The Effect And Mechanism Of Glut1 Specific Inhibitor Bay-876 On Reversing The Proliferation Of Pasmc Induced By Hypoxia

Background: Excessive proliferation of pulmonary artery smooth muscle cells((PASMC))plays an important role in the pathological process of pulmonary vascular remodeling in(PAH)with pulmonary hypertension.The metabolism of PASMC changes from oxidative phosphorylation to aerobic glycolysis to provide energy for cell proliferation during PAH which is very similar to cancer.The latest progress in the understanding of this phenotype provides a new strategy for the treatment of pulmonary hypertension.However,due to the complexity of PASMC glycolysis mechanism and the lack of specific,safe and effective glycolysis inhibitors,the treatment targeting glycolysis of PAH is not ideal.As a newly discovered glucose transporter 1(GLUT1)specific inhibitor,the role of BAY-876 in PAH has not been evaluated.As a newly discovered specific inhibitor of GLUT1,the role of BAY-876 in PAH is not clear.Objective: To investigate the expression of GLUT1 in pulmonary artery smooth muscle in vivo and in vitro and the effect of GLUT1 inhibitor BAY-876 on proliferation,apoptosis and glycolysis of pulmonary artery smooth muscle in pulmonary hypertension and its potential molecular mechanism。Methods: The model of pulmonary hypertension model induced by monocrotaline(MCT)was used and the distal pulmonary artery smooth muscle cells(PASMC)of rats were isolated and identified.The expression of GLUT1 protein in rat lung tissue,pulmonary artery and isolated PASMC were detected by Western-Blot,and the expression of GLUT1 m RNA in dissected pulmonary artery was detected by RT-PCR.Rat PASMC was treated with hypoxia(1% O2,5% CO2)for 0,3,6,12 and 24 hours,and the protein expression and m RNA level in rat PASMC were detected.Rats were given BAY-876,of different concentrations of PASMC(1.25,2.5,5,10,20,40,80,160 n M)and then treated with hypoxia(1% O2,5% CO2)for 24 hours.The cell viability was detected by CCK8 to determine the most suitable concentration of BAY-876 in PASMC.The following experiments were divided into four groups(Control,Control+BAY-876,Hypoxia and Hypoxia+BAY-876).Western-Blot method was used to detect the expression of the proliferation protein(PCNA),the apoptosis resistance protein(Survivin),the apoptosis promoting protein(cleaved Caspase3)and key glycolysis proteins(GPI,PDK1,PKM2).PASMC were also treated with autophagy inhibitors(chloroquine phosphate(CQ)and 3-methyladenine(3-MA))and the expression of GLUT1 protein in each group was detected.The expression of ROS in each group was detected by reactive oxygen species(ROS)assay kit.Results: Compared with the Control group,rats treated with MCT showed typical pulmonary artery hemodynamic changes and pulmonary vascular remodeling(m PAP increased(P<0.01),RVSP rose(P < 0.01),RVHI increased(P <0.05)).The expression of GLUT1 protein in lung tissues and its m RNA(SLC2A1)and protein expression in pulmonary artery in the MCT group were significantly increased compared with the Control group.The expression of GLUT1 protein in isolated PASMC maintained this characteristic.The expression of SLC2A1 in PASMC was time-dependent during hypoxia.It means that the expression level of SLC2A1 increased continuously with the prolongation of hypoxia time,and the expression level of GLUT1 protein was consistent with the expression trend of m RNA(P < 0.0l).After hypoxia for 24 hours,the cell viability,proliferation and apoptosis resistance of PASMC isolated from normal rats increased obviously.The use of BAY-876 could inhibit PASMC proliferation in a concentration-dependent manner.BAY-876(20n M)treatment effectively decreased the expression of PCNA(P < 0.0l),Survivin(P < 0.0l),GPI(P <0.0l),PDK1(P < 0.05)and PKM2(P < 0.05),and increased the expression of cleaved Caspase3 in PASMC treated with hypoxia.In addition,we found that autophagy inhibitors CQ and 3-MA treatment could decrease the expression of GLUT1 protein during hypoxia.BAY-876 treatment aggravated the oxidative stress of cells during hypoxia and significantly increased the production of ROS(P < 0.0l).CCK8 results showed that the cell viability decreased in hypoxia + BAY group,and this cytotoxicity was significantly alleviated by vitamin E(P < 0.0l).Conclusion: GLUT1 is essential for induced glycolysis of PASMC during hypoxia.BAY-876 blocks the glycolysis metabolism dependent on GLUT1 in PASMC,thus effectively inhibits the growth of PASMC in vitro,thus verifying that GLUT1 is an effective therapeutic target.