白芦藜醇通过抑制血管内皮生长因子受体-1-磷脂酰肌醇3激酶/蛋白激酶B信号转导通路影响HepG2细胞增殖

Effects of resveratrol on HepG2 cell proliferation by inhibiting vascular endothelial growth factor receptor-1-phosphatidylinositol 3 kinase/protein kinase B signaling pathway

目的观察白芦藜醇通过抑制血管内皮生长因子受体(VEGFR)-1-磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号转导通路对肝癌细胞增殖的影响。方法采用噻唑蓝(MTT)法检测白芦藜醇对HepG2细胞(2017年6月购自上海普诺赛生物)细胞增殖的抑制作用,流式细胞术检测细胞周期变化,反转录-聚合酶链反应(RT-PCR)检测VEGFR-1、Akt基因表达水平,蛋白质印迹法(Western blot)检测磷酸化(p)-VEGFR-1、VEGFR-1、p-Akt和Akt表达水平。采用转化后的Cochran Armitage趋势检验。结果白藜芦醇对HepG2细胞增殖具有明显的抑制作用,且呈剂量和时间依赖性,表明白藜芦醇浓度增加,作用时间越长,对HepG2细胞增殖的抑制作用越强。白藜芦醇浓度为0、20、80、160μmol/L时G0/G1分别为35.60、40.91、49.35和55.15,结果显示白藜芦醇作用HepG2细胞48 h后,可使肝癌HepG2细胞周期阻滞于G0/G1期。白藜芦醇浓度由5μmol/L逐渐增加至160μmol/L时,HepG2细胞中VEGFR-1表达水平由1.00±0.08逐渐降至0.28±0.02。HepG2细胞中Akt mRNA表达水平由2.32±0.22逐渐降至1.08±0.10。随着白藜芦醇浓度的增加,HepG2细胞中p-VEGFR-1、VEGFR-1、Akt和p-Akt蛋白表达水平均显著降低(χ2=15.183、17.842、32.628、26.163,P<0.05),其随白藜芦醇浓度变化而变化的趋势,经Cochran Armitage趋势检验,差异均有统计学意义(χ2=22.798,P<0.05)。结论白芦藜醇通过抑制VEGFR-1-PI3K/Akt信号转导通路的表达可抑制肝癌HepG2细胞增殖。

Objective To observe the effect of resveratrol on the proliferation of liver cancer cells by inhibiting vascular endothelial growth factor receptor(VEGFR)-1-phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)signal transduction pathway.Methods Methyl thiazol tetrazolium(MTT)assay was used to detect the inhibitory effect of resveratrol on the proliferation of HepG2 cells.Flow cytometry was used to detect cell cycle changes.Reverse transcriptase-polymerase chain reaction(RT-PCR)was used to detect the expression of VEGFR-1 and Akt genes.Western blotting was used to detect p-VEGFR-1 and VEGFR-1.P-Akt and Akt expression levels.Results Resveratrol significantly inhibited the proliferation of HepG2 cells in a dose-dependent and time-dependent manner,indicating that the concentration of resveratrol increased,and the longer the time of action,the stronger the inhibitory effect on the proliferation of HepG2 cells.The concentrations of resveratrol at 0,20,80 and 160μmol/L,the proportion of cells in G0/G1 phase was 35.60%,40.91%,49.35%and 55.15%,respectively.The results showed that the cycle of HepG2 cells could be blocked in the G0/G1 phase after the treatment of HepG2 cells with resveratrol for 48 h.When the concentration of resveratrol gradually increased from 5μmol/L to 160μmol/L,the expression level of VEGFR-1 in HepG2 cells gradually decreased from 1.00±0.08 to 0.28±0.02,and the expression level of Akt mRNA in HepG2 cells decreased gradually from 2.32±0.22 to 1.08±0.10.Along with the increase of the concentration of resveratrol,VEGFR-1,p-VEGFR-1,Akt and p-Akt protein expression levels in HepG2 cells were significantly reduced(χ2=15.183,17.842,32.628,26.163,P<0.05).The Cochran Armitage trend test reveald that the differences were statistically significant(χ2=22.798,P<0.05).Conclusion Resveratrol inhibits the proliferation of HCC HepG2 cells by inhibiting the expression of the VEGFR-1-PI3K/Akt signal transduction pathway.