基于网络药理学的姜黄素相关机制抗肝癌的研究与实验验证。
Investigation and experimental validation of curcumin-related mechanisms against hepatocellular carcinoma based on network pharmacology.
机构信息
College of Basic Medicine, Shaanxi University of Chinese Medicine, Xianyang 712046, China.
School of Pharmacy, Shaanxi University of Chinese Medicine, Xianyang 712046, China.
出版信息
J Zhejiang Univ Sci B. 2022 Aug 15;23(8):682-698. doi: 10.1631/jzus.B2200038.
OBJECTIVES
To determine the potential molecular mechanisms underlying the therapeutic effect of curcumin on hepatocellular carcinoma (HCC) by network pharmacology and experimental in vitro validation.
METHODS
The predictive targets of curcumin or HCC were collected from several databases. the identified overlapping targets were crossed with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) platform. Two of the candidate pathways were selected to conduct an experimental verification. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) assay was used to determine the effect of curcumin on the viability of HepG2 and LO2 cells. The apoptosis and autophagy of HepG2 cells were respectively detected by flow cytometry and transmission electron microscopy. Besides, western blot and real-time polymerase chain reaction (PCR) were employed to verify the p53 apoptotic pathway and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) autophagy pathway. HepG2 cells were pretreated with pifithrin-α (PFT-α) and GSK690693 for further investigation.
RESULTS
The 167 pathways analyzed by KEGG included apoptosis, autophagy, p53, and AMPK pathways. The GO enrichment analysis demonstrated that curcumin was involved in cellular response to drug, regulation of apoptotic pathway, and so on. The in vitro experiments also confirmed that curcumin can inhibit the growth of HepG2 cells by promoting the apoptosis of p53 pathway and autophagy through the AMPK pathway. Furthermore, the protein and messenger RNA (mRNA) of the two pathways were downregulated in the inhibitor-pretreated group compared with the experimental group. The damage-regulated autophagy modulator (DRAM) in the PFT-α-pretreated group was downregulated, and p62 in the GSK690693-pretreated group was upregulated.
CONCLUSIONS
Curcumin can treat HCC through the p53 apoptotic pathway and the AMPK/Unc-51-like kinase 1 (ULK1) autophagy pathway, in which the mutual transformation of autophagy and apoptosis may occur through DRAM and p62.
目的
通过网络药理学和体外实验验证，确定姜黄素治疗肝细胞癌（HCC）的潜在分子机制。
方法
从多个数据库中收集姜黄素或 HCC 的预测靶点，使用数据库 for Annotation、Visualization、and Integrated Discovery（DAVID）平台将鉴定的重叠靶点与基因本体论（GO）和京都基因与基因组百科全书（KEGG）分析进行交叉。选择两个候选途径进行实验验证。使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴化四唑（MTT）测定法确定姜黄素对 HepG2 和 LO2 细胞活力的影响。通过流式细胞术和透射电子显微镜分别检测 HepG2 细胞的凋亡和自噬。此外，采用 Western blot 和实时聚合酶链反应（PCR）验证 p53 凋亡途径和腺苷 5'-单磷酸（AMP）激活蛋白激酶（AMPK）自噬途径。用 pifithrin-α（PFT-α）和 GSK690693 预处理 HepG2 细胞进一步研究。
结果
KEGG 分析的 167 条途径包括凋亡、自噬、p53 和 AMPK 途径。GO 富集分析表明，姜黄素参与细胞对药物的反应、调节凋亡途径等。体外实验也证实，姜黄素通过 AMPK 通路促进 p53 通路凋亡和自噬，从而抑制 HepG2 细胞的生长。此外，与实验组相比，抑制剂预处理组两条通路的蛋白和信使 RNA（mRNA）表达均下调。PFT-α预处理组中的损伤调节自噬调节剂（DRAM）下调，GSK690693 预处理组中的 p62 上调。
结论
姜黄素可以通过 p53 凋亡途径和 AMPK/Unc-51-like kinase 1（ULK1）自噬途径治疗 HCC，其中自噬和凋亡可能通过 DRAM 和 p62 发生相互转化。
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