The Effect And Mechanism Of Isorhynchophylline On Pulmonary Arterial Hypertension And PASMCs Proliferation

Background:Pulmonary arterial hypertension (PAH) is a progressive disease, which characterized by sustained elevation of pulmonary vascular resistance induced by vasoconstriction and remodeling of small pulmonary arteries. Sustained elevations of pulmonary vascular resistance induce right ventricular failure and ultimately death, and the prognosis is very poor. Different types of PAH have similar pathological changes, like thickening of extracellular matrix accumulation, inflammatory cells infiltration, pulmonary smooth muscle cell proliferation and vascular endothelial damagement.. And pulmonary smooth muscle cells proliferation play a key role in pulmonary vascular remodeling.Therapy of PAH have made great progress in past decades, which concentrated on pulmonary vasoconstriction and dysfunction of endothelial cells. However, there is no evidence that these therapies could reverse pulmonary vascular remodeling and cor pulmonale. Isorhynchophylline (IRN) is a tetracyclic oxindole alkaloid isolated from the Chinese herbal medicine Uncaria rhynchophylla, which has long been clinically used for treatment of cardiovascular and cerebrovascular diseases. Studies have demonstrated that IRN could cause cell cycle arrest to against Angiotensin Ⅱ induced rat vascular smooth muscle cell proliferation. So, we hypothesis that IRN could inhibit pulmonary arterial smooth muscle proliferation to reverse pulmonary vascular remodeling and cor pulmonale.Objective:1. To explore the effect of IRN on MCT-induced PAH.2. To study the effect of IRN on PDGF-BB-induced PASMCs proliferation and the possible molecular mechanism.Methods:1. Eighty healthy male Wistar rat were divided into four groups:the control group, rats were given maintain feed after subcutaneous injection of normal saline; IRN group, rats were given maintain feed containing 0.1% IRN after subcutaneous injection of normal saline; PAH group, rats were given maintain feed after subcutaneous injection of MCT (40mg/ml); PAH+ IRN group, rats were given maintain feed containing 0.1% IRN after subcutaneous injection of MCT (40mg/ml).2. Three weeks later, right ventricular systolic pressure was measured with right catheterization. Then, heart, lung, and other organs were isolated. Right ventricular (RV) and left ventricular+septum (LV+S) were weight respectively to calculate right ventricle hypertrophy index (RV/LV+S). RV was stained with Sirius Red (Sigma) and HE staining to show fibrosis and cardiac myocyte size. HE and immunohistochemical staining was performed to detect the expression of a-SMA �'� PCNA.3. The effect and cytotoxicity of IRN on PDGF-BB-induced PASMCs proliferation were measured by MTT assay and trypan blue exclusion. Cell cycle progression was measured by flow cytometry. Immunoreactive bands were visualized by blotting with antibodies against Cyclin D1, CDK6, P27Kip1, PDGFR, Akt, GSK3b, ERK1/2 and STAT3.Results:1. The effect of IRN on MCT-induced PAH1) The normal control group:body weigh 259± 4.5g, right ventricular weigh 159±4.5mg, left ventricular+ septum weigh 639±13.3mg,lung weigh 1.23±0.05g; IRN group:body weigh 256±3.3g, right ventricular weigh 160±4.1mg, left ventricular+septum weigh 642±8.2mg,lung weigh 1.24±0.03g.There was no difference between the normal control group and IRN group (P<0.05)。2) The normal control group:right ventricular systolic pressure is 25.14±0.73mmHg, PAH group:right ventricular systolic pressure was 56.61±2.36mmHg. Compared with the normal control group, right ventricular systolic pressure of PAH group increased significantly (P<0.05); compared with PAH group, right ventricular systolic pressure of PAH+IRN group greatly decreased (P<0.05)3) The RVHI of the normal control group is 0.25±0.01. The RVHI of the PAH group was 0.56±0.03. Compared with the normal control group, the RVHI of PAH group increased significantly (P<0.05); compared with PAH group, the RVHI of PAH+IRN group decreased greatly (P<0.05). Sirius Red and HE analysis indicated that the degree of right ventricular fibrosis and the cardiac myocyte cross sectional area in PAH group increased significantly (P<0.05); compared with PAH group, the degree of right ventricular fibrosis and the cardiac myocyte cross sectional area of PAH+IRN group decreased greatly (P<0.05)4) Compared with the normal control group, the percentage of fully-muscularized (FM), the medial wall thickness of small artery (50-200μm) and the PASMCs proliferation of PAH group increased significantly (P<0.05); compared with PAH group, the percentage of fully-muscularized (FM), the medial wall thickness of small arteryand the PASMCs proliferation of PAH+IRN group decreased greatly (P<0.05). Western blot indicated that the expression of a-SMA and PCNA in PAH group increase greatly, compared with PAH group, the expression of a-SMA and PCNA in PAH+ IRN group decreased greatly (P<0.05)2、The effect of IRN on MCT-induced PAH and PDGF-BB-induced PASMCs proliferation.1) Compared with normal group, the number of PASMCs increased approximate 2.5 fold after culturing with 20ng/ml PDGF-BB for 24h. IRN inhibited PDGF-BB-induced PASMCs proliferation in a concentration-dependent manner without cell cytotoxicity. An investigation of the effect of IRN on PDGF-BB-stimulated PASMCs cell cycle progression showed that IRN (25μM) inhibited PDGF-BB-induced PASMCs S phase proportion.2) The results of Western Blot indicated that IRN decreased PDGF-BB induced expression of cyclin D1 and cyclin-dependent kinase (CDK) 6, and promoted p27kipl degradation. Furthermore, IRN negatively regulated PDGF-BB-induced phosphorylation of PDGF-Rβ and its downstream signaling molecules including AKT, glycogen synthase kinase (GSK) 3β and STAT3.Conclusions:These results demonstrate that IRN could inhibit PASMCs proliferation and attenuate pulmonary vascular remodeling after MCT induction. These beneficial effects were at leastthrough the inhibition of PDGF-Rβ phosphorylation and its downstream signaling pathways.