Effect Of Dabigatran On Human Airway Smooth Muscle Cells Extra Cellular Matrix Deposition Induced By Thrombin And The Mechanism Study

Background:More than ninety years ago,Huber and Koessler first put forward the the concept of airway remodeling in the classic description of fatal asthma.Airway remodeling is one of the chief culprits of irreversible ventilation dysfunction in the clinical.Studies have shown that thickened airway smooth muscle layer devote a lot to the production of asthma patients limited airflow.Extracellular matrix(ECM)plays a physical support role in the normal airway.In the asthmatic airway,ECM can not only exacerbate the airway narrowing but also promote the secretion and proliferation of airway smooth muscle cells(ASMC)to form a vicious circle of airway remodeling.Therefore,the ECM deposition of ASMC is an important therapeutic target on asthma airway remodeling.The coagulation in lung is a hot spot in the research of asthma in recent years.As the key protease in the coagulation system,thrombin can promote different noncoagulation functions including inflammation,proliferation and migration by corresponding with the specific receptors.Among the four kinds of proteinase-activated receptors(PARs),PAR-1 and PAR-2 play key roles in fibroproliferative and inflammatory lung disease,and PAR-1 is the superior.PAR-1 exists on the membranes of many airway structural cells including ASMCs,lung fibroblasts and epithelial cells.However,it is still unclear what kind of roles thrombin-PAR-1 play in airway ECM remodeling.Dabigatran,as a direct thrombin inhibtor,is now the latest oral anticoagulant drug.Recent studies have confirmed that dabigatran aslo have antiinflammatory and antifibrotic effects.Therefore,we speculate that thrombin can correspond with PAR-1 on ASMC activating the ERK1/2 signaling pathway to induce ECM deposition,and these effects can be abrogated by dabigatran.Objective:1.To culture and identify the primary human airway smooth muscle cells.2.To investigate the effect of thrombin on ASMC ECM deposition and the role of PAR-1 antagonist SCH79797,ERK signaling pathway inhibitor U0126.3.To investigate the effect of thrombin on ERK signaling pathway activation of ASMC and the role of SCH79797 and U0126.4.To investigate the effect of dabigatran on ASMC ECM deposition and ERK signaling pathway activation induced by the thrombin-PAR-1.Methods:1.Took the tissue adherence method which was skillfully grasped by our research team to separate and culture the primary human airway smooth muscle cells from the surgical specimens.The immunofluorescence stain of a-SMA was performed to identify the cells.2.Real-time PCR and western blot analysis were performed to evaluate the ECM deposition of HASMCs after treating with different agents.3.Western blot analysis was performed to evaluate the phosphorylation of ERK1/2 signaling pathway on HASMCs after treating with different agents.4.All experimental data analysis was done with SPSS 20.0.One-way analysis of variance method was used to analyze the comparisons between groups.LSD method was used to analyze the multiple comparisons when variances equal.A value of P<0.05 was considered as significant difference.Results:1.The shape of primary human airway smooth muscle cells was fusiform and they showed the "peak and valley"sign when they were confluent.More than 95%of the cells showed positive expression of ?-SMA about the immunofluorescence staining.2.Stimulated the cells by different concentration(0.1-10U/ml)thrombin,the expression of collagen ? ?1(COL-? ?1)protein was all increased compared with the control group in which lU/ml had the best effect.Stimulated the cells by thrombin for different time(12-48hours),the expression of collagen-? ?1(COL-? ?1)protein was all increased compared with the control group in which 48 hours reached the best effect.The differences were statistically significant(P<0.01).Stimulated the cells by thrombin for 12 hours,the mRNA expression of COL-? ?1,versican,fibronectin was increased compared with the control group,but the mRNA expression of collagen?,1aminin ?1,laminin ?2 was nearly equal compared with the control group.What's more,stimulated the cells by PAR-1 antagonist SCH79797 or ERK signaling pathway inhibitor U0126 30minutes early both attenuated the effects above induced by thrombin.The differences were statistically significant(P<0.05).3.Stimulated the cells by thrombin for different time(5-60minutes),the phosphorylation of ERK1/2 was all enhanced compared with the control group.The differences were statistically significant(P<0.05).The phosphorylation of ERK1/2 induced by thrombin showed early rapid model and 5 minutes was the best stimulation time.Stimulated the cells by SCH79797 or PD98059 30minutes early both attenuated the phosphorylation of ERK1/2 induced by thrombin when compared with the only thrombin stimulation group.The differences were statistically significant(P<0.01).4.It was found that dabigatran not only abrogated the ECM deposition of ASMC induece by thrombin-PAR-1 but also inhibited the rapid phosphorylation of ERK1/2 of ASMC induece by thrombin-PAR-1.Conclusions:1.Tissue adherence method can sucessfully culture primary human airway smooth muscle cells.2.Thrombin can correspond with PAR-1 on ASMC activating rapid phosphorylation of ERK1/2 signaling pathway to induce ECM deposition,and these effects can be abrogated by dabigatran.