采用网络药理学、分子对接技术和细胞实验探究华蟾素注射液(Huachansu Injection，HCSI)抗结直肠癌(colorectal cancer，CRC)的作用机制。课题组前期采用LC-MS/MS检测HCSI中16种蟾蜍内酯类成分的含量，以大于10 ng·mL-1为标准，筛选出10种蟾蜍内酯类成分，通过SwissTargetPrediction数据库收集其潜在作用靶点;利用GeneCards、OMIM、TTD、PharmGKB数据库预测CRC相关靶点，通过Venny获得HCSI治疗CRC的交集靶点，并使用Cytoscape软件构建"活性成分-靶点-疾病"网络及靶点蛋白-蛋白相互作用(PPI)网络，根据度值筛选关键靶点;对关键靶点进行基因本体论(Gene Ontology，GO)功能和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)通路富集分析，并通过AutoDock软件对主要蟾蜍内酯活性成分和关键靶点进行分子对接;检测不同浓度的HCSI、伪异沙蟾毒精(psi-bufarenogin，BUF)和蟾毒它灵(bufotalin，BFT)对结直肠癌HCT116细胞的细胞活力、迁移能力和凋亡率的影响，蛋白免疫印迹法(Western blot)检测HCT116细胞PI3K/Akt/mTOR信号通路相关蛋白的表达。筛选出HCSI的主要活性成分沙蟾毒精(arenobufagin)、BUF和BFT等8种，HCSI抗CRC的关键靶点EGFR、IL6和mTOR等20个，结合KEGG通路富集分析和分子对接结果，选择PI3K/Akt/mTOR信号通路进行验证。细胞实验结果表明，HCSI、BUF和BFT对HCT116细胞的增殖能力和迁移能力具有显著抑制作用，可诱导HCT116细胞凋亡，下调PI3K/Akt/mTOR通路相关蛋白表达，说明HCSI、BUF和BFT可通过mTOR和PIK3CA等靶点调控PI3K/Akt/mTOR信号通路，发挥抗CRC的作用。该研究为探究HCSI的抗肿瘤活性成分和作用机制提供理论依据。
Mechanism of Huachansu Injection against colorectal cancer based on network pharmacology and cellular experimental
This study aimed to elucidate the mechanism of Huachansu Injection(HCSI)against colorectal cancer(CRC)using network pharmacology,molecular docking technology,and cellular experimental.This research group initially used LC-MS/MS to detect the content of 16 bufadienolides in HCSI.Ten bufadienolide components were selected based on a content threshold of greater than 10 ng·mL-1.Their potential targets were further predicted using the SwissTargetPrediction database.CRC-related targets were obtained through GeneCards,OMIM,TTD,and PharmGKB databases.The intersection targets of HCSI in the treatment of CRC were obtained through Venny.The"active component-target-disease"network and target protein-protein interaction(PPI)network were constructed via Cytoscape software.Core targets were screened based on the degree values.Gene Ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed on these key targets.Molecular docking was conducted using AutoDock software on major bufadienolide active components and key targets.Different concentrations of HCSI,psi-bufarenogin(BUF),and bufotalin(BFT)were tested for their effects on cell viability,migration,and apoptosis rates in CRC HCT116 cells.Western blot was conducted to detect the expression of proteins related to the PI3K/Akt/mTOR signaling pathway in HCT116 cells.Eight main active components of HCSI,including arenobufagin,BUF,and BFT,as well as 20 key targets of HCSI in combating CRC,such as EGFR,IL6,and mTOR,were identified.Based on KEGG pathway enrichment and molecular docking results,the PI3K/Akt/mTOR signaling pathway was selected for further verification.Cellular experimental demonstrated that HCSI,BUF,and BFT significantly inhibited the proliferation and migration abilities of HCT116 cells,induced apoptosis in these cells,and downregulated the expression of PI3K/Akt/mTOR pathway-related proteins.This result suggests that HCSI,BUF,and BFT may exert their anti-CRC effects by regulating the PI3K/Akt/mTOR signaling pathway through targets such as mTOR and PIK3CA.This study provides theoretical evidence for exploring the active ingredients and mechanism of HCSI against CRC.
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