佛手柑内酯通过PI3K/Akt信号通路诱导人肝癌细胞HepG2和Hep3B凋亡的机制  

Effect of Bergapten on Apoptosis of HepG2 and Hep3B Cells Through PI3K/Akt Pathway

作　　者：赵丽萍 王超 田男[1] 范春雷[1] ZHAO Li-ping;WANG Chao;TIAN Nan;FAN Chun-lei(College of Life Science,Zhejiang Chinese Medical University,Hangzhou 310053,China)

机构地区：[1]浙江中医药大学生命科学学院,杭州310053

出　　处：《中国实验方剂学杂志》2020年第6期73-78,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：浙江省自然科学基金项目(LY14H290007)。

摘　　要：目的:探究佛手柑内酯通过磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)信号通路诱导人肝癌细胞Hep G2和Hep3B凋亡的机制。方法:设立佛手柑内酯(5,50,200μmol·L-1)组和空白组;采用噻唑蓝(MTT)比色法检测佛手柑内酯作用Hep G2和Hep3B细胞24,48 h细胞增殖;磷脂结合蛋白V/碘化丙啶(Annexin V-FITC/PI)双染检测细胞凋亡;实时荧光定量逆转录聚合酶链反应(Real-time PCR)及蛋白免疫印迹法(Western bolt)检测相关m RNA和蛋白的表达含量;进行平板细胞克隆形成实验评价各组Hep G2和Hep3B细胞的克隆形成率与效果。结果:MTT实验表明,佛手柑内酯能明显抑制Hep G2和Hep3B细胞的增殖活性,且呈浓度依赖性;流式细胞仪分析结果表明,佛手柑内酯200μmol·L-1组作用于Hep G2和Hep3B细胞48 h,有明显促凋亡作用(P <0. 05);Western blot结果显示,佛手柑内酯可以上调半胱氨酸蛋白酶(Caspase)-3,Caspase-8蛋白表达量(P <0. 05),并且下调B淋巴细胞瘤-2(Bcl-2),PI3K蛋白表达(P <0. 05);Real-time PCR显示PI3K,Akt m RNA相对表达量降低(P <0. 05);平板细胞克隆形成实验的结果显示,随着佛手柑内酯浓度的增加,各组细胞克隆形成率呈递减趋势,且佛手柑内酯200μmol·L-1组作用降低明显(P <0. 05)。结论:佛手柑内酯对人肝癌细胞Hep G2和Hep3B的增殖存在抑制作用,其可能是通过PI3K/Akt信号通路诱导Hep G2和Hep3B细胞的凋亡。Objective: To investigate the effect of bergapten on the apoptosis of HepG2 and Hep3 B cells through phosphatidylinositol 3 kinase( PI3 K)/protein kinase B( Akt) pathway. Method: Bergamot( 5,50,200 μmol·L-1) groups and blank group were set up. The effect of bergapten at different concentrations on proliferation of HepG2 and Hep3 B cells for 24,48 h were detected by thiazolyl blue( MTT) assay. Apoptosis was detected by Annexin V-FITC/propidium iodide double staining. Quantitative real-time fluorescence reverse transcriptional polymerase chain reaction( Real-time PCR) and Western blot assay were used to detect relevant m RNA and proteins expressions. The clone formation rate and the effect of HepG2 and Hep3 B cells in each group were evaluated by plate cell clone formation. Result: MTT assay showed that bergapten could significantly inhibit the proliferation activity of HepG2 and Hep3 B cells in a time-dependent manner. Flow cytometry analysis showed that bergapten in 200 μmol·L-1 concentration groups had significant pro-apoptotic effect on HepG2 and Hep3 B cells after 48 h( P < 0. 05). Western blot results showed that bergamolactone could up-regulate the protein expressions of Caspase-3,Caspase-8( P < 0. 05),and down-regulate protein expressions of B-lymphocytoma-2( Bcl-2),PI3 K( P < 0. 05). Real-time PCR results showed that m RNA expressions of PI3 K and Akt were decreased( P < 0. 05). The results of plate cell clone formation experiment showed that with the increase of the concentration of bergamolide,the cell clone formation rate of each group showed a decreasing trend,particularly in200 μmol·L-1 concentration group( P < 0. 05). Conclusion: Bergapten can inhibit the proliferation of HepG2 and Hep3 B cells,which may be induced through the PI3 K/Akt signaling pathway.

关 键 词：佛手柑内酯 肝细胞癌 磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt) 凋亡 信号通路 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]