基于网络药理学及体外实验探讨蛭岩消积方治疗肝癌的作用机制

The mechanism of Zhiyanxiaoji prescription in the treatment of liver cancer based on network pharmacology and in vitro experiment

目的:基于网络药理学探讨蛭岩消积方治疗肝癌的作用机制,并通过体外实验研究蛭岩消积方水提物对HepG2肝癌细胞的生物学作用及其机制。方法:利用网络药理学工具预测蛭岩消积方治疗肝癌的作用靶点和通路。将人肝癌细胞HepG2分为对照组(不含蛭岩消积方水提物)和不同浓度蛭岩消积方水提物组。采用CCK8法检测细胞增殖,Transwell检测细胞迁移和侵袭,流式细胞术检测细胞周期,western blotting法检测细胞周期相关蛋白[CDK2、CyclinA、Cdc25A、磷酸化(p-)CHK1]及信号通路蛋白的表达。结果:利用网络药理学筛选出蛭岩消积方治疗肝癌的潜在作用靶点381个,其中关键靶点30个,KEGG富集分析结果发现潜在靶点主要富集在PI3K/AKT、乙肝、丙肝、前列腺癌、AGE-RAGE、IL-17、TNF等信号通路。蛭岩消积方水提物可降低细胞增殖活性,减少迁移和侵袭细胞数,将细胞阻滞于S期;与对照组比较,蛭岩消积方水提物中、高剂量组CDK2、Cdc25A、CyclinA蛋白表达量及p-AKT与AKT比值、p-PI3K/PI3K比值降低,p-CHK1蛋白表达量升高(均P<0.05)。结论:蛭岩消积方治疗肝癌的作用机制是多成分、多靶点、多通路共同调控的结果;蛭岩消积方水提物可能通过阻断PI3K/AKT通路抑制肝癌细胞增殖、侵袭和迁移。

Objective:To explore the mechanism of Zhiyanxiaoji prescription in the treatment of liver cancer based on network pharmacology,and to study the biological effect and mechanism of aqueous extract of Zhiyanxiaoji prescription on HepG2 liver cancer cells.Methods:The target and pathway of Zhiyanxiaoji prescription in the treatment of liver cancer were predicted by network pharmacology tool.Human hepatocellular carcinoma HepG2 was divided into control group(without aqueous extract of Zhiyanxiaoji prescription)and aqueous extrac group of different concentration of Zhiyanxiaoji prescription.Cell proliferation was detected by CCK8 method Cell migration and invasion were detected by Transwell method.Cell cycle was detected by flow cytometry,and the expressions of cell cycle related proteins such as CDK2,CyclinA,Cdc25A,and phosphorylated(p-)CHK1 as well as signal pathway proteins were detected by western blotting.Results:A total of 381 potential targets of Zhiyanxiaoji prescription in the treatment of liver cancer were screened by network pharmacology,of which 30 were key targets.KEGG enrichment analysis showed that the potential targets were mainly concentrated in PI3K/AKT,hepatitis B,hepatitis C,prostate cancer,AGE-RAGE,IL-17,TNF and other signal pathways.Aqueous extract of Zhiyanxiaoji prescription can reduce the activity of cell proliferation,reduce the number of migratory and invasive cells,and block the cells in S phase.Compared with the control group,in aqueous extract of Zhiyanxiaoji prescription,the expressions of CDK2,Cdc25A,CyclinA protein and the ratio of p-AKT/AKT and p-PI3K/PI3K decreased,while the expression of p-CHK1 protein increased in the middle and high dose groups(P<0.05).Conclusion:The mechanism of Zhiyanxiaoji prescription in the treatment of liver cancer is the result of the joint regulation of multi-components,multi-targets and multi-pathways.The aqueous extract of Zhiyanxiaoji prescription may inhibit the proliferation,invasion and migration of liver cancer cells by blocking PI3K/AKT pathway.