Salidroside Blocks The Proliferation And Migration Of Pulmonary Artery Smooth Muscle Cells

Background:Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling and a progressive increase in pulmonary vascular resistance, which ultimately leads to right ventricular failure and death. PASMCs play an important role in pulmonary vascular remodeling for it is not only the effector cell of pulmonary vasoconstriction, but also the cell infrastructure of abnormal migration and proliferation of PASMCs which induce pulmonary vascular remodeling. Pathological examination showed that vessel hypertrophy and bureaucratic narrow in pulmonary hypertension. The PASMCs located in the tunica media of the vascular wall, in pathological stimuli, PASMCs can migrate from tunica media to endfomemebrane and induce a series of cell signal transduction system the start abnormal proliferation of PASMCs, which will lead to pulmonary vascular remodeling. The PAH has no effective drug treatment until now, commonly used drugs such as prostacyclin analogues; endothelin-1receptor antagonist; phosphodiesterase-5inhibitors, those drugs can only improve the patient’s symptoms but can’t delay the development process of PAH. Looking for a new drug that can inhibit or reverse the abnormal proliferation and migration of PASMCs and decrease the synthesis of extracellular matrix, improve the survival rate of the patients is very important.Objective:To establish the platelet-derived growth factor (PDGF)-BB induced model of cell proliferation and migration in rat pulmonary artery smooth muscle cells, and investigate the effects of salidroside on the proliferation and migration of PASMCs, as to search for new drugs for the treatment and prevention of pulmonary vascular remodeling.Methods:Select Sprague Dawley (SD) rats weighing between150to200g, separation pulmonary artery and then using0.2%Collagenase I digestion the PASMCs. Using20ng/ml PDGF-BB-induced proliferation and migration of PASMCs to established the cell model. Preincubation with various concentrations of salidroside (12.5-100μmol/L), then the PASMCs were treated with20ng/ml PDGF-BB for12, 24and48h in the presence of salidroside and loaded with CCK-8and BRDU to detect the proliferation and DNA synthesis; The toxicity of salidroside on PASMCs was determined by the trypan blue exclusion test. After12,24and48h incubation with salidroside from12.5to100μmol/L, the PASMCs were removed from culture and the cells that excluded0.4%trypan blue to evaluation of cell viability. PASMCs were pretreated with10μmol/L of salidroside then stimulation with20ng/mL PDGF-BB for24h, the cell cycle progression was analyzed by flow cytometry, and the mRNA expression of cyclin D1, cyclin E, CDK4, CDK2and cyclin kinase inhibitor protein p27was determined by real-time reverse transcription PCR, The cyclin D1, cyclin kinase inhibitor protein p27and matrix metalloproteinase2/9(MMP-2/9) were measured by western blot analysis. The total and phosphorylated forms of ERK1/2, p38, JNK and AKT, proto-synthase kinase3β (GSK3β) was observed in salidroside pretreated PASMCs in5,10and15min after PDGF treatment by western blot analysis; The effect of Salidroside on the migration of PASMCs induced by PDGF-BB was determined by the Transwell system.Results:CCK-8and BRDU assays showed that salidroside(12.5-100μmol/L) inhibited PDGF-induced pulmonary artery smooth muscle cell proliferation and DNA synthesis in a dose-and time-dependent manner. Trypan blue staining result indicated that salidroside did not induce cell necrosis of PASMCs with the concentration from12.5to100μmol/L compared with untreated cells, treant or untreat with salidroside, cell viability can be maintained at about95%; Flow cytometry analysis showed that Salidroside at a dose of100μ mol/L reduced the percentages of cells in S phase and increasing the G0/G1populations in PDGF-BB-stimulated PASMCs; RT-PCR results demonstrated that salidroside inhibit the cell cycle arrest was associated with an inhibition of the mRNA expression of cyclin D1, cyclin E, CDK4, CDK2, as well as an increase of the mRNA expression of cyclin kinase inhibitor protein p27in PDGF-BB-stimulated PASMCs. Further study showed that the beneficial effect of salidroside on blocking the proliferation of PASMCs is related to suppress AKT/GSK3p signaling pathway, but little affect to extracellular signal-regulated kinase(ERK)1/2, p38and JNK signaling pathway; Transwell test result indicated that the migration of PASMCs induced by PDGF-BB could be inhibit by100μ mol/L salidroside, and that is associated with regulation the expression of cyclin D1, cyclin kinase inhibitor protein p27and MMP-2/9in PDGF-BB-stimulated PASMCs.Conclusion:These results demonstrate that Salidroside can block the proliferation of PASMCs through G0/G1to S phase of the cell cycle arrest. Further study indicates that salidroside suppress PDGF-BB-induced PASMCs proliferation is associated with an inhibition of AKT/GSK3β signal pathway. Salidroside inhibit the migration of PASMCs induced PDGF-BB by regulation the expression of cyclin D1, cyclin kinase inhibitor protein p27and MMP-2/9. Salidroside, therefore, has a potential applicationin preventing pulmonary vascular remodeling.