芪贞汤对脂多糖诱导人结肠癌细胞上皮间充质转化的影响

Effects of Qizhen decoction on LPS-induced epithelial mesenchymal transition in human colon cancer cells

目的研究芪贞汤对脂多糖(lipopolysaccharide,LPS)诱导结肠癌细胞上皮间充质转化(epithelial-mesenchymal transition,EMT)的机制,为其发挥抗癌作用提供实验依据。方法将人结肠癌Lo Vo细胞分为4组,空白组不予干预,模型组、实验组和对照组分别予1μg/m L LPS、1μg/m L LPS+800μg/m L芪贞汤、1μg/m L LPS+100μg/m L 5-氟尿嘧啶(5-Fu)干预48小时。采用MTT实验计算芪贞汤诱导肿瘤细胞凋亡50%的浓度,即IC50 (half maximal inhibitory concentration);划痕实验观察细胞迁移能力;免疫荧光法检测NF-κB p65核转位情况; Western blot检测E-钙黏蛋白(E-cadherin,E-cad)和N-钙黏蛋白(N-cadherin,N-cad)蛋白的表达。结果芪贞汤在48小时诱导肿瘤细胞凋亡的IC50值为980. 855μg/m L。空白组、模型组、实验组和对照组划痕缩小面积分别为(46.38±5.15)(80.81±3.18)(17.78±9.87)和(16.95±3.45);与模型组相比,各组划痕缩小面积皆有极显著统计差异(P <0. 01)。空白组NF-κB p65多于细胞浆中表达;模型组可以明显看到NF-κB p65入核,核转位明显;对照组NF-κB p65在包浆和细胞核中都有表达;实验组NF-κB p65在细胞核中表达相对较少,核转移情况下降。相对于空白组,实验组E-cadherin表达量(1. 266±0.065)和对照组E-cadherin表达量(0.979±0.183)比模型组(0. 420±0. 071)显著升高,有极显著统计学差异(P <0. 01);实验组N-cadherin表达量(0. 518±0. 115)和对照组N-cadherin表达量(1. 275±0. 567)比模型组(2. 287±0. 257)显著降低,有极显著统计学差异(P <0. 01)。结论芪贞汤可通过抑制NF-κB p65活化而抑制结肠癌细胞EMT的发生,从而达到控制肿瘤细胞迁移的效果。

Objective To investigate the mechanism of Qizhen decoction on lipopolysaccharide(LPS)-induced epithelial mesenchymal transition(EMT)in colon cancer cells,and provide experimental evidence for its anti-cancer effects.Methods Human colon cancer LoVo cells were divided into 4 groups.The blank group was not treated.The model group,the experimental group and the control group were given 1μg/ml LPS,1μg/ml LPS+800μg/mL Qizhen decoction,and 1μg/mL LPS+100μg/mL 5-fluorouracil(5-Fu)intervention for 48h.The MTT assay was used to calculate the concentration of 50%of tumor cell apoptosis induced by Qizhen decoction,which was IC50(half maximal inhibitory concentration).The cell migration ability was observed by scratch test.The nuclear translocation of NF-κB p65 was detected by immunofluorescence.Western blot was used to detect the expression of E-cadherin and N-cadherin proteins.Results The IC50 value of Qizhen decoction induced apoptosis of tumor cells at 48h was 980.855μg/mL.The scratch reduction areas of the blank group,the model group,the experimental group and the control group were(46.38±5.15),(80.81±3.18),(17.78±9.87)and(16.95±3.45),respectively.Compared with the model group,the scratch reduction areas of each group were extremely statistically significant(P<0.01).NF-κB p65 was more expressed than in cytoplasm in the blank group.In the model group,NF-κB p65 was observed to enter the nucleus,and nuclear translocation was obvious.NF-κB p65 was expressed both in the cytoplasm and nucleus in the control group.Compared with the blank group,the expression of E-cadherin in the experimental group(1.266±0.065)and the control group(0.979±0.183)were significantly higher than those in the model group(0.420±0.071)(P<0.01).The expression of N-cadherin in the experimental group(0.518±0.115)and the control group(1.275±0.567)were significantly lower than those in the model group(2.287±0.257)(P<0.01).Conclusion Qizhen decoction can inhibit the occurrence of EMT in colon cancer cells by inhibiting the activation of NF-κB p65,thus achieving the effects of controlling tumor cell migration.