Effect Of Isoflurane On Proliferation,Apoptosis And Cell Cycle Of HepG2 Cells

ObjectiveTo investigate the influence of clinically relevant concentration isoflurane on proliferation,cell cycle and apoptosis of human liver carcinoma cell HepG2 and it's relation with p53 signal pathway.Methods1.Effect of isoflurane on the proliferation,apoptosis,cell cycle and molecule expression of HepG2 cells: HepG2 cells at logarithmic phase were selected and seeded in culture plate.After 24 h cells were randomly assigned into four groups: group control(C),group 1% isoflurane(P1),group 1.5% isoflurane(P2)and group 2% isoflurane(P3).C,P1,P2,P3 group were treated with 0%,1%,1.5%,2% isoflurane for 4h,respectively.Then the cells were cultured for another 6,18,24,72 h and cell cycle were analyzed by flow cytometer.At 24 h,EdU incorporation assay or flow cytometer,was used to detect the rate of proliferation,apoptosis of HepG2 cells,and Western blot or RT-RCR was employed to measure the expression of p53,p21,PCNA,CDK1,CDK2 proteins or p53,p21 mRNA respectively.2.Screening the target of p53-shRNA: HepG2 cells at logarithmic phase were selected and seeded in culture plate.After 24 h cells were randomly divided into six groups : Blank control group(Control group);Negative control group(Control + sh-NC group);Control+sh-p53(1802)group;Control+sh-p53(1803)group;Control+ sh-p53(1804)group;Control+sh-p53(1805)group.The HepG2 cells in the Control group were not transfected with shRNA plasmid,and in the Control+sh-NC group cells were transfected with scrambled shRNA.In the other groups,cells were transfected with corresponding shRNA.After 24 h,Western blot and RT-PCR were used to analyze the protein and mRNA expression of p53.3.The effect of isoflurane on HepG2 cells that were down-regulated p53 expression by RNAi: HepG2 cells at logarithmic phase were selected and seeded in culture plate.After 24 h cells were randomly divided into four groups: Control group;group treated with2% isoflurane(2%iso group);group transfected with sh-NC and treated with 2%isoflurane(2%iso+sh-NC);group transfected with p53-shRNA and treated with 2%isoflurane(2%iso+sh-p53 group).After 24 h,flow cytometer and EdU incorporation assay were used to detect the cell cycle and the rate of apoptosis,proliferation of HepG2 cells.The expression of p53,p21,PCNA,CDK1,CDK2 proteins and p53 mRNA,p21 mRNA were measured by Western blot or RT-RCR respectively.Results 1.Effect of isoflurane on the proliferation,apoptosis,cell cycle and molecule expression of HepG2 cells: In corresponding cell cycle phase,compared with control group,there was no significant difference in P1,P2,P3 group at 6,18,72 h.At 24 h,the percentage of cells in S phase of HepG2 cells in group P1,P2,P3 were increased as compared with group C(p<0.05).Compared with group C,the rate of proliferation of HepG2 cells in P1,P2,P3 group were inhibited as isofurane concentration increased(p<0.05).Compared with group C,the rate of apoptosis of HepG2 cells in P1,P2,P3 group were increased(p<0.05).Compared with group C,the mRNA and protein expressions of p53,p21 were upregulated in P1,P2,P3 group(p<0.05).Compared with group C,the protein expression of PCNA,CDK1,CDK2 were down-regulated in P1,P2,P3 group(p<0.05).2.Screening the target of p53-shRNA: Compared with group Control and Control+sh-NC,the expression of p53 mRNA and protein in group Control+sh-p53(1804)were down-regulated(p<0.05).3.The effect of isoflurane on HepG2 cells that were down-regulated p53 expression by RNAi: Compared with group 2%iso and 2%iso+sh-NC,the percentage of cells in S phase of HepG2 cells in group 2%iso+sh-p53 were decreased(p<0.05).The rate of proliferation of HepG2 cells in 2%iso+sh-p53 group were inceased as compared with group 2%iso and 2%iso+sh-NC(p<0.05).Compared with group 2%iso and 2%iso+sh-NC,the rate of apoptosis of HepG2 cells in 2%iso+sh-p53 group were decreased(p<0.05).Compared with group 2%iso and 2%iso+sh-NC,the mRNA and protein expressions of p53,p21 were down-regulated in 2%iso+sh-p53 group(p<0.05).Compared with group 2%iso and 2%iso+sh-NC,the protein expression of PCNA,CDK1,CDK2 were up-regulated in 2%iso+sh-p53 group(p<0.05)ConclusionIsoflurane could inhibit the proliferation and induce the apoptosis of HepG2 cells,as well as cause cell cycle arrest at S phase through p53 signal pathway.