caused by persistent inflammation and epithelial cell damage,It is now very clear that airway remodeling can lead to difficult to treat severe asthma.Many studies show that bronchial smooth muscle cells in the airway remodeling plays a key role, not only through their contractile function, more important is, through a variety of media role in asthma patients, but the mechanism has not yet been determined. At present, the main treatment of asthma is to control symptoms.Therefore, it is of great significance to the prevention and treatment of asthma by discussing the mechanism of airway remodeling in asthma.Many studies have indicated that thrombin plays an important role in the process of airway remodeling.In order to explore the relationship between thrombin activity and airway remodeling in asthma, we established a model of asthma rats, and explore the molecular mechanism of thrombin in airway remodeling in asthmatic rats.This study is divided into two parts:①Thrombin activate ERK1/2 signaling pathway through PAR-1 in asthmatic rats;②To investigate the effect of thrombin on airway inflammation in asthmatic rats.Part One Thrombin activate ERK1/2 signaling pathway through PAR-1 in asthmatic ratsObjectivesAirway remodeling is the reason of airflow obstruction and is the core of the pathophysiology of asthma, its characteristics is airway smooth muscle hypertrophy and some other changes, including basement membrane thickening, loss of epithelial cells, inflammatory cell infiltration and goblet cell hyperplasia.The precise mediators of these changes and their regulation are still not clear.And airway remodeling has a very important impact on asthma:airway hyper-reaction, irreversible airflow limitation, acute asthma and even lead to death.Therefore, it is very important and necessary to study the mechanism of airway remodeling. Thrombin is formed in the blood coagulation cascade, in addition to its proteolytic action, thrombin can activate many cells by activating protease activated receptors.In recent years, studies have indicated that thrombin plays an important role in the airway inflammation and airway remodeling, in addition to the central role of the coagulation mechanism.But the specific mechanism is still unknown.Extracellular signal-regulated kinase (ERK) is an important signal transduction pathway in mitogen-activated protein kinase(MAPK), ERK1 and ERK2 is one of the most perfectThey are activated by phosphorylation,and regulate cell proliferation, differentiation, apoptosis and so on.PERK1/2 for its phosphorylation level,It can regulate the abnormal proliferation and secretion of airway smooth muscle cells, and participate in airway remodeling。The protease activated receptor-1 (PAR-1) is a G-coupled membrane protein that can be activated by serine proteases such as thrombin.In the respiratory system, the expression of PAR-1 in epithelial cells and smooth muscle cells.Therefore, we propose a hypothesis that thrombin activates the ERK1/2 pathway through PAR-1。 which is then involved in asthmaMethods36 female rats were randomly divided into 6 groups, normal control group (SAL group), asthma model group (OVA+SAL group), thrombin group (OVA+thrombin group), hirudin group (OVA+hirudin group), thrombin+pERKl/2 inhibitor PD98059 group (OVA+thrombin+pERKl/2-inh group), thrombin+PAR-1 inhibitor ER-112780-06 group (OVA+thrombin+PAR-1-inh group),6 rats in each group.The rats were sensitized with ovalbumin to establish the asthmatic mode. Model group were injected with ovalbumin (OVA) (1 ml/kg) on day 1 and 8.From day 15,spray 2% ovalbumin every 30 minutes,3 times a week, a total of 6 weeks, SAL group with normal saline instead. OVA+thrombin group were treated with inhalation of thrombin (72u/one) before inhalation from day 15.OVA+hirudin group was given hirudin aerosol 2000AUT inhalation. OVA+thrombin+pERK1/2-inh group were treated with inhalation of thrombin (72u/one) before inhalation from day 15 and were treated with intraperitoneal injections of PD98059 (3mg/kg)。 OVA+thrombin+PAR-1-inh group was given thrombin atomization inhalation 72u/ one.and give ER-112780-06(20uM) aerosol inhalation for 30 minutes,Other groups were replaced by saline.After the model was finished, the lung tissue was taken from the rats. The expression of PAR-1 was detected by immunohistochemistry, western blot and PCR, and the expression of pERKl/2 was detected by western blot.Results1. General situationThe rats of group SAL have no dysphoria when excited, the respiratory smoothy, action sensitive and living habits normal, the coat color is shiny;OVA+SAL group have the symptoms of varying degrees of irritability, shortness of breath, abdominal convulsions, incontinence when stimulated, the severe ones’breathing become slow or uneven rhythm, the rats fell down and not move. But after many times after excitation, the above-mentioned symptoms of asthma group was slightly reduced, the coat was dull;OVA+thrombin group have the same symptoms as OVA+SAL group, but the degree was more serious. Later in the rats of group OVA+thrombin are more prone to do not move, hair color dark yellow, there is a death.When the OVA+thrombin+pERK1/2-inh group in the excited, the irritability phenomenon is lighter, faster breathing and occasional incontinence status come out, respiratory rate or rhythm have no change, coat was normal.2.Airway remodelingCompared with group SAL, the characteristics of airway remodeling were more obvious in group OVA+SAL.Compared with group OVA+SAL, the characteristics of airway remodeling were more obvious in group OVA+thrombin.Compared with OVA+SAL group, OVA+thrombin+pERKl/2-inh group had little difference in airway remodeling. Compared with OVA+thrombin group, the goblet cells in the airway epithelium were decreased.Compared with OVA+thrombin+pERKl/2-inh group and OVA+thrombin group, Airway smooth muscle thickening is not obvious.Compared with OVA+thrombin+PAR-1-inhgroup and