基于PPARG/FABP4/GPX4通路研究淫羊藿苷诱导HepG2肝癌细胞铁死亡的作用机制  

Study on mechanism of icariin-induced ferroptosis in HepG2 hepatoma carcinoma cells through PPARG/FABP4/GPX4 pathway

作　　者：李丽[1,2] 曾普华 杨仁义 邓颖[1] 贺佐梅[2] 夏鑫 朱定耀 彭清华 LI Li;ZENG Pu-hua;YANG Ren-yi;DENG Ying;HE Zuo-mei;XIA Xin;ZHU Ding-yao;PENG Qing-hua(Hunan University of Chinese Medicine,Changsha 410208,China;Hunan Provincial Hospital of Integrated Chinese and Western Medicine,Changsha 410006,China;Cancer Research Institute of Hunan Academy of Traditional Chinese Medicine,Changsha 410006,China;the First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]湖南省中西医结合医院,湖南长沙410006 [3]湖南省中医药研究院肿瘤研究所,湖南长沙410006 [4]湖南中医药大学第一附属医院,湖南长沙410007

出　　处：《中国中药杂志》2024年第5期1295-1309,共15页China Journal of Chinese Materia Medica

基　　金：湖南省教育厅中医学世界一流培育学科项目(湘教发[2022]57号);湖南省中医药科研课题(C2023030,D2022037);湖南省中医药研究院课题(202125)。

摘　　要：该研究探索了淫羊藿苷诱导肝癌细胞铁死亡的机制。通过生物信息学方法筛选淫羊藿苷的潜在靶点;构建了蛋白-蛋白互作网络与通路富集,利用分子对接技术评估了淫羊藿苷与靶点的结合能力;通过预测患者生存率来构建一套临床预测模型;同时采用cell counting kit-8(CCK-8)、EdU和克隆形成实验检测肝癌细胞的活性和增殖情况;比色法试剂盒及BODIPY 581/591 C1荧光探针用来检测细胞内铁离子和脂质过氧化水平。采用RT-qPCR和Western blot检测GPX4、xCT、PPARG和FABP4的mRNA和蛋白水平,以确定这些铁死亡相关基因在淫羊藿苷效应中的表达变化。通过生物信息学分析筛选出的6个干预靶点AR、AURKA、PPARG、AKR1C3、ALB、NQO1构成一个风险评分系统,有助于评估肝癌患者生存预后。结合患者年龄和TNM分期,开发了1个Nomogram临床预测模型,以预测肝癌患者1、3、5年的生存。实验研究结果发现,淫羊藿苷能有效地抑制肝癌细胞HepG2的活性和增殖,并能显著调节Fe^(2+)、MDA、脂质过氧化ROS水平,降低GSH水平,揭示其潜在的诱导肝癌细胞铁死亡的能力。淫羊藿苷能降低GPX4和xCT的表达(P<0.01),诱导肝癌细胞铁死亡,可能与抑制PPARG、FABP4有关(P<0.01)。综上,淫羊藿苷通过PPARG/FABP4/GPX4通路,诱导肝癌细胞铁死亡,为利用中药淫羊藿苷预防或治疗肝癌提供了实验基础。The aim of this study was to explore the mechanism of icaritin-induced ferroptosis in hepatoma HepG2 cells.By bioinformatics screening,the target of icariin′s intervention in liver cancer ferroptosis was selected,the protein-protein interaction(PPI)network was constructed,the related pathways were focused,the binding ability of icariin and target protein was evaluated by molecular docking,and the impact on patients′survival prognosis was predicted and the clinical prediction model was built.CCK-8,EdU,and clonal formation assays were used to detect cell viability and cell proliferation;colorimetric method and BODIPY 581/591 C1 fluorescent probe were used to detect the levels of Fe2+,MDA and GSH in cells,and the ability of icariin to induce HCC cell ferroptosis was evaluated;RT-qPCR and Western blot detection were used to verify the mRNA and protein levels of GPX4,xCT,PPARG,and FABP4 to determine the expression changes of these ferroptosis-related genes in response to icariin.Six intervention targets(AR,AURKA,PPARG,AKR1C3,ALB,NQO1)identified through bioinformatic analysis were used to establish a risk scoring system that aids in estimating the survival prognosis of HCC patients.In conjunction with patient age and TNM staging,a comprehensive Nomogram clinical prediction model was developed to forecast the 1-,3-,and 5-year survival of HCC patients.Experimental results revealed that icariin effectively inhibited the activity and proliferation of HCC cells HepG2,significantly modulating levels of Fe^(2+),MDA,and lipid peroxidation ROS while reducing GSH levels,hence revealing its potential to induce ferroptosis in HCC cells.Icariin was found to diminish the expression of GPX4 and xCT(P<0.01),inducing ferroptosis in HCC cells,potentially in relation to inhibition of PPARG and FABP4(P<0.01).In summary,icariin induces ferroptosis in HCC cells via the PPARG/FABP4/GPX4 pathway,providing an experimental foundation for utilizing the traditional Chinese medicine icariin in the prevention or treatment of HCC.

关 键 词：淫羊藿苷 肝细胞癌 铁死亡 临床预测模型 生物信息学 

分 类 号：R285[医药卫生—中药学]