Study On The Relationship Between TIPE2 And Coagulation Substances TF And TSP-1 In Asthmatic Patients And Its Mechanism

BackgroundBronchial asthma is a chronic airway inflammatory disease.A large number of studies have shown that bronchial asthma is closely related to hyper coagulable state.And the interaction between inflammation and coagulation disorders jointly promoted the development of bronchial asthma.Tissue factor(TF)is an important promoter of exogenous coagulation pathways and plays a key role in linking the coagulation and inflammatory network system.In addition to its procoagulant activity,TF could also bind to complementary ligand,then activating the inflammatory signal transduction pathway in vascular system.In turn,inflammatory factors could promote the expression of TF in epithelial cells and monocytes.Studies have shown that asthma patients were accompanied with increased plasma TF levels and activity,which aggravated asthma condition and promoted airway remodeling.Nevertheless,the role of TF in the mechanism between bronchial asthma hyper-coagulation and inflammation still needed further research to clarify.Thrombospondin-1(TSP-1)is a glycogen protein in platelet alpha granules.Under the stimulate of inflammatory factor,TSP-1 could be released to serum by macrophages,monocytes,vascular smooth muscle cells,endothelial cells,epithelial cells and other cells,which is involved in platelet adhesion and aggregation,and play an important role in thrombotic disease.TSP-1 could induce macrophage chemotaxis and further promote inflammatory response.Studies have shown that TSP-1 is expressed abnormally in a variety of lung diseases such as COPD and chronic pulmonary heart disease,but its role in bronchial asthma is still unknown.Tumor necrosis factor α induced protein-8 like-2(TIPE2)is a newly discovered immune response regulatory molecule,mainly expressed in immune cells such as lymphocytes and macrophages,which play an important role in regulating immune balance and inflammatory response.Decreased TIPE2 expression in peripheral blood mononuclear cells(PBMCs)was found in children with asthma,but the underline mechanism is not clear.Studies have shown that TIPE2 can regulate the expression of TF through p38 MAPK and ERK1/2 signaling pathway in umbilical vein endothelial cells.But the relationship between TIPE2 and coagulation substances in asthma remains unknown.This study aimed to investigate the expression of TIPE2,TF and serum TF and TSP-1 in PBMCs of asthmatic patients and the correlation between TIPE2 and coagulation substances TF and TSP-1.In addition,we further explored the expression of TIPE2 and TF in THP-1 cells stimulated by house dust mite extract(HDME)and the related mechanism.ObjectiveThis study amied to investigate the relationship between TIPE2 and TF、TSP-1 in bronchial asthma patients,and to explore the expression of TIPE2 and TF in THP-1 cells stimulated by house dust mite extract and the expression of related signal pathway.Then provide new ideas for the diagnosis and treatment of bronchial asthma.Materials and Methods1.According to the inclusion and exclusion criteria standards,65 asthmatic patients were collected as case group,and 40 healthy individuals were collected as control group.2.The expression of TIPE2 and TF protein in PBMCs of asthmatic patients and healthy controls were detected by Western blot.3.The levels of TF and TSP-1 in serum of asthmatic patients and healthy subjects were detected by ELISA.4.The expression of TIPE2 and TF mRNA in THP-1 cells was detected by RT-PCR after house dust mite extract stimulation.5.The changes of p38 MAPK and NF-κB signal pathway protein in THP-1 cells were detected by Western blotting after house dust mite extract stimulation.Results Part One: Study on the expression and relationship between TIPE2 and TF、TSP-1 in asthmatic patients.1.Compared with healthy controls,the expression of TIPE2 protein in PBMCs of bronchial asthma patients was decreased(p < 0.01),while the expression of TF protein was increased(p < 0.01).2.Compared with healthy control group,the levels of TF and TSP-1 in sera of patients with bronchial asthma were significantly increased(p < 0.01).3.The expression of TIPE2 protein in PBMCs of bronchial asthma patients was negatively correlated with the level of TF protein expression both in PBMCs and serum(r =-0.3828,p < 0.01;r =-0.5422,p < 0.01).In addition,the expression level of TIPE2 protein was negatively correlated with TSP-1 protein expression in serum(r =-0.3013,p < 0.05).Part Two: Expression of TIPE2 and TF and the changes of p38MAPK/NF-κB signal pathway inTHP-1 cells stimulated by HDME.1.The relative expression of TIPE2 mRNA was decreased and the relative expression of TF mRNA was increased in THP-1 after stimulation with different HDME concentrations(0.1μg/ml,1μg/ml,10μg/ml),and the most significant difference was at the stimulated concentration of 1μg/ml(p < 0.05).The expression level of TIPE2 mRNA and TF mRNA in THP-1 cells was increased or decreased at different time(1h,2h,4h,8h)after stimulation with 1μg/ml house dust mite extract and the most significant change was at 4h(p < 0.05)2.The expression of p38 MAPK and NF-κB phosphorylated protein were increased with the increase of time and was most significantly at 1h.Conclusions1.The expression of TIPE2 protein in PBMCs of asthmatic patients was decreased and the expression of TF protein was increased.Serum levels of TF and TSP-1 were elevated.TIPE2 was negatively correlated with TF and TSP-1 levels,which indicated that TIPE2 might participate in the pathological mechanism of asthma by interacting with coagulating substance TF and TSP-1.2.It was confirmed that the expression of TIPE2 mRNA was decreased and the level of TF mRNA was increased after THP-1 was stimulated by house dust mite extract,and phosphorylation of p38 MAPK and NF-κB was enhanced.