芪术抗癌方通过PCK1/Akt/p21信号轴调控肿瘤代谢重编程抑制肝细胞癌增殖的作用机制  

Mechanism of Qizhu Kang’ai Prescription for Inhibiting Cell Proliferation of Hepatocellular Carcinoma by Regulating Tumor Metabolic Reprogramming via PCK1/Akt/p21 Signaling Axis

作　　者：钟欣 胡锐 李静 彭蓝芬 刘兴宁 黄琦 孙嘉玲 孙新锋[1,2] 陈剑平[1,4] 蔡本强[1,2] 周小舟[1,2] ZHONG Xin;HU Rui;LI Jing;PENG Lanfen;LIU Xingning;HUANG Qi;SUN Jialing;SUN Xinfeng;CHEN Jianping;CAI Benqiang;ZHOU Xiaozhou(The Fourth Clinical College,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;National Key Clinical Specialties of Liver Disease(Traditional Chinese Medicine)of Shenzhen Traditional Chinese Medicine Hospital,Shenzhen,Guangdong 518033,China;Macao University of Science and Technology,Taipa Macao 999078,China;Key Laboratory of Shenzhen Hospital Chinese Medicine Preparation of Shenzhen Traditional Chinese Medicine Hospital,Shenzhen 518033,China)

机构地区：[1]广州中医药大学第四临床医学院,广州510006 [2]深圳市中医院国家临床肝病(中医)重点专科,广东深圳518033 [3]澳门科技大学,中国澳门氹仔999078 [4]深圳市中医院深圳市医院中药制剂研究重点实验室,广东深圳518033

出　　处：《中国实验方剂学杂志》2024年第3期26-36,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(82374531);国家自然科学基金青年基金项目(82205209);深圳市科技计划项目(JCYJ20210324120405015);广东省中医药局项目(20231302)。

摘　　要：目的:研究芪术抗癌方对二乙基亚硝胺(DEN)联合四氯化碳(CCl4)诱导肝癌模型小鼠肝脏及人肝癌Huh7细胞中糖异生酶磷酸烯醇丙酮酸羧激酶1(PCK1)的调控作用,探讨其调节肝癌细胞代谢重编程并抑制细胞增殖的作用机制。方法:使用DEN联合CCl4腹腔注射构建肝癌小鼠模型,设置正常组、模型组、芪术抗癌方组,每天予以芪术抗癌方(3.51 g·kg^(-1))或等体积生理盐水灌胃,干预8周后收集血清及肝脏样本。检测小鼠血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、γ-谷氨酰基转移酶(γ-GT)和甲胎蛋白(AFP)评估各组小鼠肝功能变化;苏木素-伊红(HE)染色及天狼星红染色观察肝组织病理改变。细胞实验将Huh7细胞分为空白组、芪术抗癌方低、中、高剂量组和(或)PCK1抑制剂(盐酸SKF-34288)组、索拉非尼组,给予对应含药血清和药物处理。采用细胞增殖活性检测(CCK-8)法、集落形成实验、Edu荧光标记检测、细胞内三磷酸腺苷(ATP)含量检测、细胞周期流式检测评估各组Huh7细胞增殖能力、能量代谢变化情况及对Huh7细胞周期的影响。蛋白免疫印迹法(Western blot)用于检测PCK1、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、细胞周期依赖性蛋白激酶抑制因子1A(p21)的蛋白表达水平。结果:与模型组比较,芪术抗癌方组小鼠肝癌组织中细胞异型、坏死、胶原纤维沉积等病理改变有所减轻,肝脏肿瘤数量显著减少(P<0.01),血清ALT、AST、γ-GT和AFP水平显著降低(P<0.01)。细胞水平上,与空白组比较,芪术抗癌方含药血清低、中、高剂量组和索拉非尼组均可显著降低Huh7细胞存活率(P<0.01),显著减少Edu标记的阳性细胞数(P<0.01),显著抑制其克隆增殖能力(P<0.01);芪术抗癌方组还可降低细胞内ATP含量(P<0.05),增加细胞周期G0/G1期分布比例(P<0.05),且呈剂量依赖性。与模型组和空白组比较,芪术抗癌方组小鼠肝癌组织及Huh7细胞�Objective:To study the effect of Qizhu Kang’ai Prescription(QZAP)on the gluconeogenesis enzyme phosphoenolpyruvate carboxykinase 1(PCK1)in the liver of liver cancer murine model induced by diethylnitrosamine(DEN)combined with carbon tetrachloride(CCl4)and human liver cancer Huh7 cells,so as to explore the mechanism on regulating metabolic reprogramming and inhibiting cell proliferation of liver cancer cells.Methods:Induction method containing DEN and CCl4 was used to construct a mouse model of liver cancer via intraperitoneal injection.A normal group,a model group,and a QZAP group were set up,in which QZAP(3.51g▪kg^(-1))or an equal volume of normal saline was administered daily by gavage,respectively.Serum and liver samples were collected after 8 weeks of intervention.Detect serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),γ-glutamyltransferase(γ-GT)and alpha-fetoprotein(AFP)in mice to evaluate liver function changes of mice in each group.Hematoxylineosin(HE)staining and sirius red staining to observe pathological changes of liver tissue.In the cell experiment,Huh7 cells were divided into blank group,QZAP low,medium and high dose group and/or PCK1 inhibitor(SKF-34288 hydrochloride)group,and Sorafenib positive drug group,given the corresponding drug-containing serum and drug treatment respectively.Cell proliferation-toxicity detection kit(CCK-8 method),colony formation experiment,Edu fluorescent labeling detection,intracellular adenosine triphosphate(ATP)content detection,and cell cycle flow cytometry detection were used to evaluate the proliferation ability,energy metabolism changes and change of cell cycle of Huh7 cells in each group.Western blot was used to detect the protein expression levels of PCK1,serine/threonine kinase(Akt),phosphorylated Akt(p-Akt),and cell cycle-dependent protein kinase inhibitor 1A(p21)in mouse liver tissue samples and Huh7 cell samples.Results:Compared with the model group,the pathological changes such as cell atypia,necrosis,and collagen fiber deposition in l

关 键 词：肝细胞癌 芪术抗癌方 糖异生 代谢重编程 细胞周期依赖性蛋白激酶抑制因子1A(p21) 

分 类 号：R2-0[医药卫生—中医学] R22R242R285.5R735.7