蟾酥活性成分协调索拉非尼通过下调Akt/NF-κB信号途径抑制肝癌HepG2细胞生长  

Synergy with the active ingredients of toad venom and sorafenib suppresses hepatocellular carcinoma HepG2 cells proliferation through down-regulating Akt/NF-κB signaling pathways

作　　者：王志美 徐忠伟[2] 王佳宝 代二庆[3] 徐瑞成[2,4] 

机构地区：[1]锦州医科大学中国人民武装警察部队后勤学院附属医院研究生培养基地,天津300162 [2]中国人民武装警察部队后勤学院中心实验室,天津300162 [3]中国人民武装警察部队后勤学院附属医院军人医疗保健中心,天津300162 [4]天津市职业与环境危害生物标志物重点实验室,天津300309

出　　处：《中国药理学通报》2017年第11期1510-1516,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81673651;81273552;81273745);武警后勤学院博士启动金项目(No WHB201501)

摘　　要：目的探讨蟾酥活性成分蟾蜍灵和华蟾毒配基协调索拉非尼抑制肝癌HepG2细胞生长的机制。方法 MTT法检测HepG2细胞经药物作用12、24、48 h后的增殖活性;Hoechst 33342荧光染色检测细胞形态学变化;流式细胞仪检测药物对HepG2细胞周期的影响;Western blot检测Akt、p-Akt(Ser473)、IκB、NF-κB、p-NF-κB p65、Bcl-2、Bax、cyclin A、PCNA蛋白表达水平。结果蟾蜍灵、华蟾毒配基和索拉非尼作用组均能抑制HepG 2细胞增殖,且药物联合作用组的增殖抑制率明显高于单独药物作用组,呈时间依赖性,用金氏公式得出在24 h具有协同作用(P<0.01);荧光染色结果显示,HepG 2细胞经药物处理24 h后,细胞呈现出核染色质凝集的凋亡形态特征;细胞周期检测结果显示,索拉非尼作用组使细胞周期阻滞于G0/G1期(P<0.01),蟾蜍灵、华蟾毒配基作用组使细胞周期阻滞于S期(P<0.01),药物联合作用组使细胞周期阻滞于S期(P<0.01);Western blot结果显示,蟾蜍灵、华蟾毒配基和索拉非尼单独药物作用组和联合作用组Akt、NF-κB总蛋白表达均无明显变化,p-Akt(Ser473)、p-NF-κB p65、Bcl-2、cyclin A、PCNA蛋白表达水平逐渐降低,联合作用组比单独药物作用组降低更为明显(P<0.01)。IκB、Bax蛋白表达水平逐渐升高,联合作用组比单独药物作用组升高更加明显(P<0.01)。结论蟾酥活性成分蟾蜍灵和华蟾毒配基协调索拉非尼通过下调Akt/NF-κB信号通路,抑制肝癌HepG 2细胞增殖。Aim To investigate the effect of the active ingredients of toad venom （ bufalin and cinobufagin） combined with sorafenib on the growth of hepatocellular carcinoma HepG2 cells , and to explore the possible mechanism. Methods The rates of inhibition aftertreated with drugs 12 , 24 , 48 h wem detected by MTT assay. The changes of cell morphology were detected by Hoechst 33342 fluorescent staining. The changes of cell cycle were detected by flow cytometry. The expres-sions of proteins such as Akt , p-Akt （ Ser473 ）,IkB , NF- κB , p- INF-KB p65 , Bcl-2 , Bax , cyclin A , PCNA were detected by Western blot. Results Bufalin,cinobufagin and sorafenib could inhibit the proliferation of HepG2 cells,presenting a dose- and time-dependent manner. Meanwhile,it could significantly increase the inhibitory rate of cells compared with those of single treatment,and they performed a synergistic activity in sorafenib combined with cinobufagin or bufalin by Jin Formula after 24 h treatment （P 〈0. 01 ） . The results of fluorescence staining showed the observation of the morphological features of nuclear condensation. Sorafenib induced the cell cycle G0/Gi phase arrest （P 〈 0. 0 1 ）, and bufalin, cinobufagin and the combination treatment generated the cell cycle S phase arrest （ P 〈 0. 01 ） . The results of Wester blot showed that the ex-pressions of Akt, NF- kB were not obviously changed between control and all other treatment. The expression levels of p-Vkt （ Ser473 ）,p- NF-κB p65,Bcl-2,PC-NA and cyclin A in combination treatment significantly decreased, and the expression levels of I kB and Bax significantly increased compared to those in single treatment （ P 〈 0. 01） . Conclusion The active ingre-dients of toad venom （ bufalin and cinobufagin） com-bined with sorafenib performs a synergetic effect on the anti-cancer of HepG2 cells by down-regulating Akt/ NF- κB signaling pathway.

关 键 词：索拉非尼 蟾酥 蟾蜍灵 华蟾毒配基 HEPG2 Akt NF-κB 

分 类 号：R282.74[医药卫生—中药学] R329.24[医药卫生—中医学]