摘要
目的探讨蛇莓醇提物通过铁死亡抑制肾癌细胞OS-RC-2增殖的作用机制。方法蛇莓醇提物给药,CCK-8试验检测细胞增殖;活性氧(reactive oxygen species,ROS)检测试剂盒、GSH试剂盒、MDA试剂盒、细胞凋亡试剂盒分别检测细胞ROS、GSH、MDA含量及凋亡水平;网络药理学分析蛇莓成分-靶点;分子对接检测蛇莓成分与铁死亡核心靶点GPX4的结合能力;Western blot对铁死亡信号通路关键蛋白进行检测;利用铁死亡的抑制剂Fer-1和ROS抑制剂NAC进行回复试验,检测细胞增殖及铁死亡蛋白的变化;蛇莓醇提物中的Dulcitol(卫矛醇)给药,检测细胞增殖及GPX4蛋白的变化。结果蛇莓可在一定程度上呈时间-浓度依赖性抑制细胞增殖,显著增加ROS、MDA水平而降低GSH水平;流式细胞术显示蛇莓可显著促进细胞凋亡,但并没有呈浓度依赖方式;网络药理学和分子对接结果显示蛇莓中的卫矛醇与GPX4结合稳定(-6.5 kJ·mol^(-1));Western blot结果显示蛇莓明显降低GPX4、SLC7A11并升高HO-1及Transferrin蛋白水平;回复试验结果显示,Fer-1与蛇莓联合给药、NAC与蛇莓联合给药均可显著逆转蛇莓单独给药引起的细胞活力降低和铁死亡关键蛋白GPX4、SLC7A11、HO-1及Transferrin的蛋白水平变化。卫矛醇给药可抑制OS-RC-2细胞增殖并降低GPX4蛋白表达水平。结论综上所述,蛇莓醇提物可能通过降低GPX4的表达,促使肾癌细胞发生增殖抑制及ROS积累,进一步驱动细胞铁死亡,且卫矛醇可能是蛇莓中潜在的直接作用于GPX4的药效物质。
Aim To investigate the effect of ethanol extract of Duchesnea Indica on the proliferation of renal cell carcinoma OS-RC-2 cells induced by ferroptosis and the related mechanism.Methods Cell proliferation was detected by the CCK-8 assay.The levels of ROS,GSH,MDA and apoptosis rate were detected by ROS detection,GSH kit,MDA kit and apoptosis Kit,respectively.The components and targets of Duchesnea Indica were analyzed by network pharmacology.The binding ability of Duchesnea Indica components to GPX4,the core target of ferroptosis,was detected by molecular docking.The key proteins in the ferroptosis signaling pathway were detected by Western blot.Next,ferroptosis inhibitor Fer-1 and ROS inhibitor NAC were used to detect the changes in cell proliferation and ferroptosis related proteins.The cell proliferation and the protein level of GPX4 were detected by the administration of Dulcitol.Results Duchesnea Indica inhibited cell proliferation in a time-concentration dependent manner,upregulated the level of ROS and MDA while down regulated GSH Flow cytometry showed that Duchesnea Indica significantly promoted cell apoptosis,but not in a concentration-dependent manner.The results of network pharmacology and molecular docking showed that dulcitol had a stable binding with GPX4(-6.5 kJ·mol^(-1)).Western blot results showed that Duchesnea Indica significantly decreased GPX4 and SLC7A11,while increased HO-1 and Transferrin protein levels.The results of the rescue experiment showed that the co-administration of Fer-1/NAC and Duchesnea Indica could significantly reverse the cell viability and the changes in the protein levels of GPX4,SLC7A11,HO-1 and Transferrin,when compared to treated with Duchesnea Indica alone in OS-RC-2.Dulcitol in the alcohol extract of Duchesnea Indica could inhibit the proliferation of OS-RC-2 cells and reduce their GPX4 protein expression level.Conclusion In summary,Duchesnea Indica could reduce the expression of GPX4,which further promotes proliferation inhibition and ROS accumulation in renal cancer cells,driving cell iron death.Moreover,Dulcitol may be a potential pharmacodynamic substance that directly acts on GPX4.
作者
游赣花
安群英
何娟
李凯
杨萌
文周
钱城
朱建国
YOU Gan-hua;AN Qun-ying;HE Juan;LI Kai;YANG Meng;WEN Zhou;QIAN Cheng;ZHU Jian-guo(Dept of Scientific Research,the Second People′s Hospital of Guizhou Province,Guiyang 550004,China;Dept ofGeneral Medicine,the Sixth People′s Hospital of Hengshui,Hengshui Hebei 053200,China;Dept of Stomatology,the Second Affiliated Hospital of Guizhou Traditional Chinese Medicine University,Guiyang 550003,China;College of Biological and Environmental Engineering,Guiyang University,Guiyang 550005,China;Graduate School,Zunyi Medical University,Zunyi,Guizhou 563000,China;Dept of Medical Quality Management,the 925th Hospital of the Chinese People′s Liberation Army Joint Logistic Support Force,Guiyang 550000,China;Dept of Urology Surgery,Guizhou Province People′s Hospital,Guiyang 550000,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2024年第4期662-670,共9页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 82160551)
贵州省高层次创新型人才(No黔科合平台人才[2018]5639)。
