哇巴因和华蟾毒配基通过调节ERK信号通路诱导人肝癌HepG2细胞凋亡的研究  

Apoptosis of HepG2 Cell Induced by Ouabain and Cinobufogenin through ERK Signaling Pathway

作　　者：高默杰[1,2] 徐忠伟[2] 王凤梅[3] 陈小义[2] 呼文亮[2] 徐瑞成[2] 

机构地区：[1]中国人民武装警察部队医学院附属医院,天津市300162 [2]中国人民武装警察部队医学院/天津市职业和环境危害生物标志物重点实验室,天津市300162 [3]天津市第三中心医院肝内科,天津市300162

出　　处：《中国药房》2010年第27期2500-2503,共4页China Pharmacy

基　　金：国家自然科学基金资助项目(30840010);天津市应用基础研究计划面上项目(06YFJMJC10400)

摘　　要：目的:通过检测钠钾泵(Na+/K+-ATPase)抑制剂哇巴因和华蟾毒配基对肿瘤细胞存活的影响,研究其通过调节ERK信号通路诱导肿瘤细胞凋亡的机制。方法:以人肝癌细胞HepG2为靶细胞,检测Na+/K+-ATPase的活性变化,Hochest33342荧光染色检测细胞形态学变化;单细胞电泳检测细胞DNA损伤程度,钙离子荧光探针检测Ca2+浓度变化;Westernblot检测Caspase-3、ERK的表达变化。结果:哇巴因和华蟾毒配基可抑制Na+/K+-ATPase的活性;可使HepG2细胞呈典型的凋亡形态特征;单细胞电泳显示其可损伤HepG2细胞DNA双链;Fluo-3AM检测显示哇巴因和华蟾毒配基促进了Ca2+浓度增高;Western blot显示其可促进Caspase-3的活化,上调ERK磷酸化水平。结论:Na+/K+-ATPase抑制剂可诱导肝癌HepG2细胞凋亡,导致细胞DNA损伤,诱导细胞内游离Ca2+浓度增高,促进细胞凋亡途径中主要蛋白Caspase-3的活化。而ERK信号通路在哇巴因和华蟾毒配基诱导的HepG2细胞凋亡过程中发挥着关键调控作用。OBJECTIVE：To investigate the effect of ouabain and cinobufogenin on the apoptosis of HepG2 cell in order to study its apoptosis mechanism through ERK signaling pathway. METHODS：The activity of Na^＋/K^＋-ATPase was detected with HepG2 cell as target. The morphological changes of HepG2 cell and DNA injury were determined by Hoechst 33342 fluorescent staining and single cell gel electrophoresis （SCGE） respectively. The changes of the concentration of calcium ion were investigated by Fura-3 AM, and the expressions of Caspase-3 and ERK were detected by Western blotting assay. RESULTS：Ouabain and cinobufogenin inhibited the activity of Na^＋/K^＋-ATPase in HepG2 cell. Hoechst 33342 fluorescent staining showed that the clear apoptotic characteristics of HepG2 cell. The results of SCGE demonstrated that DNA double-strand of HepG2 cell was injured by ouabain and cinobufogenin. The concentration of calcium ion also increased after the treatment of ouabain and cinobufogenin according to the result of Fura-3 AM assay. The results of Western blot proved that ouabain and cinobufogenin promoted the production of p-ERK and the activition of caspase-3. CONCLUSION：Ouabain and cinobufogenin can induce the apoptosis of HepG2 cell and DNA damage, the increase of free calcium concentration and the activity of Caspase-3. ERK signaling pathway plays an important role in the apoptosis of HepG2 induced by ouabain and cinobufogenin.

关 键 词：哇巴因 华蟾毒配基 HEPG2细胞 细胞凋亡 Na+/K+-ATP酶 游离钙离子 

分 类 号：R322.123[医药卫生—人体解剖和组织胚胎学] R329.24[医药卫生—基础医学]