摘要
[目的]探讨中药补骨脂活性成分补骨脂酚(BAK)诱导人肝癌Hep3B细胞生长抑制的机制。[方法]采用噻唑蓝法(MTT法)检测BAK对人肝癌Hep3B细胞生长抑制作用;相差显微镜观察BAK处理后Hep3B细胞形态变化;Hoechst/Mitotracker/Rhodamine荧光探针染色结合高内涵细胞成像系统分析BAK对线粒体功能的影响;免疫印迹法(Westernblot法)考察BAK处理后自噬标志性蛋白溶酶体相关蛋白1(LAMP1)、微管相关蛋白1轻链3(LC3)、SQSTM1蛋白(p62)以及线粒体自噬标志性蛋白PTEN诱导假定激酶1(PINK1)、帕金蛋白(Parkin)、线粒体融合蛋白2(MFN2)、线粒体膜蛋白(Tom20)的表达情况;MTT法考察线粒体自噬抑制剂氯喹(CQ)、巴弗洛霉素A1(Baf-A1)对BAK诱导的肝癌细胞生长抑制的影响。[结果]BAK时间剂量依赖性抑制Hep3B细胞生长;高内涵实验结果表明BAK能够升高线粒体质量,降低线粒体膜电位;BAK可以上调LAMP1、LC3、PINK1、Parkin表达量,下调p62、MFN2及Tom20蛋白表达;加入线粒体自噬抑制剂可显著逆转BAK诱导的Hep3B细胞生长抑制。[结论]补骨脂酚通过线粒体自噬抑制人肝癌Hep3B细胞生长。
[Objective]To investigate the effect of bakuchiol(BAK)on the mitophagy of human hepatocellular carcinoma Hep3B cells and its Mechanism.[Methods]MTT assay was used to detect the effect of BAK and mitophagy inhibitors CQ/Baf-A1 on the proliferation of Hep3B cells;Morphological changes of Hep3B cells after BAK treatment observed by phase contrast microscopy;Hoechst/Mitotracker/Rhodamine fluorescent probe staining combined with high connotation cell imaging system was used to analyze the effect of BAK on mitochondrial function;The expression of autophagy-related proteins LAMP1,LC3,p62,and mitophagy-related proteins PINK1,Parkin,MFN2,Tom20 at different time points of BAK were detected by Western blotting.[Results]BAK inhibited the growth of Hep3B cells in the time-and dose-dependent manners;High Content Analysis results suggest that BAK can elevate mitochondrial mass and decrease mitochondrial membrane potential;The addition of mitophagy inhibitors markedly reverse the death rate of Hep3B cells induced by BAK;The proteins LAMP1,LC3,PINK1,Parkin were significantly increased by BAK in the time-dependent manners,while the p62,MFN2,Tom20 were reduced;Addition of mitochondrial autophagy inhibitor could significantly reverse the growth inhibition of Hep3B cells induced by BAK.[Conclusion]Bakuchiol induces inhibition of cell growth in Hep3B cells by activating mitophagy.
作者
赵浩楠
张强
温中庆
康宁
ZHAO Haonan;ZHANG Qiang;WEN Zhongqing;KANG Ning(School of Intergrative Medicine,Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China;School of Medical Technology,Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China)
出处
《天津中医药大学学报》
CAS
2024年第5期400-406,共7页
Journal of Tianjin University of Traditional Chinese Medicine
基金
天津市研究生科研创新项目(2021YJSS169)
天津中医药大学研究生科研创新项目(YJSKC-20212019)
天津中医药大学中西医结合学院研究生创新基金资助项目(ZXYCXLX202013)。
关键词
肝癌
补骨脂酚
线粒体自噬
细胞生长抑制
hepatocellular carcinoma
bakuchiol
mitophagy
cell growth inhibition
