Mechanism Of Shenfuyixin Granule In Improving Pulmonary Arterial Hypertension

BackgroundChina is a developing country in which the economic development is uneven between regions.Currently,since targeted drug of pulmonary hypertension(PH)is not listed in National Basic Medical Care Insurance Medicine Catalogue,patients bear a heavy economic burden.In China,only about 20% PH patients receive targeted drug therapy,and even in some well-developed cities and regions,the rate of using PAH targeted drugs just reaches 75%.PAH is a highly fatal disease in heart,lung and blood vessel featured with resistance progressive increase by pulmonary vascular remodeling which eventually results in death from right heart failure,and its clinical manifestations include dyspnea,fatigue,dizziness,chest pain,hemoptysis,and so on,which are similar to "lung distension" and “heart failure” in the traditional medicine.Traditional drug therapies for PAH mainly treat right ventricular dysfunction and primary thrombus formation in pulmonary artery,namely,targeted drug therapy for oxygen inhalation,diuresis,cardiac and anticoagulation as well as pulmonary vascular dilatation.Though targeted drugs can not only significantly improve symptoms and life quality of patients,but also extend their life span,they are expensive and have high incidenceof drug resistance and strong side effects.Therefore,it is of great practical significance to study the prevention and treatment of PAH by traditional Chinese medicine which can overcome the above disadvantages.ObjectiveThis study is to observe the effect of the Shenfuyixin granule on hemodynamics and angiogenesis in PAH rats and further explore the relationship between hemodynamics and angiogenesis in PH rats;to investigate the effect of Shenfuyixin granule on the proliferation and migration of bone marrow-derived late endothelial progenitor cells in rats;to establish a primary PH regulatory network through GO function analysis and KEGG signaling pathway analysis,thereby providing new ideas and novel targets for drug therapy against PAH.Material and methods1.Thirty male Wistar rats were randomly divided into blank group,model group and Shenfuyixin granule group,with 10 rats in each group.And rats in model group and Shenfuyixin granule group were hypodermically injected with monocrotaline(60mg/kg)to establish PAH model.The parameters of pulmonary hemodynamics in rats after 28-day drug gavage were determined by modified right heart catheterization and the mean pulmonary arterial pressure was calculated.Rats were sacrificed to measure their right ventricular hypertrophy indexes which were represented by RV/(LV+S).Finally,the right lower lung tissues were taken to measure the microvessel density(MVD).2.Sixty male Wistar rats were randomly divided into model group(50 rats)and blank group(group K,10 rats).And rats in model group were hypodermically injected with monocrotaline in the neck and back to establish the PH model.Then,rats in model group were randomly divided into five groups,saline group(group N),PI3 K inhibitor group(group Y),low-dose Shenfuyixin granule group(group D),medium-dose Shenfuyixin granule group(group Z)and high-dose Shenfuyixingranule group(group G),and their serums containing medicine were prepared.The late endothelial progenitor cells were cultured in serum containing medicine of each group for 24 hours and detected by flow cytometry,and then the ModFitLT software was employed to analyze the cell cycle situations.Cells at logarithmic growth phase were obtained to examine the number of migrating cells by Transwell method;the levels of Ang-II,IGF,HGF and VEGF in blank group,model group and Shenfuyixin granule group were detected by ELISA.3.GO and KEGG databases were used to perform GO analysis and KEGG signaling pathway analysis on TGF,ECM,HGF,STAT and VEGF genes and a primary PAH regulatory network was established.Results1.Shenfuyixin granule has significant effect on the hemodynamic index of PAH rat induced by monocrotaline.The MVDs of Shenfuyixin granule group and model group were both significantly higher than that of blank group(P<0.05),and that in Shenfuyixin granule group was dramatically higher than that in model group(P<0.05),which indicated that the intervention of Shenfuyixin granule prevented the MVD of lung tissue in PH rat from reduction.2.Cell proliferation experimental results: compared with group Y,cells at G1 phase in group K,group N,group D,group Z and group G decreased(P<0.05)while at G2/M and S phases increased(P<0.05);compared with group K,cells at G1 phase in group D,group Z and group G decreased(P<0.05)while at G2/M and S phases increased(P<0.05);compared with group N,cells at G1 phase in group D,group Z and group G decreased(P<0.05)while at G2/M and S phases increased(P<0.05);and these above results indicated that the distribution of cells in different phase of cell cycles was dose-dependent within certain dose range.Cell migration experimental results: compared with group Y,the number of migrating cells in group K,group N,group D,group Z and group G rose(P<0.05);compared with group K,the number of migrating cells in group D,group Z and group G notably rose(P<0.05);from the results described above,the numbers of migrating cells in group D,group Z andgroup G all rose in a dose-dependent manner within certain dose range.ELISA test:compared with blank group,the levels of Ang-II,IGF,HGF and VEGF in model group were elevated remarkably(P<0.05);similarly,compared with model group,the levels of Ang-II,IGF,HGF and VEGF in Shenfuyixin granule were also elevated significantly(P<0.05).3.Through GO function analysis and KEGG signaling pathway analysis,the primary PAH regulatory network based on TGF,ECM,HGF,STAT,VEGF and other related genes was established.Conclusions1.Shenfuyixin granule can significantly improve the hemodynamic indexes and symptoms of PH rat induced by monocrotaline and promote angionesis.2.Shenfuyixin granule can promote the survival,proliferation and migration of late endothelial progenitor cells and improved their paracrine effect,providing reliable experimental data for further study on mechanism of Shenfuyixin granule.3.The primary PAH regulatory network was established,which lays foundation for further exploration on pathway of Shenfuyixin granule in treating PAH.