Mechanisms Of Berberine In The Treatment Of Pulmonary Arterial Hypertension By Regulating PP2A Signaling Pathway

Background:Pulmonary arterial hypertension is a chronic,progress disease which was mainly characterized by persisient increase of pulmonary vascular resistance.Secondary to Coronary heart disease and hypertension,the current incidence of PAH is in the third place of cardiovascular diseases.Thus,further identifying the mechanism of PAH,exploring possible intervention to improve the survival quality of PAH patients is urgently needed.Protein phosphatase 2A(PP2A),one of the major serine/threonine phosphatases involved in the regulation of many cellular processes such as cell proliferation and migraton PP2A acts as an anti-tumor gene so it may be a potential target for PAH treatment since PAH and tumors share the similar mechanisms.Previous data show that BBR could reverse the activity of PP2A via inhibiting the phosploryration level of PP2A,whether BBR is effective in the treatment of PAH and the related mechanism was unknown.Object:To investigate whether BBR could attenuate pulmonary vascular remodeling via PP2A signaling pathway both in vivo and in vitroMethods:In vivo:Male Sprague-Dawley(SD)rats weighing 200-250 g at 6 weeks of age were obtained from the Animal Center of Nanjing Medical University and randomly divided into three groups:the control group(n=6),the PAH group(n=7)and the treatment group(n=7).These rats received basal cardiac function examination using echocardiography.The control group and the PAH group received normal saline(1ml/day,ig),the treatment group received BBR(100mg/kg/day,ig),the PAH group and the treatment group were exposed to chronic hypoxia(10%oxygen).28 days later,right heart-catheter was taken to measure the right ventricular systolic pressure(RVSP),HE and EVG staining to examine the pulmonary vascular remodeling,Western blotting to examine the expression of related proteinsIn vivo:Primanary pulmonary arterial smooth muscle cells(PASMCs)isolated from male SD rats were incubated with NE(10-5 mmol/L)or hypoxia(5%O2)in the absence or presence of berberine(10-5 mmol/L),12 hours later,western blotting were taken to investigate protein expression.24 hours later MTT,proliferating cell nuclear antigen(PCNA)and cell counting kit-8(CCK-8)assay taken to evaluate the proliferation of PASMCs,Boyden chamber migration and wound-healing assays to assess migration.Overexpression of PP2A in the absence or presence of NE,related protein and proliferation and migration were assessed.PASMCs treated with NE Followed by inhibition the activity of PP2A with siRNA then in the absence or presence of berberine for 12h and related protein and proliferation and migration were assessed.Results:(1)The phosphorylation of PP2A was upregulated in PAH patients and two experimental PAH models;(2)Phosphorylation of PP2A was involved in the proliferation and migration of PASMCs induced by NE or hypoxia while PP2A overexpression reversed this effect;3)BBR could alleviated PAH both in vivo and in vitro;and 4)Genetic ablation of PP2A reproduced the PAH features relieved by BBR in vitro.Conculsion:These findings demonstrated that phosphorylation of PP2A(Y307)plays an important role in the progression of PAH and that reconstitution of PP2A activity may provide a potentially effective drug target for the treatment of PAH.