鱼腥草对肝癌细胞生物学行为的影响及网络药理学分析

Effects of Herba Houttuyniae on the biological behavior of liver cancer cells and a network pharmacology analysis

目的探讨鱼腥草(Herba Houttuyniae)对肝癌细胞生物学行为的影响及其潜在的分子机制。方法使用不同浓度的鱼腥草提取物处理肝癌细胞SMMC-7721和SK-Hep-1,利用CCK-8检测细胞的活力,使用Transwell实验检测肝癌细胞的迁移和侵袭能力,细胞免疫荧光实验检测转移相关蛋白MMP-9的表达情况,活性氧(ROS)检测试剂盒检测细胞ROS水平,TUNEL染色检测细胞凋亡情况,Western blot检测细胞中凋亡相关蛋白表达水平。采用网络药理学筛选鱼腥草的活性成分并构建蛋白间相互作用(PPI)网络识别鱼腥草治疗肝癌的作用靶点,通过GO和KEGG通路富集分析探索与鱼腥草抗肝癌作用靶点相关的信号通路,利用qRT-PCR检测关键靶点的mRNA表达水平。结果CCK-8、Transwell、细胞免疫荧光实验结果显示鱼腥草提取物可抑制SMMC-7721和SK-Hep-1细胞的活力、迁移和侵袭能力以及转移相关蛋白MMP-9的表达(均P<0.05)。TUNEL染色、ROS检测、Western blot结果显示鱼腥草提取物可促进SMMC-7721和SK-Hep-1细胞凋亡及升高细胞中ROS水平和促凋亡蛋白BAX的表达水平(均P<0.05)。网络药理学预测鱼腥草治疗肝癌主要通过作用于TNF、AKT1、TP53等靶点,涉及癌症、TNF、PI3K-AKT等信号通路。qRT-PCR证实鱼腥草提取物可降低SMMC-7721和SK-Hep-1细胞中TNF-α、AKT1基因的mRNA表达水平,增加TP53基因的mRNA表达水平(均P<0.05)。结论鱼腥草可能通过调节TNF、AKT1、TP53等基因抑制肝癌细胞的细胞活力及迁移和侵袭能力,且促进肝癌细胞凋亡。

Objective To investigate the effects of Herba Houttuyniae on the biological behavior of liver cancer cells and related potential molecular mechanisms.Methods Liver cancer cells SMMC‐7721 and SK‐Hep‐1 were treated with different concentrations of Herba Houttuyniae extract.Cell viability was detected by CCK‐8.Transwell assays were used to evaluate the migration and invasion ability of the liver cancer cells.The expression of the metastatic‐related protein MMP‐9 was detected by cellular immunofluorescence assay.The reactive oxygen species(ROS)levels in the cells were measured by using ROS detection kit,and cell apoptosis was assessed by TUNEL staining.The expression levels of apoptosis‐related proteins were analyzed by Western blot.Network pharmacology was employed to screen the active components of Herba Houttuyniae and to construct a protein‐protein interaction(PPI)network to identify the therapeutic targets of Herba Houttuyniae in liver cancer.GO and KEGG pathway enrichment analyses were performed to explore the signaling pathways associated with the target of Herba Houttuyniae in anti‐liver cancer.The qRT‐PCR was used to detect the mRNA expression levels of key targets.Results The CCK‐8,Transwell,and cellular immunofluorescence assays indicated that Herba Houttuyniae extracts inhibited the viability,migration,and invasion abilities of SMMC‐7721 and SK‐Hep‐1 cells,as well as the expression of the metastasisrelated protein MMP‐9(all P<0.05).TUNEL staining,ROS detection,and Western blot results showed that Herba Houttuyniae extracts promoted apoptosisinSMMC‐7721and SK‐Hep‐1 cells,elevated intracellular ROS levels,and increased the expression of the pro‐apoptotic protein BAX(all P<0.05).Network pharmacology predicted that Herba Houttuyniae treated liver cancer primarily by targeting TNF,AKT1,TP53,etc.involving cancer,TNF,PI3K‐AKT and other signaling pathways.The qRT‐PCR confirmed that Herba Houttuyniae extracts decreased the mRNA expression levels of TNF⁃αand AKT1 genes,while increased the mRNA expression level of the TP53 gene in SMMC‐7721 and SK‐Hep‐1 cells(all P<0.05).Conclusions Herba Houttuyniae may inhibit the viability,migration,and invasion abilities of liver cancer cells and promote the apoptosis by regulating genes such as TNF,AKT1,and TP53.