扁蒴藤素调节Akt/NF-κB/m TOR信号通路对肝癌细胞自噬和凋亡的影响  

Influence of pristimerin on autophagy and apoptosis in hepatocellular carcinoma cells by regulating Akt/NF-κB/mTOR signaling pathway

作　　者：符丽娜 邢博真 陈瑶 潘相静 符丽云 孙少清 Fu Lina;Xing Bozhen;Chen Yao;Pan Xiangjing;Fu Liyun;Sun Shaoqing(Department of Hepatology and Gastroenterology,the First Affiliated Hospital of Hainan Medical University,Haikou 570102,China;Department of Infectious Diseases,the First Affiliated Hospital of Hainan Medical University,Haikou 570102,China;Department of Radiology,the First Affiliated Hospital of Hainan Medical University,Haikou 570102,China)

机构地区：[1]海南医科大学第一附属医院,肝胆科,海口570102 [2]海南医科大学第一附属医院,感染科,海口570102 [3]海南医科大学第一附属医院,放射科,海口570102

出　　处：《解剖学杂志》2024年第5期404-409,共6页Chinese Journal of Anatomy

摘　　要：目的:探讨扁蒴藤素(PRI)调节丝氨酸/苏氨酸蛋白激酶(Akt)/核因子-κB(NF-κB)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对肝癌细胞自噬和凋亡的影响。方法:MTT法检测不同浓度PRI处理肝癌HEPG2细胞的存活率;将HEPG2细胞分为对照组(常规培养),5、10、20μmol/L PRI组(5、10、20μmol/L PRI干预细胞24 h)和Recilisib组(20μmol/L PRI+10μmol/L Akt激活剂Recilisib共同干预细胞24 h);克隆形成实验检测细胞克隆数;MDC染色法和透射电镜检测细胞自噬;流式细胞术检测细胞凋亡;免疫印迹检测p62、微管相关蛋白1轻链3-Ⅰ/Ⅱ(LC3-Ⅰ/Ⅱ)、B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Akt、p-Akt、NF-κB p65、p-NF-κB p65、mTOR、p-mTOR蛋白表达。结果:HEPG2细胞存活率在PRI处理下呈剂量依赖性显著降低,IC50值为(13.07±1.20)μmol/L,因此选用5、10、20μmol/L PRI处理HEPG2细胞进行后续实验。与对照组相比,5、10、20μmol/L PRI组细胞克隆数和p62、Bcl-2、p-Akt、p-NF-κB p65、p-mTOR蛋白表达显著下降,而细胞绿色荧光平均光密度值、自噬小体数量、凋亡率、LC3-Ⅱ/LC3-Ⅰ和Bax蛋白表达显著升高;与20μmol/L PRI组相比,Recilisib组细胞克隆数和p62、Bcl-2、p-Akt、p-NF-κB p65、p-mTOR蛋白表达显著上升,而细胞绿色荧光平均光密度值、自噬小体数量、凋亡率、LC3-Ⅱ/LC3-Ⅰ和Bax蛋白表达显著下降。结论:PRI可通过抑制Akt/NF-κB/mTOR信号通路促进肝癌细胞自噬和凋亡。Objective:To investigate the influence of pristimerin(PRI)on the autophagy and apoptosis of hepatocellular carcinoma by regulating the serine/threonine kinase(Akt)/nuclear factor-κB(NF-κB)/mammalian target of rapamycin(mTOR)signaling pathway.Methods:MTT method was applied to detect the survival rate of HEPG2 cells treated with different concentrations of PRI(0,1.25,2.5,5,10,20,40μmol/L);HEPG2 cells were separated into CON group(conventional cultured cells),the 5,10 and 20μmol/L PRI group(5,10 and 20μmol/L PRI intervened the cells for 24 h,respectively)and Recilisib group(20μmol/L PRI+10μmol/L Akt activator Recilisib co-intervention for 24 h).Colony formation assay was used to detect the number of cell colonies.MDC staining and transmission electron microscopy were used to detect cell autophagy.Flow cytometry was applied to detect cell apoptosis.Western blotting was applied to detect the protein expressions of p62,microtubule-associated protein light chain 3-Ⅰ/Ⅱ(LC3-Ⅰ/Ⅱ),B cell lymphoma(Bcl-2),Bcl2-associated X(Bax),Akt,p-Akt,NF-κB p65,p-NF-κB p65,mTOR,and p-mTOR in cells.Results:Compared with 0μmol/L,the survival rate of H EPG2 cells was obviously decreased in a dose-dependent manner under the P RI treatments.IC50 value was(13.07±1.20)μmol/L,therefore,in this study,HEPG2 cells were treated with 5,10 and 20μmol/L PRI for subsequent experiments.Compared with the CON group,the number of cell colonies,the protein expressions of p62,Bcl-2,p-Akt,p-NF-κB p65,and p-mTOR in the 5,10 and 20μmol/L PRI groups were obviously decreased,while the average optical density of green fluorescence,the number of autophagosomes,the apoptosis rate,the protein expressions of LC3-Ⅱ/LC3-Ⅰand Bax were obviously increased.Compared with the 20μmol/L PRI group,the number of cell colonies,the protein expressions of p62,Bcl-2,p-Akt,p-NF-κB p65,and p-mTOR in the Recilisib group were obviously increased,however,the average optical density of green fluorescence,the number of autophagosomes,the apoptosis rate,the protein

关 键 词：扁蒴藤素 Akt/NF-κB/mTOR信号通路 肝癌细胞 自噬 凋亡 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]