金荞麦提取物通过ROS/MAPK信号通路途径对结直肠癌细胞抗肿瘤作用

Antitumor Effect of Fagopyri Dibotryis Rhizoma Extract on Colorectal Cancer Cells Through ROS/MAPK Signaling Pathway

目的:探讨金荞麦对人结直肠癌HCT-116细胞增殖、凋亡及自噬的影响,明确其可能的作用机制。方法:采用细胞增殖与活性检测(CCK-8)法检测细胞活性,计算半数抑制率(IC50);设置空白组、金荞麦组(2、4、8 mg·L^(-1)),采用克隆形成实验观察金荞麦对HCT-116细胞增殖的影响;用流式细胞术检测金荞麦对细胞凋亡的影响;蛋白免疫印迹法(Wester blot)检测自噬相关蛋白表达;Wester blot检测p38丝裂原活化蛋白激酶(p38 MAPK)、细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)、磷酸化(p)-p38、p-ERK、p-JNK等蛋白的表达;流式细胞仪和荧光显微镜分析活性氧(ROS)的变化,并用ROS清除剂(NAC)进行验证。结果:与空白组比较,金荞麦组HCT-116细胞活性明显降低(P<0.05,P<0.01);金荞麦组HCT-116细胞凋亡率显著升高(P<0.01);自噬相关蛋白泛素结合蛋白(p62)表达降低,自噬特异性基因(Beclin1)、自噬微管相关蛋白1轻链3B(LC3B)表达明显增强(P<0.05,P<0.01)。与金荞麦组比较,金荞麦+NAC组细胞凋亡率显著降低(P<0.01),相关自噬蛋白Beclin1表达显著降低(P<0.01)、LC3B蛋白表达显著降低(P<0.01)。金荞麦组MAPK通路相关蛋白ERK、JNK表达明显降低(P<0.05,P<0.01),p-ERK、p-JNK表达明显增强(P<0.05,P<0.01)。结论:金荞麦可抑制HCT-116细胞增殖,诱导细胞凋亡及自噬,作用机制与ROS/MAPK信号通路途径有关。

Objective:To observe the effects and underlying mechanisms of Fagopyri Dibotryis Rhizoma extract on the proliferation,apoptosis,and autophagy of human colorectal cancer HCT-116 cells.Method:Firstly,cellular activity was detected by the cell proliferation and activity-8(CCK-8)assay,and the half inhibition rate(IC50)was calculated.Blank group and Fagopyri Dibotryis Rhizoma group(2,4,8 mg·L^(-1))were set.The effect of Fagopyri Dibotryis Rhizoma on the proliferation of HCT-116 cells was observed by cloning experiments,and that of Fagopyri Dibotryis Rhizoma on apoptosis was observed by flow cytometry.The expressions of autophagy-related proteins,p38 mitogen-activated protein kinase(p38 MAPK),extracellular signal-regulated kinase(ERK),c-Jun amino-terminal kinase(JNK),phosphorylated(p)-p38,p-ERK,p-JNK,and other proteins were detected by Western blot.Finally,flow cytometry instrumentation and fluorescence microscopy were used to analyze the changes in reactive oxygen species(ROS),and a ROS scavenger(NAC)was adopted for verification.Result:Compared with the blank group,the activity of HCT-116 cells was significantly decreased in the Fagopyri Dibotryis Rhizoma group(P<0.05,P<0.01).The apoptosis rate of HCT-116 cells in the Fagopyri Dibotryis Rhizoma group was significantly increased(P<0.01).The expression of autophagy-related protein ubiquitin-binding protein(p62)was decreased,but that of autophagyspecific genes(Beclin1)and autophagic microtubule-associated protein 1 light-chain 3B(LC3B)was enhanced(P<0.05,P<0.01).Compared with the Fagopyri Dibotryis Rhizoma group,the apoptosis rate of HCT-116 cells in the Fagopyri Dibotryis Rhizoma+NAC group was significantly reduced(P<0.01).The expression of related autophagy protein Beclin1 was significantly reduced(P<0.01),and that of LC3B protein was reduced(P<0.01).In addition,the expression of MAPK pathway-related proteins ERK and JNK was decreased in the Fagopyri Dibotryis Rhizoma group(P<0.05,P<0.01),and that of p-ERK and p-JNK was enhanced(P<0.05,P<0.01).Conclusion:Fagopyri Dibotryis Rhizoma can inhibit the proliferation of HCT-116 cells and induce apoptosis and autophagy through the ROS/MAPK signaling pathway.