The Inhibitory Effect And Mechanism Of Dioscin On The Proliferation And Migration Of Vascular Smooth Muscle Cells And Intimal Hyperplasia

Objective: To explore the effect and mechanism of dioscin on the proliferation and migration of vascular smooth muscle cells and intimal hyperplasia.Method: 1.The effects of dioscin on proliferation of vascular smooth muscle cells(VSMCs)and intimal hyperplasia after carotid artery injury in rats: The rat's right carotid artery was injuried by balloon dilatation,leading to intimal thickening and then establishing restenosis model.2 weeks later,the artery were harvested and stained by hematoxylin-elsin(HE)staining and immunofluorescence of PCNA,?-SMA and CD31.The morphological changes were checked under microscope.The area of the intimal and medial layer of the vessels,and their ratio was analyzed and calculated.The expression level of PCNA,?-SMA and CD31 were expressed as the positive index and fluorescence area,respectively.2.The effect of dioscin on proliferation,migration and phenotype switching of vascular smooth muscle cells in vitro: Primary rat aortic vascular smooth muscle cells were cultured in vitro.The effect of dioscin on VSMCs proliferation was observed by MTT test.PCNA protein expression was examined by western blot to further explore the effect of dioscin on VSMCs proliferation.The effect of dioscin on VSMCs migration was detected by wound healing.The effect of dioscin on VSMCs phenotype switching was explored by western blot and RT-q PCR.The effect of dioscin on HUVECs proliferation and migration was detected by MTT and wound healing.3.The mechanism of dioscin inhibiting proliferation and migration of vascular smooth muscle cells: The expression level of phosphorylated extracellular signal-regulated kinase 1/2(ERK1/2)and Forkhead box M1(Fox M1)was examined by western blot.The expression level of Fox M1 and its downstream target genes was determined by RT-q PCR.The effect of Fox M1 on proliferation and migration of vascular smooth muscle cell was examined by MTT and wound healing after overexpression and knockdown of Fox M1 in smooth muscle cells.Result: 1.Dioscin inhibited proliferation of vascular smooth muscle cells and neointimal hyperplasia after carotid artery injury in rats: The lumen area was reduced significantly,while the intimal area and intima/media area increased significantly,indicating that the model was successful.Compared with the model group,the lumen area of dioscin high-dose group increased significantly,and the intimal area and intima/media ratio decreased significantly(P < 0.05).The expression of PCNA and ?-SMA in the injured artery was significantly increased.Compared with the model group,high dose of dioscin significantly decreased the positive indice of PCNA and the fluorescence area of ?-SMA(P < 0.05).The immunofluorescence of CD31 showed that dioscin did not affect re-endothelialization after vascular injury(P > 0.05).2.Dioscin inhibited proliferation,migration and phenotype switching of vascular smooth muscle cells in vitro: MTT assay showed that dioscin inhibited VSMCs proliferation induced by 10% FBS in a dose-dependent manner.Western blot analysis showed that dioscin significantly reduced PCNA protein expression level.Wound healing showed that dioscin inhibited VSMCs migration induced by 20ng/ml PDGF.In addition,dioscin increased the expression level of VSMCs contractile phenotype markers,?-SMA and SM22?,and decreased the expression level of a synthetic phenotype marker,OPN.However,Dioscin at the same dose did not significantly affect HUVEC proliferation and migration.3.Dioscin inhibited proliferation and migration of vascular smooth muscle cells by the inhibition of ERK1/2-Fox M1 pathway: Western blot and RT-q PCR showed that dioscin inhibited the phosphorylation of ERK1/2 and decreased the expression level of Fox M1 and its downstream target genes.MTT and wound healing revealed overexpression of Fox M1 in vascular smooth muscle cells promoted cells proliferation and migration,and knockdown of Fox M1 in vascular smooth muscle cells inhibited cells proliferation and migration.Conclusion:1.Dioscin inhibits intimal hyperplasia in rat carotid artery injury model,which is caused by the inhibitory effect of dioscin on proliferation of vascular smooth muscle cells,suggesting that dioscin may have a positive preventive and therapeutic effect on restenosis after PCI.2.Dioscin inhibits proliferation,migration and phenotype switching of vascular smooth muscle cells induced by FBS or PDGF.3.Dioscin inhibits proliferation and migration of vascular smooth muscle cells through ERK1/2-Fox M1 pathway.