扶正软坚抗癌方调控Akt/MDM2/P53信号通路对肝癌HepG2细胞恶性生物学行为的影响  

Effect of Fuzheng Ruanjian Anticancer Formula on malignant biological behaviors of hepatocellulars carcinoma HepG2 cells by regulating Akt/MDM2/P53 signaling pathway

作　　者：娄静[1] 赵雷[1] 朱岩洁 袁帅强 王菲[1] 张杭洲 徐娇娇 余晓珂[2] 侯留法[1] LOU Jing;ZHAO Lei;ZHU Yanjie;YUAN Shuaiqiang;WANG Fei;ZHANG Hangzhou;XU Jiaojiao;YU Xiaoke;HOU Liufa(Department of Hepatobiliary Splenogastric,Affiliated Hospital,Henan Provincial Academy of Traditional Chinese Medicine,Zhengzhou 450003,China;Department of Geriatrics,Third Affiliated Hospital,Henan University of Traditional Chinese Medicine,Zhengzhou 450003,China)

机构地区：[1]河南省中医药研究院附属医院肝胆脾胃科,河南郑州450003 [2]河南中医药大学第三附属医院老年病内科,河南郑州450003

出　　处：《吉林大学学报（医学版）》2024年第6期1654-1663,共10页Journal of Jilin University：Medicine Edition

基　　金：河南省中医药科学研究专项课题(2023ZY2139,2022ZY1147)。

摘　　要：目的:探讨扶正软坚抗癌方调控蛋白激酶B(Akt)/鼠双微体2(MDM2)/P53信号通路对肝癌HepG2细胞恶性生物学行为的影响。方法:采用0、0.05、0.10、0.20、0.40、0.80、1.60、3.20和6.40 g·mL^(-1)扶正软坚抗癌方分别处理HepG2细胞48 h,CCK-8法检测HepG2细胞存活率,筛选扶正软坚抗癌方浓度用于后续实验。将HepG2细胞分为对照组、低剂量扶正软坚抗癌方组(0.2 g·mL^(-1))、中剂量扶正软坚抗癌方组(0.4 g·mL^(-1))、高剂量扶正软坚抗癌方组(0.8 g·mL^(-1))、SC79组(8 mg·L^(-1)SC79)和高剂量扶正软坚抗癌方+SC79组(0.8 g·mL^(-1)扶正软坚抗癌方+8 mg·L^(-1)SC79)。CCK-8法检测各组HepG2细胞增殖活性,克隆形成实验检测各组HepG2细胞克隆形成率,流式细胞术检测各组HepG2细胞凋亡率,Transwell小室实验检测各组HepG2细胞迁移和侵袭细胞数,Western blotting法检测各组HepG2细胞中增殖细胞核抗原(PCNA)、含半胱氨酸的天冬氨酸蛋白酶3(Caspase-3)、基质金属蛋白酶(MMP)-2、MMP-9、磷酸化Akt(p-Akt)、磷酸化MDM2(p-MDM2)和P53蛋白表达水平。结果:随着扶正软坚抗癌方浓度(0、0.05、0.10、0.20、0.40、0.80、1.60、3.20和6.40 g·mL^(-1))的升高,HepG2细胞存活率逐渐降低(P<0.05),选取0.2、0.4和0.8 g·mL^(-1)扶正软坚抗癌方用于后续实验。CCK-8法检测,与对照组比较,低、中和高剂量扶正软坚抗癌方组HepG2细胞增殖活性均明显降低(P<0.05),并呈剂量依赖性,SC79组HepG2细胞增殖活性明显升高(P<0.05);与高剂量扶正软坚抗癌方组比较,高剂量扶正软坚抗癌方+SC79组HepG2细胞增殖活性明显升高(P<0.05)。克隆形成实验检测,与对照组比较,低、中和高剂量扶正软坚抗癌方组HepG2细胞克隆形成率均明显降低(P<0.05),并呈剂量依赖性,SC79组HepG2细胞克隆形成率明显升高(P<0.05);与高剂量扶正软坚抗癌方组比较,高剂量扶正软坚抗癌方+SC79组HepG2细胞克隆形成率明显升高(P<0.05)。流式细Objective:To discuss the effect of Fuzheng Ruanjian Anticancer Formula on the malignant biological behaviors of the hepatocellular carcinoma HepG2 cells by requlating protein kinase B(Akt)/murine double minute 2(MDM2)/P53 signaling pathway.Methods:The HepG2 cells were treated with 0,0.05,0.10,0.20,0.40,0.80,1.60,3.20,and 6.40 g·mL−1 Fuzheng Ruanjian Anticancer Formula for 48 h.CCK-8 method was used to detect the survival rates of the HepG2 cells in various groups,and the concentrations of Fuzheng Ruanjian Anticancer Formula for the subsequent experiments were screened.The HepG2 cells were divided into control group,low dose of Fuzheng Ruanjian Anticancer Formula group(0.2 g·mL−1),medium dose of Fuzheng Ruanjian Anticancer Formula group(0.4 g·mL−1),high dose of Fuzheng Ruanjian Anticancer Formula group(0.8 g·mL−1),SC79 group(8 mg·L−1 SC79),and high dose of Fuzheng Ruanjian Anticancer Formula+SC79 group(0.8 g·mL−1 Fuzheng Ruijian Anticancer Formula+8 mg·L−1 SC79).CCK-8 method was used to detect the proliferation activities of the HepG2 cells in various groups;clone formation assay was used to detect the clone formation rates of the HepG2 cells in various groups;flow cytometry was used to detect the apoptotic rates of the HepG2 cells in various groups;Transwell chamber assay was used to detect the numbers of migration and invasion HepG2 cells in various groups;Western blotting method was used to detect the expression levels of proliferating cell nuclear antigen(PCNA),cysteine aspartate specific proteinase(Caspase-3),matrix metalloproteinase(MMP)-2,MMP-9,phosphorylated Akt(p-Akt),phosphorylated MDM2(p-MDM2),and P53 proteins in the HepG2 cells in various groups.Results:As the increasing of concentrations of Fuzheng Ruanjian Anticancer Formula(0,0.05,0.10,0.20,0.40,0.80,1.60,3.20,and 6.40 g·mL−1),the surival rates of the HepG2 cells were gradually decreased(P<0.05),and 0.2,0.4,and 0.8 g·mL−1 Fuzheng Ruanjian Anticancer Formula were selected for the subsequent experiments.The CCK-8 assay resul

关 键 词：扶正软坚抗癌方 蛋白激酶B 鼠双微体2 P53 肝肿瘤 细胞增殖 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤]