基于PI3K/Akt信号通路探讨华良姜素抗肝癌细胞HepG2凋亡的作用机制  

Anti-hepatocarcinoma HepG2 Cell Mechanism of Jaranol:Based on PI3K/Akt Signaling Pathway

作　　者：姚红 侯宇芯 任晋宏 王禹璇 薛慧清 李青山 YAO Hong;HOU Yu-xin;REN Jin-hong;WANG Yu-xuan;XUE Hui-qing;LI Qing-shan(Shanxi Key Laboratory of Innovative Drugs for the Treatment of Serious Diseases Basing on the Chronic Inflammation,Shanxi University of Chinese Medicine,Taiyuan 030619,China)

机构地区：[1]山西中医药大学,基于炎性反应的重大疾病创新药物山西省重点实验室,太原030619

出　　处：《中国实验方剂学杂志》2022年第9期80-87,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家国际合作专项项目(2013DFA30700);山西省应用基础研究项目(201801D221437);山西省“百人计划”第六批引进人才项目(晋组函字[2013]号)。

摘　　要：目的:观察华良姜素的体外抗肝癌细胞HepG2凋亡的作用机制。方法:噻唑蓝(MTT)比色法检测华良姜素(0、5、10、25、50、100、150、300μmol·L^(-1))对HepG2细胞不同时间(24、48、72 h)的增殖抑制作用;异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)凋亡试剂盒检测华良姜素(0、3、15、75μmol·L^(-1))对HepG2细胞的凋亡作用;蛋白免疫印迹法(Western blot)检测华良姜素对HepG2细胞中B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)表达的影响;转录组学分析华良姜素(15μmol·L^(-1))对HepG2细胞处理后基因差异表达与信号通路改变情况;实时荧光定量聚合酶链式反应(Real-time PCR)验证差异基因[TEK、血小板源性生长因子α受体(PDGFRA)、脾酪氨酸激酶(SYK)、磷酸肌醇-3-激酶催化亚基G肽(PIK3CG)、Janus激酶3(JAK3)、膜相关鸟苷酸激酶转化蛋白2(MAGI2)]mRNA相对表达。结果:与空白组比较,华良姜素组HepG2细胞的增殖降低(P<0.05,P<0.01);凋亡率升高(P<0.01);Bax蛋白表达水平升高(P<0.05,P<0.01),Bcl-2的蛋白表达水平降低(P<0.01);转录测序得到59000个受调控的表达基因,受调控显著差异表达基因125个,其中表达上调差异基因47个,表达下调差异基因74个,京都基因和基因组数据库(KEGG)分析显示,加华良姜素后磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路中与凋亡相关的差异基因变化较大。与空白组比较,华良姜素组(15μmol·L^(-1))TEK、PDGFRA、SYK、PIK3CG、JAK3、MAGI2的mRNA表达水平降低(P<0.01)。结论:采用体外细胞学实验研究华良姜素能抑制HepG2细胞增殖,初步验证其凋亡,可能是影响了PI3K/Akt信号通路中部分差异基因的表达。为后续华良姜素抗肿瘤提供实验基础,同时有助于促进黄酮类化合物的开发利用及潜在的应用价值。Objective:To study the in vitro anti-hepatocarcinoma HepG2 cell mechanism of Jaranol.Method:The methyl thiazolyl tetrazolium(MTT)assay was employed to examine the inhibition of Jaranol(0,5,10,25,50,100,150,300μmol·L^(-1))on HepG2 cell proliferation at different time(24,48,72 h),annexin V-fluorescein isothiocyante/propidium iodide(Annexin V-FITC/PI)kit to detect the effect of Jaranol(0,3,15,75μmol·L^(-1))on HepG2 cell apoptosis,and Western blot to determine the influence of Jaranol on the expression of B-cell lymphoma 2(Bcl-2)and Bcl-2-associated X protein(Bax)in HepG2 cells.Transcriptome sequencing was performed to analyze the differential expression of genes and changes of related signaling pathways after the treatment of HepG2 cells with Jaranol(15μmol·L^(-1)).Real-time PCR was carried out to verify the relative mRNA content of differential genes[TEK,platelet-derived growth factor receptorα(PDGFRA),spleen tyrosine kinase(SYK),phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma(PIK3CG),Janus kinase 3(JAK3),membrane-associated guanylate kinase inverted 2(MAGI2)].Result:Compared with the blank group,Jaranol decreased HepG2 proliferation(P<0.05,P<0.01),increased apoptosis rate of HepG2 cells(P<0.05,P<0.01),raised Bax expression(P<0.05,P<0.01),and reduced Bcl-2expression(P<0.05,P<0.01).Transcriptome sequencing yielded 59000 regulated genes,125 of which showed significantly different expression,with 47 up-regulated and 74 down-regulated.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed that the differential genes related to apoptosis in the phosphatidylinositol3-kinase/protein kinase B(PI3K/Akt)signaling pathway changed significantly after drug addition.The mRNA expression of TEK,PDGFRA,SYK,PIK3CG,JAK3,and MAGI2 decreased in Jaranol(15μmol·L^(-1))group compared with that in the control group(P<0.05).Conclusion:In vitro cytological experiment verified that Jaranol inhibited the proliferation of HepG2 cells and promoted the apoptosis,possibly by influencing the expression of some

关 键 词：华良姜素 HEPG2 凋亡 转录组学 磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路 

分 类 号：R933/937[医药卫生—生药学] R289[医药卫生—药学]