冬凌草甲素通过抑制亚甲基四氢叶酸脱氢酶2诱导肝癌细胞凋亡  

The inhibiting effect of rubescensine A on hepatocellular carcinoma cells induced by methylene tetrahydrofolate dehydrogenase 2

作　　者：黄应文 黄岩屹 王瑶瑶 刘宏元 朱明阳 刘晓宁[2] HUANG Yingwen

机构地区：[1]岑溪市人民医院,广西梧州543200 [2]黄河科技学院医学院,河南郑州450063

出　　处：《中医临床研究》2022年第17期6-9,共4页Clinical Journal Of Chinese Medicine

基　　金：国家级大学生创新创业训练计划项目(201911834007),河南省创新型科技团队--河南省区域性中药抗肿瘤活性研究创新型科技团队(豫科人组【2017】1号)。

摘　　要：目的:探讨亚甲基四氢叶酸脱氢酶2(Methylene Tetrahydrofolate Dehydrogenase 2,MTHFD2)在冬凌草素诱导人肝癌细胞HepG2细胞凋亡中的作用及其机制。方法:通过冬凌草甲素处理HepG2细胞24 h,蛋白质免疫印迹技术检测MTHFD2、Ras基因(Ras)、磷酸激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、胞外调节蛋白激酶(ERK)、胞外调节蛋白激酶磷酸化(p-ERK)和C-Myc癌基因(c-Myc)蛋白的表达;以酶联免疫吸附剂测定(Enzyme-linked Immunosorbent Assay,ELISA)试剂盒检测MTHFD2的酶活性;采用Annexin V-FITC/碘化丙啶凋亡检测试剂盒检测凋亡比例;Giemsa染色观察细胞克隆;通过分子对接研究MTHFD2与冬凌草甲素可能的结合模型。结果:冬凌草甲素对MTHFD2的抑制呈剂量依赖性;MTHFD2的抑制引起线粒体活性氧物质(Reactive Oxygen Species,ROS)升高,表现为线粒体ROS清除剂N-乙酰-L-半胱氨酸(N-Acetyl-L-Cysteine,NAC)的处理使细胞克隆形成和细胞凋亡比例逆转;冬凌草甲素通过Ras、p-Akt和c-Myc抑制MTHFD2。结论:冬凌草甲素通过Ras-Akt-Myc信号通路抑制MTHFD2,介导线粒体ROS升高,从而导致HepG2细胞凋亡。Objective:To investigate the effect and underlying mechanisms of rubescensine on MTHFD2-induced apoptosis of HepG2 cells.Methods:The HepG2 cells were treated with oridonin for 24 h.Western blot was used to detect the expression levels of MTHFD2,Ras,Akt,p-Akt,ERK,p-ERK,and c-Myc proteins in HepG2 cells.ELISA kit was used to determine the enzyme activity of MTHFD2 in HepG2 cells.Annexin V-FITC/PI apoptosis detection kit was used to identify the apoptotic ratio.Giemsa staining was used to observe the cell clone.Molecular docking was carried out to investigate the possible binding mode of MTHFD2 and rubescensine A.Results:Rubescensine A inhibited MTHFD2 in a dose-dependant manner.The inhibition of MTHFD2 led to the elevation of mitochondrial ROS,which was evidenced by the reverse of the proportion of cell clone formation and apoptosis after the application of mitochondrial ROS scavenger NAC.Rubescensine A inhibited MTHFD2 via Ras,p-Akt,and c-Myc.Conclusion:Rubescensine A inhibits MTHFD2 through the Ras-Akt-Myc signaling pathway,mediates the generation of mitochondrial ROS,and leads to HepG2 cells apoptosis.

关 键 词：亚甲基四氢叶酸脱氢酶2 冬凌草甲素 线粒体活性氧物质 肝癌 HEPG2细胞 

分 类 号：R73[医药卫生—肿瘤]