丹酚酸B对结肠癌HCT116细胞增殖的抑制作用机制研究

Inhibition effect and mechanism of the salvianolic acid B on the proliferation of human colon cancer HCT116 cells

目的探究丹酚酸B(Sal B)对结肠癌细胞发生自噬性死亡的作用机制。方法将细胞分为4组:空白对照组(结肠癌细胞以等量2%胎牛血清处理)和低、中、高3个浓度实验组(结肠癌细胞分别以50,100,200μmol·L^-1的Sal B处理),均干预24 h。通过转染自噬双标腺病毒(Ad-m RFP-GFP-LC3)检测HCT116细胞自噬情况;CCK-8法检测HCT116细胞增殖并计算细胞增值抑制率;蛋白质印迹法检测微管相关蛋白I轻链3(LC3)、核孔蛋白p62、自噬相关蛋白5同源物(Atg5)表达和蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路蛋白磷酸化水平。结果干预HCT116细胞24 h后,空白对照组和高浓度实验组中FITC-LC3阳性细胞百分比分别为(5.32±0.35)%和(40.12±1.45)%,组间差异有统计学意义(P<0.01)。空白对照组和低、中、高3个浓度实验组细胞增殖抑制率分别为(2.32±0.45)%,(12.84±1.97)%,(28.48±0.46)%和(48.77±2.71)%;这4组的自噬相关蛋白LC3-Ⅱ的相对表达量分别为0.11±0.02,0.21±0.03,0.38±0.07和0.62±0.06;这4组的LC3-Ⅰ蛋白相对表达量分别为0.97±0.09,0.93±0.08,0.86±0.13和0.71±0.05;这4组的Atg5蛋白相对表达量分别为0.42±0.08,0.67±0.07,0.85±0.13和0.90±0.09;这4组的p62蛋白相对表达量为0.84±0.11,0.75±0.07,0.56±0.09和0.31±0.06;这4组的p-AKT相对表达量为0.81±0.13,0.37±0.05,0.16±0.07和0.14±0.03;这4组的p-mTOR相对表达量为0.74±0.14,0.41±0.08,0.31±0.05和0.27±0.05。上述指标:3个浓度实验组与空白对照组比较,差异均有统计学意义(均P<0.05);且各指标逐渐增加或降低与浓度呈正相关。结论Sal B可抑制HCT116结肠癌细胞增殖,自噬可能介导了Sal B诱导HCT116结肠癌细胞死亡的过程,其作用机制可能与其降低AKT/mTOR信号通路相关蛋白表达有关。

Objective To investigate the effect and mechanism of salvianolic acid B(Sal B)on autophagy death of colon cancer cells.Methods The cells were divided into four groups:Blank control group(with the same amount of 2%fetal bovine serum)and low,middle and high concentration experimental groups(with Sal B of 50,100 and 200 mol·L^-1),for 24 h.Autophagy of HCT116 cells was detected by transfection with Ad-m RFP-GFP-LC3.The proliferation of HCT116 cells was detected by CCK-8 method and the inhibition rate of cell proliferation was calculated.The expression of microtubule associated protein I light chain 3(LC3),nucleoporin p62,autophagy associated protein 5 homologue(ATG5)and protein kinase B(Akt)/mammalian rapamycin target protein(mTOR)pathway protein phosphorylation were detected by Western blot.Results After intervention for 24 h,the percentage of FITC-LC3 positive cells in blank control group and high concentration experimental group were(5.32±0.35)% and(40.12±1.45)%,respectively;compared between high concentration experimental group and blank control group,the difference was significant(P<0.01).The proliferation inhibitory rate in blank control group and low,middle and high concentration experimental groups were(2.32±0.45)%,(12.84±1.97)%,(28.48±0.46)% and(48.77±2.71)%,respectively;the relative expression of microtubule associated protein I light chain 3-II type(LC3-II)protein in the 4 groups were 0.11±0.02,0.21±0.03,0.38±0.07,0.62±0.06,respectively;the relative expression of microtubule associated protein I light chain 3-I type(LC3-Ⅰ)protein in the 4 groups were 0.97±0.09,0.93±0.08,0.86±0.13,0.71±0.05,respectively;the relative expression of ATG5 in the 4 groups were 0.42±0.08,0.67±0.07,0.85±0.13,0.90±0.09,respectively;the relative expression of p62 protein in the 4 groups were 0.84±0.11,0.75±0.07,0.56±0.09,0.31±0.06;the relative expression of p-Akt in the 4 groups were 0.81±0.13,0.37±0.05,0.16±0.07,0.14±0.03;and the relative expression of p-mTOR in the 4 groups were 0.74±0.14,0.41±0.08,0.31±0.05,0.27±0.05.Compared between three concentration experimental groups and normal group,the difference of the factors were significant(all P<0.01),and each index increased or decreased gradually and was positively correlated with the concentration.Conclusion Sal B can inhibit the proliferation of HCT116 colon cancer cells,and autophagy may mediate the process of Sal B inducing the death of HCT116 colon cancer cells,which may be related to the decreased expression of AKT/mTOR signaling proteins.