Effects And Mechanisms Of Ginsenoside Re On Proliferation Of Vascular Smooth Muscle Cell Induced By PDGF-BB

Objective:To investigate the effects of ginsenoside Re（GS-Re）on the proliferation of vascular smooth muscle cell（VSMCs）induced by platelet derived growth factor-BB（PDGF-BB）,and explore the mechanism whether via eNOS-NO-cGMP-ERK-TGF-β1signaling pathway.Methods:VSMCs were cultured by enzyme digestion method from the thoracic artery of SD male rats.The cells were made following groups:Normal group（Normal）,Normal+GS-Re-H group（N+GS-Re）,Model group(PDGF-BB 25 ng·mL^-1),GS-Re Low,Middle,High groups(GS-Re-L,M,H:PDGF-BB 25 ng·mL^-1+GS-Re 0.05μM,0.2μM,0.8μM),the proliferation model of VSMCs was induced by PDGF-BB which was added in 1 h before GS-Re,and GS-Re was incubated for 24 h;CCK8 assay was used to evaluate the effects of GS-Re（0.05μM,0.2μM,0.8μM）on PDGF-BB induced VSMCs proliferation,the cycle of VSMCs were detected by flow cytometry.To analysis the effect of GS-Re which inhibited VSMCs proliferation whether refer to eNOS-NO-cGMP-ERK-TGF-β1signal pathway,based on the effect of GS-Re on inhibiting VSMCs proliferation,we using eNOS inhibitor L-NAME and TGF-β1 agonist SIR011381（SIR）to interfere the pathway.Thus the cells were divided as follow:（1）Normal group,Model group(PDGF-BB 25ng·mL^-1),GS-Re-H group,L-NAME group(PDGF-BB 25 ng·mL^-1+GS-Re 0.8μM+L-NAME 20μM);（2）:Normal group,Model group(PDGF-BB 25 ng·mL^-1),GS-Re-H group,SIR group(PDGF-BB 25 ng·mL^-1+GS-Re 0.8μM+SIR 10μM).L-NAME,SIR and PDGF-BB were added to the preconditioned cells 1 h ahead of GS-Re,and the cells were treated with GS-Re for 24 h.The protein expression of PCNA,CyclinD1,CDK4,P21,SM22α,SM-MHC,eNOS,p-eNOS^ser1177,Ras,MEK1/2,p-MEK,ERK1/2,p-ERK1/2,TGF-β1,Smad2,p-Smad2,Smad3,p-Smad3 were detected by Western Blot.The levels of NO in cell culture medium were measured by Nitric Oxide Colorimetric Assay and cGMP in VSMCs supernatant were detected by Enzyme-linked immunosorbent assay（ELISA）.Results:The results of CCK8 showed that compared with Normal group,PDGF-BB promoted the proliferation of VSMCs（P<0.01）;But the phenomenon of proliferation is blocked by GS-Re（P<0.05 or P<0.01）,which also promote the VSMCs proliferation cycle to stagnate during the G0/G1 period.The levels of PCNA,CyclinD1,CDK4,Ras,MEK1/2,p-ERK1/2,TGF-β1,p-Smad2,p-Smad3 in Model group significantly increased,compared with Normal group（P<0.05 or P<0.01）;P21,SM22α,SM-MHC,p-eNOS^ser1177er1177 on the contrary.The addition of GS-Re lead to lower leverls of PCNA,CyclinD1,CDK4,Ras,p-MEK1/2,p-ERK1/2,TGF-β1,p-Smad2,p-Smad3（P<0.01）,and up-regulate the expressions of P21,SM22α,SM-MHC,p-eNOS^ser1177.The effects of GS-Re（treatment at the optimal concentration（0.8μM））mentioned above was inhibited by L-NAME or SIR（P<0.05）.The results of colorimetry showed that the content of NO in VSMCs medium was decreased comparing with Normal groups,this effect would be attenuated after treating with GS-Re.The content of cGMP and NO increased or decreased in the same way according to the results of ELISA.Conclusions:GS-Re could significantly inhibit the VSMCs proliferation induced by PDGF-BB;The action mechanisms was related to the activation of NO system and the inhibition of ERK-TGF-β1 signaling pathway.