ROS/JNK/FoxO3a信号通路在钩吻素子抑制结肠癌细胞增殖和诱导凋亡中的作用  

Effect of Koumine on Proliferation and Apoptosis of Colorectal Cancer Cells via ROS/JNK/FoxO3a Signaling Pathway

作　　者：赵斌 吴紫陆 席作武[2] ZHAO Bin;WU Zi-lu;XI Zuo-wu(Second Clinical Medical College,Henan University of Chinese Medicine,Zhengzhou 450002,China;Henan Province Hospital of Traditional Chinese Medicine,Zhengzhou 450002,China)

机构地区：[1]河南中医药大学第二临床医学院,郑州450002 [2]河南省中医院,郑州450002

出　　处：《中国实验方剂学杂志》2021年第24期100-108,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：河南省高等学校重点科研项目(14104691-2019);河南省中医药科学研究专项(2019ZY2025)。

摘　　要：目的:以人结肠癌细胞HCT-116细胞为研究对象,通过在培养液中加入不同浓度的钩吻素子(Kou,0,100,200,400μmol·L^(-1)),进一步探讨Kou对结肠癌细胞增殖和凋亡的影响及其可能的分子作用机制。方法:Kou体外干预HCT-116细胞24 h后,用细胞增殖与活性检测(CCK-8)法检测其对细胞增殖的影响;采用流式细胞术检测细胞周期、细胞凋亡及活性氧(ROS)的表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测叉头蛋白O3a(FoxO3a)mRNA表达情况,采用小干扰核糖核酸(siRNA)进行细胞转染,蛋白免疫印迹法(Western blot)检测FoxO3a靶基因蛋白的表达情况。结果:与空白组比较,Kou处理明显降低了HCT-116细胞的增殖率(P<0.05,P<0.01),呈剂量依赖性。与空白组比较,Kou处理引起细胞周期阻滞于G0/G1期并诱导HCT-116细胞的凋亡(P<0.05,P<0.01),其作用与Kou的浓度呈正相关;FoxO3a siRNA干扰显著降低了FoxO3a及其下游相关细胞周期蛋白依赖性激酶抑制剂1A(p21),细胞周期蛋白依赖性激酶抑制剂1B(p27)和细胞死亡调解子(Bim)蛋白的表达(P<0.01)。与空白组比较,Kou处理诱导了HCT116细胞中氨基末端激酶(JNK)的活化(P<0.01),JNK特异性抑制剂(SP600125)处理抑制了Kou诱导的FoxO3a活化及其下游相关蛋白的表达(P<0.01);乙酰半胱氨酸(NAC)处理显著降低了Kou诱导的ROS水平和JNK信号的活化(P<0.01)。结论:Kou能有效抑制结肠癌细胞HCT-116的增殖,促进HCT-116细胞的凋亡,其机制可能与其作用于ROS/JNK/FoxO3a途径有关。Objective:To explore the effect and underlying mechanism of koumine(Kou)at different concentrations(0,100,200,400 μmol·L^(-1))on the proliferation and apoptosis of colorectal cancer HCT-116 cells.Method:After 24 hours of in vitro intervention with HCT-116 cells by Kou,cell counting kit-8(CCK-8)assay was used to detect its effect on cell proliferation.Flow cytometry was used to detect cell cycle,apoptosis,and reactive oxygen species(ROS)expression.Real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression of forkhead box O3a(FoxO3a).Cells were transfected with small interfering ribonucleic acid(siRNA).Western blot was employed to detect the protein expression of the FoxO3a target gene.Result:Compared with the conditions in the blank group,Kou treatment reduced the proliferation rate of HCT-116 cells(P<0.05,P<0.01)in a dose-dependent manner,caused cell cycle arrest in the G0/G1 phase,and induced the apoptosis of HCT-116 cells(P<0.05,P<0.01),which was positively correlated with the concentration of Kou.FoxO3a siRNA interference reduced the expression of FoxO3a and its downstream target genes cyclin-dependent kinase inhibitor 1 A(p21),cyclin-dependent kinase inhibitor 1 B(p27),and Bcl-2 interacting mediator of cell death(Bim)(P<0.01).Kou treatment induced the activation of c-Jun N-terminal kinase(JNK)in HCT116 cells.SP600125(JNK specific inhibitor)treatment inhibited the Kouinduced FoxO3a activation and the expression of its downstream target genes.N-acetyl cysteine(NAC)treatment reduced Kou-induced ROS levels(P<0.01) and JNK signal activation.The above results were significantly different from those in the blank group(P<0.01).Conclusion:Kou can effectively inhibit the proliferation of HCT-116 cells and promote apoptosis,and the mechanism may be related to the regulation of the ROS/JNK/FoxO3a pathway.

关 键 词：钩吻素子 细胞凋亡 活性氧 氨基末端激酶 叉头蛋白O3a 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]