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-?1signaling 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 0.05?M,0.2?M,0.8?M),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?0.05?M,0.2?M,0.8?M?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-?1signal pathway,based on the effect of GS-Re on inhibiting VSMCs proliferation,we using eNOS inhibitor L-NAME and TGF-?1 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 0.8?M+L-NAME 20?M);?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 0.8?M+SIR 10?M).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,SM22?,SM-MHC,eNOS,p-eNOS^ser1177,Ras,MEK1/2,p-MEK,ERK1/2,p-ERK1/2,TGF-?1,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-?1,p-Smad2,p-Smad3 in Model group significantly increased,compared with Normal group?P<0.05 or P<0.01?;P21,SM22?,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-?1,p-Smad2,p-Smad3?P<0.01?,and up-regulate the expressions of P21,SM22?,SM-MHC,p-eNOS^ser1177.The effects of GS-Re?treatment at the optimal concentration?0.8?M??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-?1 signaling pathway.