Shikonin Improves Pulmonary Vascular Remodeling In Monocrotaline Pulmonary Arterial Hypertension Rats Via Regulating PKM2

Purpose and significance: Pulmonary arterial hypertension(PAH)is a fatal disease,aerobic glycolysis plays an important role in the development of PAH,pyruvate kinase 2(PKM2)is a key enzyme in the glycolytic pathway and can promote the occurrence of aerobic glycolysis,which has been found upregulation in PAH and a variety of tumor diseases.Traditional Chinese medicine Shikonin can specifically inhibit the expression of PKM2.This study intends to determine whether shikonin can inhibit or reverse vascular remodeling in animal models of pulmonary arterial hypertension via regulating PKM2 and downstream pathways,with which we expect to elucidate new mechanism in PAH development and find new target medicine.Methods:(1)Animal model construction: twenty-four male SD rats were randomly divided into control group(CON),monocrotaline pulmonary arterial hypertension group(MCT-PAH),and monocrotaline pulmonary arterial hypertension rats with shikonin treatment group(SH+MCT-PAH).CON group was intraperitoneal injection of the same amount of saline,three weeks later,the same amount of vegetable oil was given by gavage for one week;MCT-PAH group was treated with intraperitoneal injection of monocrotaline(MCT)once,in dose of 60 mg·kg-1,three weeks later,the same amount of vegetable oil was given by gavage for one week;MCT-PAH+SH group was treated with MCT and shikonin,shikonin was administered orally at a dose of 10 mg·kg-1 for one week.(2)pulmonary artery acceleration time(PAAT),right ventricular internal diameter(RVID)and tricuspid annulus systolic displacement(TAPSE)were measured by doppler echocardiography;pulmonary artery mean pressure,systolic pressure and diastolic pressure were meteraged cardiac catheterization;HE staining was used to detect the morphology of pulmonary tissue and the thickness of pulmonary arterial blood vessels.(3)Western blotting was used to detect the expression of PKM2,ERK1/2,pERK,Ser 37 PKM2,GULT1 and LDHA proteinases in rats lung tissues.(4)Immunofluorescence method and laser confocal microscope were used to detect the expression and distribution of PKM2.Results:(1)Compared with CON group,PAAT shortened,RVID increased,TAPSE decreased and RV/(LV+S)ratio increased in the MCTPAH group.Right heart catheterization showed pulmonary systolic blood pressure,diastolic pressure and mean pressure were significantly increased in MCT-PAH group;HE staining indicated that the pulmonary artery vessel wall was significantly thickened and vascular remodeling was present.(2)Shikonin intervention can reduce pulmonary pressure in rats of the MCTPAH group.Compared with MCT-PAH group,PAAT prolonged,RVID slightly reduced,TAPSE improvedand RV/(LV+S)ratio decreased in the MCT-PAH+SH group.Right heart catheterization showed that the pulmonary artery systolic pressure,diastolic pressure,and average pressure decreased post shikonin treatment;HE staining results showed that vascular remodeling was improved,and the thickness of media of pulmonary artery became thinner.(3)Compared with CON group,the expression of PKM2,p-PKM2,p-ERK,GLUT1,and LDHA increased in lung tissue of rats in MCT-PAH group,however,expression of ERK1/2 protease slightly changed without statistical significance.Compared with MCT-PAH group,expressions of PKM2,p-PKM2,p-ERK,GLUT1,and LDHA were reduced in MCT-PAH+SH group,and the expression of ERK1/2 protease was not significantly changed.(4)PKM2 fluorescence intensity in pulmonary vessels of CON group was similar to surrounding tissue.Compared with CON group,PKM2 fluorescence intensity in pulmonary vessels of rats in MCT-PAH group was significantly higher than that in surrounding tissues.PKM2 fluorescence intensity in pulmonary vessels in MCT-PAH+SH group was significantly weakened,but still higher than that in surrounding tissues.Conclusions:(1)Upregulation of PKM2 existed in pulmonary arteries of PAH,and the upregulation of PKM2 promotes activation of ERK1/2 phosphorylation.ERK1/2 phosphorylation may cause the upregulation of GLUT1,LDHA and PKM2 via promoting PKM2 phosphorylation and nuclear transfer,with which aerobic enhanced glycolysis may be provoked and play a role in pulmonary vascular remodeling.(2)Shikonin treatment alleviated PAH via improving pulmonary vascular remodeling in MCT-PAH rats.The mechanism may be related to inhibition of PKM2 expression,ERK1/2 phosphorylation and aerobic glycolysis.