运用网络药理学和体外实验探究黄芩苷是否诱导HepG2细胞发生铁死亡并探讨潜在机制。体外培养HepG2细胞，CCK-8法检测细胞活力。TCGA数据库获取肝癌转录组测序数据，FerrDb V2数据库获取铁死亡基因数据。使用DEG2程辑包筛选差异表达基因，并与铁死亡基因取交集，得到介导铁死亡调节肝癌进程的靶向基因。通过基因本体数据库(Gene Ontology，GO)与京都基因与基因组百科全书数据库(Kyoto Encyclopedia of Genes and Genomes，KEGG)以 P＜0。05、| log2(fold change)|＞0。5为标准，分析模块相关的分子功能及结构。DCFH-DA探针检测细胞活性氧(reactive oxygen spe-cies，ROS)水平变化。试剂盒检测细胞还原型谷胱甘肽(glutathione，GSH)、Fe2+水平。实时荧光定量聚合酶链式反应(real-time quantitative PCR，RT-PCR)检测谷胱甘肽过氧化物酶 4(glutathione peroxidase 4，GPX4)、溶质载体家族 7 成员 11(solute carrier family 7 member 11，SLC7A11)mRNA 表达。蛋白质印迹法检测 GPX4、SLC7A11、PI3K、p-PI3K、Akt、p-Akt、FoxO3a、p-FoxO3a蛋白表达。CCK-8检测结果显示，200 μmol·L-1黄芩苷干预48 h，能显著抑制HepG2细胞活力。网络药理学结果显示，可通过PI3K/Akt信号通路调节肝癌中铁死亡的发生。细胞实验结果显示，黄芩苷可下调SLC7A11，降低GSH水平和GPX4表达，诱导HepG2细胞ROS累积，增加Fe2+产生。铁死亡抑制剂(ferrostatin-1，Fer-1)可减少黄芩苷诱导的ROS累积，上调SLC7A11、GSH和GPX4表达，减弱PI3K、Akt、FoxO3a磷酸化。综上，黄芩苷可通过抑制ROS介导的PI3K/Akt/FoxO3a通路诱导HepG2细胞铁死亡。
Baicalin induces ferroptosis in HepG2 cells by inhibiting ROS-mediated PI3K/Akt/FoxO3a signaling pathway
This study aims to investigate whether baicalin induces ferroptosis in HepG2 cells and decipher the underlying mechanisms based on network pharmacology and cell experiments.HepG2 cells were cultured in vitro and the cell viability was detected by the cell counting kit-8(CCK-8).The transcriptome data of hepatocellular carcinoma were obtained from the Cancer Genome Atlas(TCGA),and the ferroptosis gene data from FerrDb V2.The DEG2 package was used to screen the differentially expressed genes(DEGs),and the common genes between DEGs and ferroptosis genes were selected as the target genes that mediate ferroptosis to regulate hepatocellular carcinoma progression.The functions and structures of the target genes were analyzed by Gene Ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment with the thresholds of P＜0.05 and|log2(fold change)|＞0.5.DCFH-DA probe was used to detect the changes in the levels of cellular reactive oxygen species(ROS)in each group.The reduced glutathione(GSH)assay kit was used to measure the cellular GSH level,and Fe2+assay kit to determine the Fe2+level.Real-time quantitative PCR(RT-PCR)was employed to measure the mRNA levels of glutathione peroxidase 4(GPX4)and solute earrier family 7 member 11(SLC7A11)in each group.Western blot was employed to determine the protein levels of GPX4,SLC7A11,phosphatidylinositol 3-kinase(PI3K),p-PI3K,protein kinase B(Akt),p-Akt,forkhead box protein O3a(FoxO3a),and p-FoxO3a in each group.The results showed that treatment with 200 μmol·L-1 baicalin for 48 h significantly inhibited the viability of HepG2 cells.Ferroptosis in hepatocellular carcinoma could be regulated via the PI3K/Akt signaling pathway.The cell experiments showed that baicalin down-regulated the expression of SLC7A11 and GPX4,lowered the GSH level,and increased ROS accumulation and Fe2+production in HepG2 cells.However,ferrostatin-1,an ferroptosis inhibitor,reduced baicalin-induced ROS accumulation,up-regulated the expression of SLC7A11 and GPX4,elevated the GSH level,and decreased PI3K,Akt,and FoxO3a phosphorylation.In summary,baicalin can induce ferroptosis in HepG2 cells by inhibiting the ROS-mediated PI3K/Akt/FoxO3a pathway.
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