基于凋亡和自噬研究氧化克班宁抑制肝癌HepG2细胞增殖的机制  

Oxocrebanine inhibits proliferation of hepatoma HepG2 cells by inducing apoptosis and autophagy

作　　者：王正文 潘探艳 魏昌龙 廖辉 张小坡 张彩云 于蕾[4] WANG Zheng-wen;PAN Cai-yan;WEI Chang-long;LIAO Hui;ZHANG Xiao-po;ZHANG Cai-yun;YU Lei(Department of Hepatobiliary and Pancreatic Surgery/Hainan Clinical Medical Research Center for Liver Disease and Critical Liver Care,Hainan Cancer Hospital/Hainan Medical University Affiliated Cancer Hospital,Haikou 570312,China;Hainan Pharmaceutical Research and Development Science and Technology Park,Haikou 570312,China;School of Pharmacy,Hainan Medical University,Haikou 570312,China;College of Pharmacy/Drug Engineering Technology Research Center,Harbin University of Commerce,Harbin 150076,China)

机构地区：[1]海南省肿瘤医院海南医科大学附属肿瘤医院肝胆胰外科/海南省肝病与肝脏危重症临床医学研究中心,海南海口570312 [2]海南省药物研究与开发科技园,海南海口570312 [3]海南医科大学药学院,海南海口570312 [4]哈尔滨商业大学药学院/药物工程技术研究中心,黑龙江哈尔滨150076

出　　处：《中国中药杂志》2025年第6期1618-1625,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82060778);海南省自然科学基金项目(820RC776);海南省卫生健康科技创新联合项目(WSJK2024MS162);黑龙江省中医药科研课题(ZHY2024-098)。

摘　　要：研究海南地不容抗肝癌活性成分氧化克班宁,抑制肝癌细胞增殖的具体机制。首先以噻唑蓝(MTT)法、5-溴脱氧尿嘧啶核苷(BrdU)标记、平板克隆法探究氧化克班宁是否造成HepG2和Hep3B2.1-7细胞增殖的抑制,经碘化丙啶(PI)单染观察氧化克班宁诱导HepG2和Hep3B2.1-7细胞凋亡,并以Western blot法验证凋亡效应蛋白半胱氨酸蛋白酶蛋白-3(c-caspase-3)、聚腺苷二磷酸核糖聚合酶1(PARP1)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Bcl-2同源杀手蛋白(Bak)和髓样细胞白血病1(Mcl-1)是否参与凋亡过程。其次氧化克班宁与自噬抑制剂3-甲基腺嘌呤(3-MA)同时作用于HepG2细胞,检测细胞自噬标记物LC3和自噬相关蛋白真核翻译起始因子4E结合蛋白1(4EBP1)、磷酸化真核翻译起始因子4E结合蛋白1(p-4EBP1)、核糖体蛋白S6激酶(P70S6K)、磷酸化核糖体蛋白S6激酶(p-P70S6K)的变化。MTT法、BrdU标记、平板克隆法检测结果均显示,氧化克班宁可抑制HepG2和Hep3B2.1-7细胞增殖,且呈剂量依赖性。流式细胞术检测结果显示,氧化克班宁作用后,HepG2和Hep3B2.1-7细胞凋亡率显著升高。Western blot结果显示,氧化克班宁作用于HepG2细胞后,可上调c-caspase-3、Bax、Bak蛋白表达,下调PARP1、Bcl-2、Mcl-1蛋白表达,表明氧化克班宁可引发肝癌细胞凋亡,进而抑制肝癌细胞增殖;氧化克班宁抑制HepG2细胞增殖可能与诱导肝癌细胞发生保护性自噬有关;氧化克班宁还可促使细胞自噬标记物LC3-Ⅰ向LC3-Ⅱ转化,降低4EBP1、P70S6K蛋白的磷酸化水平,自噬抑制剂3-MA可逆转此过程,表明氧化克班宁可通过诱导细胞自噬达到抑制肝癌细胞增殖作用。综上所述,氧化克班宁可通过诱导肝癌细胞凋亡和细胞自噬,进而抑制其细胞增殖。The study investigated the specific mechanism by which oxocrebanine,the anti-hepatic cancer active ingredient in Stephania hainanensis,inhibits the proliferation of hepatic cancer cells.Firstly,methyl thiazolyl tetrazolium(MTT)assay,5-bromodeoxyuridine(BrdU)labeling,and colony formation assay were employed to investigate whether oxocrebanine inhibited the proliferation of HepG2 and Hep3B2.1-7 cells.Propidium iodide(PI)staining was used to observe the oxocrebanine-induced apoptosis of HepG2 and Hep3B2.1-7 cells.Western blot was employed to verify whether apoptotic effector proteins,such as cleaved cysteinyl aspartate-specific protease 3(c-caspase-3),poly(ADP-ribose)polymerase 1(PARP1),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),Bcl-2 homologous killer(Bak),and myeloid cell leukemia-1(Mcl-1)were involved in apoptosis.Secondly,HepG2 cells were simultaneously treated with oxocrebanine and the autophagy inhibitor 3-methyladenine(3-MA),and the changes in the autophagy marker LC3 and autophagy-related proteins[eukaryotic translation initiation factor 4E-binding protein 1(4EBP1),phosphorylated 4EBP1(p-4EBP1),70-kDa ribosomal protein S6 kinase(P70S6K),and phosphorylated P70S6K(p-P70S6K)]were determined.The results of MTT assay,BrdU labeling,and colony formation assay showed that oxocrebanine inhibited the proliferation of HepG2 and Hep3B2.1-7 cells in a dose-dependent manner.The results of flow cytometry suggested that the apoptosis rate of HepG2 and Hep3B2.1-7 cells increased after treatment with oxocrebanine.Western blot results showed that the protein levels of c-caspase-3,Bax,and Bak were up-regulated and those of PARP1,Bcl-2,and Mcl-1 were down-regulated in the HepG2 cells treated with oxocrebanine.The results indicated that oxocrebanine induced apoptosis,thereby inhibiting the proliferation of hepatic cancer cells.The inhibition of HepG2 cell proliferation by oxocrebanine may be related to the induction of protective autophagy in hepatocellular carcinoma cells.Oxocrebanine still promoted the conversion

关 键 词：海南地不容 氧化克班宁 肝癌细胞 细胞凋亡 细胞自噬 

分 类 号：R285[医药卫生—中药学]