叶下珠复方Ⅱ号对肝癌Huh7细胞增殖、凋亡与自噬的影响  

Effect of Compound Phyllanthus urinaria Ⅱ(CPU Ⅱ) on Proliferation,Apoptosis and Autophagy of Hepatocellular Carcinoma Huh7 Cells

作　　者：杜欣芸 李常青[1] 陈滨 李小翚[1] DU Xin-yun;LI Chang-qing;CHEN Bing;LI Xiao-hui(Institute of Tropical Medicine,School of Chinese Materia Medica,Guangzhou University of Chinese Medicine,Guangzhou 510405,China)

机构地区：[1]广州中医药大学热带医学研究所中药学院

出　　处：《中国实验方剂学杂志》2019年第2期48-54,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81673861,81373524)

摘　　要：目的:观察叶下珠复方Ⅱ号对肝癌Huh7细胞增殖、凋亡与自噬的影响,进一步探讨其抗肝癌的作用机制。方法:体外培养肝癌Huh7细胞,设空白组,叶下珠复方Ⅱ号高、低质量浓度组(40,20 g·L^-1)和5-氟尿嘧啶(5-FU)(0. 04 g·L^-1)组,采用噻唑蓝(MTT)比色法检测叶下珠复方Ⅱ号对肝癌Huh7细胞增殖的影响,流式细胞仪检细胞凋亡变化,单丹磺酰尸胺(MDC)染色观察药物作用后细胞自噬体的变化,实时荧光定量聚合酶链式反应(Real-time PCR)检测各组磷脂酰肌醇3-激酶(PI3K),丝氨酸-苏氨酸蛋白激酶2(Akt2),B淋巴细胞瘤-2(Bcl-2)和微管相关蛋白1轻链3(LC3Ⅱ) mRNA表达的改变,蛋白免疫印迹法(Western blot)检测Akt2和LC3Ⅱ蛋白表达的变化。结果:叶下珠复方Ⅱ号(40,20 g·L^-1)可显著抑制肝癌Huh7细胞,并诱导其凋亡,凋亡率分别为51. 72%,19. 74%,显著高于空白组(P <0. 01)。采用MDC染色后,叶下珠复方Ⅱ号(40,20 g·L^-1)组可见大量自噬体的形成。与空白组比较,叶下珠复方Ⅱ号高、低剂量组PI3K,Akt2,Bcl-2 mRNA表达均显著下降,LC3ⅡmRNA表达均显著增加(P <0. 01)。与空白组比较,叶下珠复方Ⅱ号(40,20 g·L^-1)组处理后的Akt2蛋白表达显著下降,LC3Ⅱ蛋白表达明显增加(P <0. 05,P <0. 01)。结论:叶下珠复方Ⅱ号对肝癌Huh7细胞增殖有明显的抑制作用,其作用机制可能与抑制PI3K/Akt信号通路活化从而诱导细胞凋亡和自噬有关。Objective: To observe the effect and investigate the mechanism of compound Phyllanthus urinaria Ⅱ( CPU Ⅱ) on proliferation,apoptosis and autophagy of human hepatoma cell line Huh7. Method:Huh7 cells were cultured in vitro and divided into blank control group,high-dose CPU Ⅱ group( 40 g·L^-1),lowdose CPU Ⅱ group( 20 g·L^-1),and 5-FU group( 0. 04 g·L^-1). Methye thiazolye telrazlium( MTT) assay was used to detect the inhibitory effect of CPU Ⅱ on proliferation of human hepatoma Huh7 cells. The apoptosis rate was observed by Annexin V-FITC/PI flow cytometry;the changes of autophagosomes in each group were observed by monodansylcadaverin( MDC) staining;Real-time fluorescence quantitative polymerase chain reaction( Realtime PCR) was used to detect the mRNA expression of phosphatidylinositol 3-kinase( PI3 K),Serine-threonine protein kinase 2( Akt2),B-cell lympoma-2( Bcl-2) and Microtubule-associated protein 1 light chian 3( LC3Ⅱ)and Western blot was used to detect the protein expression of Akt2 and LC3Ⅱ. Result: CPU Ⅱ( 40,20 g·L^-1)significantly inhibited the hepatoma cell line Huh7 and induced apoptosis,with an apoptosis rate of 51. 72% and19. 74% respectively,significantly higher than that of control group( P < 0. 01). After MDC staining,it was observed that a large number of fluorescent particles were distributed in the cytoplasm and nucleus of liver cancer cells under the effect of CPU Ⅱ. As compared with blank control group,the mRNA expression levels of PI3 K,Akt2,and Bcl-2 were decreased significantly while the mRNA expression of LC3 Ⅱ was increased significantly in CPU II high-and low-dose groups( P < 0. 01);the protein expression of Akt was significantly decreased while protein expression of LC3Ⅱwas significantly increased in CPU Ⅱ high-dose and low-dose groups( P < 0. 05,P <0. 01). Conclusion: CPUⅡ had obvious inhibitory effect on the proliferation of hepatoma cell line Huh7,and the mechanism may be related to inhibiting the activation of PI3 K/Akt signaling pathway to induce apop

关 键 词：叶下珠复方Ⅱ 增殖 凋亡 自噬 HUH7细胞 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]