基于Raf/MEK/ERK信号通路及ROS水平研究石蒜碱调控肝癌HepG-2细胞凋亡的机制  

Mechanism of Lycorine Regulating hepatocarcinoma HepG-2 cells Apoptosis Based on Ras/raf/Mek/Erk Signaling Pathway and ROS Level

作　　者：任丽平[1] 李先佳[2] 金少举[1,3] 马永超 徐松涛 Ren Liping;Li Xianjia;Jin Shaoju;Ma Yongchao;Xu Songtao(Department of Pharmacy,Luohe Medical College,Luohe 462002;Department of Basic Medicine,Luohe Medical College,Luohe 462002;Tumor Occurrence and Prevention research innovation team of Henan,Luohe 462002;Department of Medical Science,Luohe Medical College,Luohe 462002)

机构地区：[1]漯河医学高等专科学校药学系,漯河462002 [2]漯河医学高等专科学校基础医学部,漯河462002 [3]河南省肿瘤发生与防治研究创新型科技团队,漯河462002 [4]漯河医学高等专科学校医疗系,漯河462002

出　　处：《中药药理与临床》2021年第2期28-32,共5页Pharmacology and Clinics of Chinese Materia Medica

基　　金：河南省高等学校重点科研项目(编号:18A31001);漯河医学高等专科学校2019年度创新创业发展能力提升工程科研类项目(编号:2020-LYZKYYB002)。

摘　　要：目的:探讨石蒜碱诱导肝癌HepG-2细胞凋亡的的机制。方法:裸鼠成瘤实验检测石蒜碱对体内肝癌细胞增殖能力的影响;MTT法分析石蒜碱对肝癌HepG-2细胞增殖的抑制作用;将25、50及100μmol/L石蒜碱作用于HepG-2细胞48 h,倒置荧光显微镜观察细胞形态;流式细胞仪检测细胞凋亡;激光共聚焦观察活性氧荧光强度;测定SOD的活性,MDA的水平;Western blot法检测细胞Raf蛋白激酶(Raf)、p-Raf、丝裂原活化蛋白激酶(MEK)、p-MEK、细胞外调节蛋白激酶(ERK2)、p-ERK2、半胱天冬酶3(cleaved Caspase-3)、cleaved Caspase-9蛋白表达水平。结果:裸鼠成瘤实验显示石蒜碱对肝癌HepG-2细胞裸鼠皮下移植瘤生长具有抑制作用;MTT结果显示,石蒜碱对肝癌HepG-2细胞的增长具有抑制作用;Hoechest 33342染色和流式细胞仪检测,细胞凋亡率明显增加(P<0.05或P<0.01);与模型对照组比较,25、50、100μmol/L石蒜碱能增加ROS、MDA含量,降低SOD活性;上调凋亡相关蛋白cleaved Caspase-3、cleaved Caspase-9表达,50、100μmol/L的石蒜碱能降低HepG-2细胞中p-Raf/Raf、p-MEK/MEK、p-ERK2/ERK2比值(P<0.05或P<0.01)。结论:石蒜碱能抑制肝癌HepG-2细胞增殖,促进细胞凋亡,其机制可能和调控Ras/raf/MEK/ERK信号通路和ROS水平有关。Objective: To investigate the mechanism of lycorine-induced apoptosis of HepG-2 cells. Methods: Nude mouse tumorigenesis test was used to detect the proliferation of hepatoma cells in vivo. MTT assay was used to analyze the inhibitory effect of lycoline on the proliferation of HepG-2 cells. 25, 50, and 100 μmol/L lycoline were incubated with HepG-2 cells for 48 h, respectively. The cell morphology was observed with an inverted fluorescence microscope. Flow cytometry was used to detect the cell apoptosis. Confocal laser was used to observe the intensity of reactive oxygen fluorescence, the activity of SOD and MDA level were measured. Western blot was used to detect the cell protein expressions of Raf, p-Raf, Mek, p-Mek, Erk2,p-Erk2, Cleaved caspase-3 and cleaved caspase-9. Results: Nude mouse tumorigenesis experiments showed that lycorine inhibited the growth of subcutaneou transplantation HepG-2 cells in nude mice. MTT results suggested that lycorine had an inhibitory effect on the growth of HepG-2 cells. Hoechest 33342 staining and flow cytometry results showed the apoptotic rate was significantly increased. Compared with the model group, 25, 50, and 100 μmol/L lycorine increased the contents of ROS and MD, and decrease SOD activity, it also up-regulated the expressions of apoptosis related protein like cleaved caspase-3 and cleaved caspase-9. In 50 and 100 μmol/L of lycorine groups, the levels of p-Raf/Raf, p-Mek/Mek, p-Erk2/Erk2 in HepG-2 cells were down-regulated. Conclusion: Lycorine can inhibit the proliferation of HepG-2 cells and promote the apoptosis, and its mechanism may be regulating the Ras/raf/Mek/Erk signaling pathway and ROS level.

关 键 词：石蒜碱 Raf蛋白激酶/丝裂原活化蛋白激酶/细胞外调节蛋白激酶信号通路 活性氧水平 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]