A Study Of The Role On Cell Proliferation And The Formation Of Neointima Mediated By Rac1b And Its Specific Inhibitor-sanguinarine

Objective: To examine Rac1b’s function in cell proliferation and migration and to determine Rac1b’expression in carotid neoitima after balloon injured.To investigate sanguinarine’s potential inhibitory effects on cell proliferation in Rac1 b expressing cell-lines and the formation of carotid neoitima after balloon injured.Methods: 1)Three 293 T cell-lines expressing GFP,Rac1,or Rac1 b were made by lentivirus packaging constructs,named as Lenti-GFP,Lenti-Rac1-GFP,and Lenti-Rac1b-GFP.Cell proliferation was serially-recorded by cell number counting in the conditions of 10% serum and 0% serum respectively,and migration followed by scratching assay in the marked locations.2)AKT,phosphorylated AKT,PARP fragment and cyclin D1 were detected in Lenti-Rac1b-GFP cultured with mediums containing either 10% or 0% serum as compared to Lenti-GFP via Western Blot.3)293T cell-lines infected with Lenti-GFP,Lenti-Rac1-GFP,or Lenti-Rac1b-GFP were treated with and without berberine(10umol/L)or sanguinarine(1umol/L),cell number were counted.HT29 cells expressing Rac1 b endogenousely was also treated with sanguiarine at different levels of concentration(0,0.1,0.3,1.0,3.0 u M)controlled by HCT116 cells not expressing Rac1 b,CCK-8 kit was used to assay inhibition ratio of these two cell lines.4)Rat carotid balloon injury model was build to investigate the expression of Rac1 b in neointima and inhibitory effect on neoitima formation by sanguiarine.Results: 1)Neither over-expressed Rac1 nor Rac1 b could influence cell proliferation in the circumstance of 10% serum,as compared to control cells expressing GFP;but in the serum-deprived condition,cells over-expressing Rac1 b demonstrated a highest and cells over-expressing Rac1 showed a moderate increase in survival rate,as compared to the control cells;Over-expression of Rac1 or Rac1 b could not promote cell migration among three cell-lines.2)The level of activated AKT was significantly higher in Lenti-Rac1b-GFP than in Lenti-GFP under the condition of 0% serum.The levels of PARP fragment and cyclin D1 were similar in these 2 cell lines under the condition of 0% serum.3)Sanguinarine(1 umol/L),but not berberine(1 umol/L),significantly inhibited cell proliferation only in cells exogenously over-expressing Rac1b;sanguinarine in concentration of 1.0 u M could also significantly inhibit cell proliferation in HT29 cells compared to HCT116 cells.4)De novo Rac1 b was expressed in the neoitima of injured carotid and sanguinarine could decrease the ratio of area of intima and media(I/M).Conclusion: De novo Rac1 b was expressed in the neointima of injured carotid and renders cells with enhanced ability to survive under growth-limiting condition through the mechanism of upregulating phosphorylated AKT.Sanguinarine possesses specific inhibitory effects on cell proliferation of cells expressing either exogenous or endogenous Rac1 b,and could also inhibit the vessel neoitima formation after balloon injured.