翼首草提取物对结肠癌LoVo细胞增殖、凋亡的影响

Effects of Pterocephalus hookeri extracts on cell proliferation and apoptosis rates of colon cancer LoVo cell line

[目的]研究翼首草提取物对结肠癌LoVo细胞增殖抑制、凋亡的影响。[方法]结肠癌LoVo细胞处理后分为4组,一组加含有10%胎牛血清培养基设为对照组;其余三组采用不同浓度翼首草提取物培养基处理,按质量浓度分3组:低剂量(5~10μg/mL)、中剂量(20~40μg/mL)、高剂量(80~100μg/mL)。采用噻唑蓝比色法、流式细胞仪、反转录-聚合酶链反应法和蛋白质印迹法检测翼首草提取物对LoVo细胞增殖抑制率、凋亡率及细胞周期、Bcl-2、Caspase-3、Bax的mRNA与蛋白表达。[结果]翼首草提取物低、中高剂量组的细胞抑制率分别为(30.39±1.27)%、(51.47±1.21)%、(69.15±1.43)%,高剂量组对LoVo细胞增殖抑制作用最强,各组间差异具有统计学意义(P<0.05)。对照组、低、中高剂量组对LoVo细胞凋亡率分别为(2.32±0.11)%、(5.96±0.14)%、(20.39±0.34)%、(29.88±0.36)%,高剂量对LoVo细胞的凋亡作用最强,各组间差异具有统计学意义(P<0.05)。低、中、高剂量组能抑制细胞S期与G1/S转换,S、G2期细胞比例下降,S期细胞比例分别为(45.20±0.72)%、(27.41±1.58)%、(11.43±0.97)%;相对于对照组,低中高剂量组出现S期细胞占比逐渐下降,且三组间差异具有统计学意义(P<0.05)。对照组Bcl-2、Caspase-3、Bax mRNA表达量分别为(1.84±0.61)、(0.36±0.07)、(0.68±0.41),低剂量组为(1.52±0.54)、(0.67±0.14)、(0.88±0.54),中剂量组为(1.27±0.48)、(0.85±0.36)、(1.02±0.66),高剂量组为(1.06±0.37)、(0.98±0.41)、(1.25±0.70),随着翼首草提取物浓度上升,细胞Bcl-2 mRNA表达量下降,Caspase-3与Bax mRNA表达量上升,三组间差异具有统计学意义(P<0.05)。各组对LoVo细胞中Bcl-2作用的条带密度低于对照组,Caspase-3、Bax强于对照组;且随着翼首草提取物浓度上升,其Bcl-2条带密度下调,Caspase-3、Bax条带密度上调。[结论]翼首草提取物能抑制结肠癌LoVo细胞增殖,诱导其凋亡,作用机制与Bcl-2下调、Caspase-3与Bax上调表达有关。

[Objective]To investigate the effects of Pterocephalus hookeri extracts on proliferation inhibition and apoptosis rates of colon cancer LoVo cell line.[Method]J Colon cancer LoVo cells were classified into four groups.Cells cultured in regular medium containing 10%fetal bovine serum were set as control group.The rest cells were cultured in medium containing different concentrations of Pterocephalus hookeri extracts,and were divided into three groups according to the mass concentration,low-dose group(5-10μg/mL),medium-dose group(20-40μg/mL)and high-dose group(80-100μg/mL).Thiazole blue colorimetry,flow cytometry,reverse transcription polymerase chain reaction and Western Blot were adopted to detect the inhibition rate of proliferation,apoptosis rate,cell cycle,mRNA and protein expression of Bcl-2,Caspase-3 and Bax in LoVo cells.[Result]The inhibition rate of proliferation was(30.39±1.27)%,(51.47±1.21)%and(69.15±1.43)%in low-dose group,medium-dose group and high-dose group respectively,indicating that the high-dose group exhibited the strongest inhibitory effect on the proliferation of LoVo cells,with statistical difference in paired comparison(all P<0.05).The cell apoptosis rate was(2.32±0.11)%,(5.96±0.14)%,(20.39±0.34)%and(29.88±0.36)%in control group,low-dose group,medium-dose group and high-dose group respectively,suggesting that the high-dose group exhibited the strongest apoptosis effect on LoVo cells,with statistical difference in paired comparison(all P<0.05).Pterocephalus hookeri extracts of low,medium and high doses could inhibit the transition of cells in S phase and G1/S phase,and reduce the proportion of cells in S and G2 phases.The proportions of cells in S phase were(45.20±0.72)%,(27.41±1.58)%and(11.43±0.97)%in low-,medium-and high-dose groups,respectively.Compared with control group,the proportion of S-phase cells in the low-,middle-and high-dose groups gradually decreased,with statistical difference in paired comparison(all P<0.05).The mRNA expression levels of Bcl-2,Caspase-3 and Bax in LoVo cells were(1.84±0.61),(0.36±0.07)and(0.68±0.41)in control group,(1.52±0.54),(0.67±0.14)and(0.88±0.54)in low-dose group,(1.27±0.48),(0.85±0.36)and(1.02±0.66)in medium-dose group,and were(1.06±0.37),(0.98±0.41)and(1.25±0.70)in high-dose group.With the increase of the concentration of Pterocephalus hookeri extracts,the mRNA expression of Bcl-2 in cells decreased,while the mRNA expression of Caspase-3 and Bax increased,with statistical difference(P<0.05).The optical density value of Bcl-2 protein in Pterocephalus hookeri extract treatment groups was smaller than control group,while that of Caspase-3 and Bax proteins were larger than control group(all P<0.05).Moreover,the density of Bcl-2 proteins was down regulated while Caspase-3 and Bax proteins were up-regulated as the concentration of Pterocephalus hookeri extracts increased.[Conclusion]Pterocephalus hookeri extracts inhibit the proliferation and induce apoptosis of colon cancer LoVo cells,which may be related to the down-regulation of Bcl-2 and up-regulation of Caspase-3 and Bax expression.