PDGF-BB Regulates Phenotype Transformation Of Pulmonary Artery Smooth Muscle Cells Via SIRT3 Affecting Glycolytic Pathway

ObjectiveTo investigate whether platelet-derived growth factor-BB(PDGF-BB)stimulated phenotypic transformation of pulmonary artery smooth muscle cells(PASMCs)was associated with aerobic glycolysis and its mechanism.Methods1.Cell models and group dividingUsing tissue explant method in primary cultured rat pulmonary artery smooth muscle cells(PASMCs).Platelet-derived growth factor-BB(PDGF-BB)was used as stimulator to interfere with 4-6 cells for 12 hours,which in good growth state,to induce cell phenotype from a contractile to a synthetic type of replication cell model.Primary cultured rat PASMCs were divided into three groups by using 2-deoxyglucose(2-DG),an inhibitor of the glycolytic pathway: normal control group,PDGF-BB group(30ng/ml)and PDGF-BB(30ng/ml)+2-DG(10mmol/l)group.Lentiviral vector was uesd to overexpress SIRT3,and the experiment was divided into control group,PDGF-BB group(30ng/ml),PDGF-BB+deacetylase sirtuin-3(SIRT3)overexpression group and PDGF-BB+empty vector group.2.Observation targetsThe expression levels of phenotype related index such as smooth muscle myosin heavy chain(SM-MHC),?-smooth muscle actin(?-SMA),calponin,vimentin were detected by Western Blot and q RT-PCR.The expression level of ?-SMA was detected by cellular immunofluorescence staining.EDU staining was used to detecte the proliferation of PASMCs.The expression level of SIRT3 was detected by Western Blot.The expression levels of glucose transporter 1 and aerobic glycolytic enzymes were detected by Western Blot and q RT-PCR in lentivirus-mediated overexpression assay.ResultsPDGF-BB stimulation for 12 h,the expression levels of vimentin m RNA and proteinwere upregulated(P<0.05)while ?-SMA,SM-MHC,calponin m RNA and protein expression levels were significantly downregulated(P<0.05)compare to control group,and 2-DG reversed the above-mentioned effects of PDGF-BB.Cellular immunofluorescence and EDU proliferation assay showed that PDGF-BB significantly decreased the number of ?-SMA positive cells and increased cell proliferation,while 2-DG reversed the processes.Moreover,PDGF-BB significantly reduced the expression level of SIRT3 protein compared with the control group(P<0.05).In lentivirus-mediated overexpression assay,PDGF-BB significantly increased the expression levels of glucose transporter 1(Glut1),hexokinase 2(HK2),6-phosphfructo-2-kinase 3(PFKFB3)m RNA and protein(P<0.05)compared with the blank control group,which was reserved by overexpression of SIRT3.There were no significant difference in m RNA and protein expression levels between PDGF-BB group and PDGF-BB+empty vector group(P>0.05).Conclusion:PDGF-BB regulates phenotypic transformation of pulmonary artery smooth muscle cells via SIRT3 affecting glycolytic pathway.