Effect Of Puerarin On The Proliferation Of PASMCs Induced By Hypoxia By Inhibiting Autophagy

Pulmonary arterial hypertension(PAH)is defined as a rise in flat pulmonary arterial pressure to ≥25 mmHg in the low-pressure pulmonary circulatory system.If it isn’t properly diagnosed and treated in time,it will cause right heart failure and the prognosis is poor.Puerarin has many biological activities such as anti-tumor,anti-inflammatory,antioxidant and anti-angiogenic properties and has been widely used to treat cardiovascular and cerebrovascular diseases and diabetes.However,how puerarin mediates autophagy in pulmonary hypertension remains is unknown.To explore whether puerarin can prevent and protect hypoxic pulmonary hypertension rats by inhibiting autophagy,and further reveal the protective mechanism of puerarin on hypoxia-induced PAH to provide theoretical basis.In order to explore the protective role of puerarin in hypoxia-induced PAH and how it affects autophagy levels,this study established a PAH rat model through hypoxia induction.Puerarin and an equal dose of 0.5% sodium carboxymethylcellulose vehicle were administered intragastrically for 21 days.The Doppler ultrasound system measured the pulmonary artery acceleration time(PAAT),pulmonary artery velocity time integral(PAVTI),right ventricular anterior wall;diastole(RVAW;d)and right ventricular anterior wall;systole(RVAW;s).The right ventricular catheter method continuously records the right ventricular systolic pressure(RVSP),and the ratio of the right ventricle /(left ventricle + ventricular septum)weight is used to indicate the degree of right ventricular hypertrophy.The pathological and morphological changes of pulmonary artery tissue were observed by HE staining and immunohistochemistry.Using immunohistochemistry to observe the expression of autophagy-related proteins LC3B-II and BECN-1 in pulmonary arteries.The expression of LC3B-II,BECN-1,ATG5 and SQSTM1 in pulmonary arteries are detected by using Western blotting.The expression of LC3B-II in PASMCs was measured by immunofluorescence analysis,and autophagy flux was detected by bafilomycin A1.Autophagy flow was monitored by transfection of PASMCs with autophagy double-labeled adenovirus(Ad-mRFP-GFP-LC3).The results show that puerarin can down-regulate the expression of autophagy-related proteins in vivo and in vitro.In order to further investigate whether puerarin inhibited the hypoxia-induced abnormal proliferation of PASMCs in an autophagy-dependent manner,the autophagy inhibitor bafilomycin A1 was intervened on the basis of puerarin administration.The level of cell proliferation was detected by using CCK-8 method,and the expression levels of PCNA,Cyclin A,Cyclin D1 and Cyclin E in cells are detected by using Western blotting method.The results show that bafilomycin A1,an autophagy inhibitor,has similar effects to puerarin,further proving that puerarin may reduce hypoxia-induced abnormal proliferation of PASMCs by inhibiting autophagy.In conclusion,we reveal that the preventive effect of puerarin is significant,and that the autophagy inhibitory effect of puerarin significantly eliminates the effects of hypoxia-induced vascular remodeling.To our knowledge,this study found for the first time that puerarin reduces the abnormal proliferation of hypoxia-induced PASMCs by inhibiting autophagy,and prevents the development of PAH,providing a new drug platform and treatment strategy for the treatment of PAH.