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

ZHONG Xin; HU Rui; LI Jing; PENG Lanfen; LIU Xingning; HUANG Qi; SUN Jialing; SUN Xinfeng; CHEN Jianping; CAI Benqiang; ZHOU Xiaozhou

doi:10.13422/j.cnki.syfjx.20232424

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

更新时间：2023-12-28

- Mechanism of Qizhu Kang'ai Prescription for Inhibiting Proliferation of Hepatocellular Carcinoma by Regulating Tumor Metabolic Reprogramming via PCK1/Akt/p21 Signal Axis
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 30, Issue 3, Pages: 26-36(2024)
- 作者机构：
  
  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，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
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20232424
  CLC： R2-0;R22;R242;R285.5;R735.7
- Received：21 August 2023，
  
  Published Online：03 November 2023，
  
  Published：05 February 2024
- 稿件说明：
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钟欣,胡锐,李静等.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[J].中国实验方剂学杂志,2024,30(03):26-36.

ZHONG Xin,HU Rui,LI Jing,et al.Mechanism of Qizhu Kang'ai Prescription for Inhibiting Proliferation of Hepatocellular Carcinoma by Regulating Tumor Metabolic Reprogramming via PCK1/Akt/p21 Signal Axis[J].Chinese Journal of Experimental Traditional Medical Formulae,2024,30(03):26-36.
钟欣,胡锐,李静等.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[J].中国实验方剂学杂志,2024,30(03):26-36. DOI： 10.13422/j.cnki.syfjx.20232424.

ZHONG Xin,HU Rui,LI Jing,et al.Mechanism of Qizhu Kang'ai Prescription for Inhibiting Proliferation of Hepatocellular Carcinoma by Regulating Tumor Metabolic Reprogramming via PCK1/Akt/p21 Signal Axis[J].Chinese Journal of Experimental Traditional Medical Formulae,2024,30(03):26-36. DOI： 10.13422/j.cnki.syfjx.20232424.

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（CCl

）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CCl

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）、

-谷氨酰基转移酶（

-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（

0.01），血清ALT、AST、

-GT和AFP水平显著降低（

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（

0.01），显著减少Edu标记的阳性细胞数（

0.01），显著抑制其克隆增殖能力（

0.01）；芪术抗癌方组还可降低细胞内ATP含量（

0.05），增加细胞周期G

期分布比例（

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细胞的PCK1、p21蛋白水平明显降低（

0.05，

0.01），p-Akt蛋白水平显著增加（

0.01）。与空白组比较，盐酸SKF-34288组Huh7细胞ATP含量、细胞存活率明显升高（

0.05），但Edu阳性细胞率、G

期分布比例变化差异无统计学意义；与盐酸SKF-34288组比较，芪术抗癌方联合盐酸SKF-34288组可明显降低Huh7细胞的ATP含量、细胞存活率及Edu阳性细胞率（

0.05），增加G

期分布比例（

0.05）。

结论

芪术抗癌方可能通过激活PCK1诱导肝癌细胞代谢重编程促进Akt/p21介导的抑癌作用，从而发挥抗肝癌增殖的作用机制。

Abstract

Objective

To study the effect of Qizhu Kang'ai prescription （QZAP） on the gluconeogenesis enzyme phosphoenolpyruvate carboxykinase 1 （PCK1） in the liver of mouse model of liver cancer induced by diethylnitrosamine （DEN） combined with carbon tetrachloride （CCl

） and Huh7 cells of human liver cancer， so as to explore the mechanism on regulating metabolic reprogramming and inhibiting cell proliferation of liver cancer cells.

Method

DEN combined with CCl

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 （3.51 g·kg

-1

） or an equal volume of normal saline was administered daily by gavage， respectively. Serum and liver samples were collected after eight weeks of intervention. Serum alanine aminotransferase （ALT）， aspartate aminotransferase （AST），

-glutamyltransferase （

-GT）， and alpha-fetoprotein （AFP） in mice were detected to evaluate liver function changes of mice in each group. Hematoxylin-eosin （HE） staining and Sirius red staining were used to observe pathological changes in liver tissue. In the cell experiment， Huh7 cells were divided into blank group， QZAP low， medium， and high dose groups and/or PCK1 inhibitor （SKF-34288 hydrochloride） group， and Sorafenib group. The corresponding drug-containing serum and drug treatment were given， respectively. Cell counting kit-8 （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 in the 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）.

Result

Compared with the model group， the pathological changes such as cell atypia， necrosis， and collagen fiber deposition in liver cancer tissue of mice in the QZAP group were alleviated， and the number of liver tumors was reduced （

0.01）. The serum ALT， AST，

-GT， and AFP levels were reduced （

0.01）. At the cell level， compared with the blank group， low， medium， and high-dose groups of QZAP-containing serum and the Sorafenib group could significantly reduce the survival rate of Huh7 cells （

0.01） and the number of positive cells with Edu labeling （

0.01） and inhibit clonal proliferation ability （

0.01）. The QZAP groups could also reduce the intracellular ATP content （

0.05） and increase the distribution ratio of the G

phase of the cell cycle （

0.05） in a dose-dependent manner. Compared with the model group and blank group， PCK1 and p21 protein levels of mouse liver cancer tissue and Huh7 cells in the QZAP groups were significantly reduced （

0.05，

0.01）， and the p-Akt protein level was significantly increased （

0.01）. Compared with the blank group， the ATP content and cell survival rate of Huh7 cells in the SKF-34288 hydrochloride group were significantly increased （

0.05）， but there was no statistical difference in the ratio of Edu-positive cells and the proportion of G

phase distribution. Compared with the SKF-34288 hydrochloride group， the QZAP combined with the SKF-34288 hydrochloride group significantly reduced the ATP content， cell survival rate， and Edu-positive cell ratio of Huh7 cells （

0.05） and significantly increased the G

phase distribution proportion （

0.05）.

Conclusion

QZAP may induce the metabolic reprogramming of liver cancer cells by activating PCK1 to promote Akt/p21-mediated tumor suppression， thereby exerting an anti-hepatocellular carcinoma proliferation mechanism.

关键词

Keywords

references

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