The Studies On Effects Of Toremifene On The Biological Behavior Of Vascular Endothelial And Smooth Muscle Cells And Its Effect Mechanism

Cardiovascular diseases are one of the major causes of human death in the world.Drug-eluting stent(DESs)implantation are the best treatment,which can reduce the chance of restenosis.However,the rate of restenosis(RS)is not completely eliminated.The prevention and treatment of restenosis is one of the focus area of cardiovascular research.The inhibition of VSMCs proliefartion and rapid re-endothelialization are the key for the repairment of injured vessel.In this present study,using human umbilical vein endothelial cells(HUVECs)and VSMCs as cell model in vitro,we investigated the effect of toremifene(TOR),a new generation of antineoplastic drugs,on biology behavior of HUVECs and VSMCs and its effect mechanism.The main research contents and results are as follows:(1)Effects of TOR treament on biological behavior of HUVECs: Studies showed that the TOR can inhibit cells proliferation and its inhibiting effect gradually enhanced with increase of TOR concentration.TOR can significantly promote the early cell apoptosis,late cell apoptosis and cell necrosis,alter cell cycle distribution and arrest cells at the G0/G1 cell cycle phase.TOR can delay the cell adhesion time but did not affect the number of adherent cells.TOR can also significantly inhibit the group cell movement and migration behavior.High concentration of TOR can significantly change the cell morphology.(2)The mechanism of the effects of TOR treament on HUVEC:Immunocytochemistry and immunoblotting was used to examine the expression of PCNA and P53,which can reflect cell proliferation and apoptosis,respectively.The results showed that the higher concentration of TOR reatment inhibited cell growth and induced cell apoptosis.With the increae of TOR concentration,the expression of PCNA decreased,and the expression of P53 increased,which indicated that TOR accelerated cell apoptosis by promoting expression of apoptosis gene P53.The research of migration mechanism showed that the TOR significantly inhibited the expression of Integrin?1 and Rho,and decreased the level of MLC and pMLC,which pointed out that TOR inhibited cell migration behavior by the Integrin ?1-Rho-ROCK-(p)MLC signaling pathway.(3)Effects of TOR treament on biological behavior of VSMCs: The research results showed that the high concentration of TOR can inhibit cell proliferation and itsinhibiting effect gradually enhanced with increase of TOR concentration.TOR can significantly promote VSMCs apoptosis.TOR can delay the cell adhesion time but did not affect the number of adherent cells.TOR can also significantly inhibit the group cell movement and migration behavior.High concentration of TOR can significantly change the morphology of VSMCs.(4)The mechanism of the effects of TOR treatment on VSMCs:Immunocytochemistry and immunoblotting were used to examine the expression of PCNA,P53 and protein of Rho/ROCK signal transmission pathway,which can reflect cell proliferation,apoptosis and migration mechanism,respectively.Flow cytometry was used to further test the cell apoptosis.The results showed that the higher concentration of TOR reatment inhibited cell growth and induced cell apoptosis.With the increase of TOR concentration,the expression of PCNA decreased,and the expression of P53 increased,which indicated that TOR accelerated cell apoptosis by promoting expression of apoptosis gene P53.Compared with the control,high concentration of TOR can significantly increased mitochondrial membrane potential(MMP)level in TOR treament cells,which led to the increase of reactive oxygen species(ROS),and caspase-3 and-9 activities.The decrease of MMP,increase of ROS and activities of caspase-3 and-9 may be signals of cell apoptosis.TOR significantly inhibited the expression of Integrin ?1 and Rho,and decreased the expression level of ROCK signaling pathways factor MLC and pMLC,which pointed out that TOR may inhibit cell migration behavior by the Integrin ?1-Rho-ROCK-(p)MLC signaling pathway.