OVA+thrombin group, Airway smooth muscle thickening is not obvious.4.Detection of pERK1/2 expression in lung tissue of each group with Western blotThe expression level of pERKl/2 in group OVA+SAL was higher than that in group SAL, and there was statistical significance (P< 0.05).The expression level of pERK1/2 in the OVA+thrombin group was statistically significant compared with the OVA+SAL group.The expression level of pERKl/2 in OVA+thrombin+ pERK1/2-inhgroup was statistically significant compared with the OVA+thrombin group. Asthma rats treated with PAR-1 inhibitors,decreased expression of pERKl/2 and TGF-β,and there was statistical significance compared with the OVA+thrombin group (P< 0.05),5.PAR-1 protein expressionThe expression level of PAR-1 positive cells in SAL group was very low,the expression level in OVA+SAL group was very high, and there was statistical significance compared with the SAL group (P< 0.05),The highest expression level of OVA+thrombin group,and there was statistical significance compared with the OVA+SAL group (P< 0.05),The expression in OVA+hirudin group decreased,and there was statistical significance compared with the OVA+thrombin group (P< 0.05),The expression level in OVA+thrombin+PAR-1-inh group was significantly lower,and there was statistical significance compared with the OVA+thrombin group (P< 0.05),Conclusions1. Increased thrombin activity can upregulate the expression of PAR-1 gene and protein in respiratory;2. Thrombin activates ERK1/2 signaling pathway to participate in airway remodeling in asthmatic rats by PAR-1 way.3..Thrombin participates in airway remodeling in asthmatic rats through pERKl/2 signaling pathPart Two The Research Of Relationship Of Thrombin Activity, Airway Inflammation and Airway Remodeling In Asthmatic RatsObjectiveAirway inflammation is the pathogenesis of urgent and chronic lung disease,asthma is included. Nociceptive stimuli can activate a variety of cells, including eosinophils, macrophages, mast cells, fibroblasts, smooth muscle cells, endothelial cells, making them release of vasoactive substances, toxic metabolite and cytokines to participate in acute and chronic bronchial contraction. IL-6 is one of the cytokines involved, and it is also an indicator of inflammation in the presence of inflammation. The study confirmed that thrombin was involved in the airway inflammation through the activation of NF-kB. Repeated inflammatory stimulation can cause tissue damage and subsequent tissue structural changes, namely airway remodeling. In this study, we established a standard rat model of asthma by exogenous thrombin and thrombin inhibitor. By ELISA, HE staining, Immunofluorescence, Immunohistochemistry, Western blot and other test methods to explore the effects of thrombin on the airway inflammation in asthmatic rats, thus to provide new ideas for thrombin to promote discussion on the mechanism of airway remodeling in asthma.Method24 female rats were randomly divided into 4 groups:normal control group (group SAL), asthma group (OVA+SAL group), thrombin group (OVA+thrombin group), thrombin inhibitor group (OVA+hirudin group),6 rats in each group. The 15 day began with 402 ultrasonic atomizer treated with 2% ova suspension 50ml,30 minutes each time, three times a week, continuous excitation 6 weeks. Before atomization30 minutes,in OVA+thrombin group, each rat treated with atomizing inhalation of thrombin 72u, and in OVA+hirudin group, each rat treated with atomizing inhalation of hirudin (500 U/kg),and SAL group with physiological salt spray. After the model was successfully made, the rat alveolar lavage fluid was taken and the thrombin activity was measured by ELISA method. Lung tissue samples were collected, using HE staining to observe the effect of inflammatory cells infiltration and immunofluorescence to detect rat airway claudin-1 expression, immune group of method to detect the IL-6 expression, Western blot determination of NF-kB protein expression.Result1. Inflammatory infiltration of lung tissueIn group OVA+SAL, there was a large number of inflammatory cell infiltration in the bronchial wall, septum and blood vessels, which was statistically significant compared with the SAL group(P<0.05);The inflammatory infiltration in OVA+thrombin group was more obvious than that in the OVA+SAL group (P<0.05);The inflammatory cell infiltration in OVA+hirudin group was less than that in OVA+thrombin group (P<0.05).2.The expression of claudin-1 was detected by immunofluorescence.In group OVA+SAL, the expression of Claudin-1 in airway epithelial cells was decreased, and the epithelial integrity was slightly damaged, compared with the SAL group there were differences.The expression of Claudin-1 in the airway epithelial cells of OVA+thrombin group was weak, and the epithelial integrity was severely damaged, and there was a difference between the OVA+SAL group and the SAL group.The expression of claudin-1 positive cells in OVA+hirudin group was more than that in OVA+thrombin group.3.The expression of IL-6 in lung tissue was detected by immunohistochemistry.The expression of IL-6 in OVA+SAL group was higher than that in group SAL (P<0.05).The positive expression of IL-6 in OVA+thrombin group was significantly higher than that in group OVA+SAL (P<0.05).The expression of IL-6 was decreased (P<0.05) in the OVA+hirudin group compared with the OVA+thrombin group.4.Western bolt to detect the expression of NF-kB protein in lung tissue.The expression of NF-kB protein was increased (P<0.05) in OVA+SAL group compared with SAL group.The expression of NF-kB protein in OVA+thrombin group was higher than that in OVA+SAL group (P<0.05). The expression of NF-kB protein in group OVA+hirudin was lower than that in OVA+thrombin group (P<0.05).Conclusion1.Thrombin can promote inflammatory response in the airway of asthmatic rats;2.Thrombin can lead to the decrease of claudin-1 expression in airway epithelial cells of asthmatic rats;...