缺氧条件下白花丹醌对肝癌HepG2细胞增殖、凋亡与侵袭及HIF-1α表达的影响  

Effects of Plumbagin on Proliferation, Apoptosis, Invasion and Expression of HIF-1α in Hepatocellular HepG2 Cells Under Hypoxia Condition

作　　者：韦燕飞 吕贝贝 金丽杰 刘莎莎 成桃 刘欢[2,3] WEI Yanfei;LYU Beibei;JIN Lijie;LIU Shasha;CHENG Tao;LIU Huan(College of Basic Medicine of Guangxi University of Chinese Medicine,Nanning 530200,China;Guangxi Key Laboratory of Molecular Biology of Preventive Medicine of Traditional Chinese Medicine,Nanning 530024,China;The First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530024,China)

机构地区：[1]广西中医药大学基础医学院,南宁530200 [2]广西中医药防治医学分子生物重点实验室,南宁530024 [3]广西中医药大学第一附属医院,南宁530024

出　　处：《中国现代应用药学》2022年第14期1789-1795,共7页Chinese Journal of Modern Applied Pharmacy

基　　金：国家自然科学基金项目(81760757);广西自然科学基金项目(2019GXNSFBA245041,2019JJD140032);广西中医药大学第二批“岐黄工程”高层次人才团队培育项目(2021001)。

摘　　要：目的 研究白花丹醌在缺氧条件下对人肝癌细胞HepG2增殖、凋亡、侵袭和低氧诱导因子1α(hypoxia induced factor1α,HIF-1α)及其下游基因表达的影响。方法 采用二氯化钴(CoCl2)化学诱导法建立HepG2细胞体外缺氧模型,经不同浓度(2,4,8μmol·L^(–1))白花丹醌处理24 h后,MTT、平板克隆形成试验观察HepG2细胞增殖水平变化;使用Annexin V/PI双染流式细胞术检测HepG2细胞凋亡情况;使用Transwell试验观察HepG2细胞侵袭能力的变化;使用qRT-PCR检测HepG2细胞内HIF-1A mRNA转录水平变化;使用Western blotting检测细胞内HIF-1α及其下游基因c-Myc、VEGFA、MMP9和TWIST1的蛋白表达水平。结果 当CoCl2处理浓度为150μmol·L^(–1)时,HepG2细胞增殖水平未见显著变化,而HIF-1α蛋白表达水平显著升高(P<0.01),提示体外缺氧模型建立成功。与常氧对照组相比,MTT、平板克隆形成试验结果显示不同浓度白花丹醌作用HepG2细胞后,其增殖水平受到显著抑制(P<0.05或P<0.01);Transwell试验结果显示白花丹醌可显著抑制HepG2细胞侵袭(P<0.05或P<0.01);流式细胞术结果表明白花丹醌能显著诱导HepG2细胞凋亡(P<0.05或P<0.01);Westernblotting及qRT-PCR检测结果显示,白花丹醌能显著下调HIF-1α蛋白及HIF-1AmRNA表达水平(P<0.05或P<0.01)。Western blotting检测结果显示HIF-1α及其下游基因c-Myc、VEGFA、MMP9和TWIST1的蛋白表达水平均显著下调(P<0.05或P<0.01)。结论 CoCl2体外模拟肝癌HepG2细胞缺氧的适宜浓度为150μmol·L^(–1);在缺氧条件下,白花丹醌可显著抑制肝癌HepG2细胞的增殖与侵袭,同时诱导细胞凋亡,其作用机制可能与下调HIF-1α蛋白及其下游靶基因蛋白表达有关。OBJECTIVE To investigate the effects of plumbagin on proliferation, apoptosis, invasion and expression of hypoxia induced factor 1α(HIF-1α) as well as its target genes in HepG2 cells under hypoxia condition. METHODS Cobalt chloride(CoCl2) was used to induce a chemical hypoxia condition for HepG2 cells. Under this hypoxia condition, HepG2 cells were treated with plumbagin at 2, 4, 8 μmol·L^(–1) for 24 h respectively. The proliferation of HepG2 cells were measured by MTT and plate clone formation assay. The apoptotic of HepG2 cells were detected by flow cytometry to detect labeled Annexin V/PI.Transwell experiment was conducted to analyze the invasion ability of HepG2 cells after plumbagin treatment. qRT-PCR were used to detect the transcription level of its coding gene, HIF-1A. Western blotting analysis was further used to detect the protein expression level of HIF-1α and its downstream target genes such as c-Myc, VEGFA, MMP9 and TWIST1 in cells. RESULTS The concentration of CoCl2 were optimized to 150 μmol·L^(–1) without interference with cell proliferation and the expression level of HIF-1α was significantly increased(P<0.01), indicating successfully establishment of the hypoxia model by Co Cl2 treatment. Compared to the normoxia control group, MTT and plate clone formation assay results showed that proliferation of Hep G2 cells was significantly inhibited by treatment with different concentration of plumbagin(P<0.05 or P<0.01). In addition, Transwell experiment result showed that the invasion ability of Hep G2 cells was also decreased by plumbagin(P<0.05 or P<0.01). Flow cytometry results indicated that plumbagin could significantly induce the apoptosis of Hep G2 cells(P<0.05 or P<0.01). The results of Western blotting and q RT-PCR showed that plumbagin could significantly down-regulate the expression levels of HIF-1α protein and HIF-1A m RNA(P<0.05 or P<0.01). Western blotting analysis results further showed that the protein expression levels of HIF-1α and its downstream genes c-Myc, VEGFA, MMP9

关 键 词：白花丹醌 肝癌 细胞凋亡 肿瘤侵袭 氯化钴 低氧诱导因子1Α 缺氧 

分 类 号：R285.5[医药卫生—中药学]