Cyclophosphamide And Etanercept In The Monocrotaline-induced Rat Model Of Pulmonary Hypertension And Its Related Mechanisms Discussed

BackgroundPulmonary arterial hypertension(PAH) is defined as a group of disease with complicated etiology.Especially connective tissue disease(CTD) associated PAH,the prognosis in clinic is still poor.One of the researching obstacles is that we are lacking of a good animal model which can appropriately mimic the pathogenesis of CTD associated PAH,if we could get one,it will be greatly helpful to the study of CTD associated PAH. Monocrotaline(MCT) induced PAH in rat is a model which maybe most close to our wish among all the current PAH model,because it's the only one that inflammation plays an important role in it.Investigating the efficacy of some anti-inflammation drugs or immunosuppressive agents intervened in this model and exploring the mechanism of action,will surely improve the understanding of CTD associated PAH and may lead to new drug therapies in clinic.Objective1.Establish the MCT induced rat PAH model.2.Administrate Etanercept(ECP) and Cyclophosphamide(CTX) to the rats for prophylactic intervention and remedial intervention,and investigate the efficacy of these two drugs by determining the mean pulmonary arterial hypertension (mPAP).3.Assess the role of inflammation in this model by determining the expression of TNF-α,IL-1αand IL-6 in the rat serum and lung tissue,and then combine the results of drug intervention study,to assume the mechanism of these drug therapies on this model.Method1.Rats received MCT 60mg/kg i.p.once.At the end of week 2 and week 4, determine the mPAP by the right heart catheterization technique.Compare the mPAP of the model groups with normal control group to inspect whether we successfully built the PAH model or not. 2.Administrate ECP and CTX to the rats for prophylactic intervention and remedial intervention.At the end of week 2 and week 4,determine the mPAP of each group by the right heart catheterization technique.Compare the mPAP of the treatment groups respectively with the relative model group(2w or 4w) to inspect the efficacy of each treatment group.And we introduced the R value which indicated the stenosis severity of the small artery's lumen in the lung to assistantly assess the mPAP determination.3.Using Enzyme linked immunosorbent assay(ELISA) to determine the expression of TNF-α,IL-1αand IL-6 in the rat serums;Using Immunohistochemistry staining to determine the expression of TNF-αand IL-6 in the rat lung tissues.Results1.After administration of MCT 60mg/kg i.p.once,the mPAP of rats in the 2w model group significantly increased(compared with the normal control group, P＜0.01).At the end of Week 4,the mPAP of the 4w model group was remarkably higher.2.The prophylactic intervention of ECP can significantly reduce the mPAP in experimental group(compared with the 2w model group,P＜0.01);The remedial intervention of CTX can significantly reduce the mPAP in experimental group (compared with the 4w model group,P＜0.01).3.The expression of TNF-α,IL-1αand IL-6 in the rat serums of all groups was too low to be detected by ELISA.4.The slides of the rat lung tissues in model group under light microscope showed increased thickness of alveolar septum with macrophages and lymphocytes infiltration and the wall of small arteries by the side of bronchioles,swelling endothelial cells and stenotic lumen of these small artries,some rats showed remarkable pulmonary edema.5.The statistical analysis of the R value,showed the mPAP was proportional to the stenosis severity of the lumen of small arteries.Using R value to assess the efficacy of the two drugs,we got the same results. 6.The immunohistochemistry staining showed there were massive expressions of TNF-αand IL-6 in the lung tissues of model groups and all treatment groups. The staining was predominatly distribute in the cytoplasm of lung macrophages.Conclusion1.Receiving MCT 60mg/kg i.p.once can successfully establish the rat PAH model.2.Prophylactic intervention of ECP and remedial intervention of CTX showed good efficacy.3.As a consequence,we think the mechanism of the effectiveness of ECP intervention in MCT induced PAH in rat is:ECP can block TNF-αreceptor, inhibit the lung macrophages to secret the inflammatory factors TNF-α,IL-6 et al, which can then inhibit the activation of the mononuclear-macrophage system, then can attenuate the infiltration of inflammatory cells in alveolar septum and small artery wall and thickening of them,finally lead to prevent the pulmonary arterial pressure elevation.4.The mechanism of the effectiveness of CTX intervention in MCT induced PAH in rat is:On one side CTX can suppress immunocytes such as macrophage and lymphocyte to attenuate imflammation and immune reaction;On the other side, CTX can suppress the post-injury proliferation of the endothelial cells of small arteries in the lung,so can attenuate the lumen stenosis,finally lead to prevent the pulmonary arterial pressure elevation.