基于蛋白组学探讨灵芝酸X治疗肝母细胞瘤的作用机制  

Mechanism of ganoderic acid X in treating hepatoblastoma based on proteomics

作　　者：叶婷 高航 葛洋 沈瑞 余鸿雁 陈峰远 宋航 YE Ting;GAO Hang;GE Yang;SHEN Rui;YU Hong-yan;CHEN Feng-yuan;SONG Hang(Graduate School,Anhui University of Chinese Medicine,Hefei 230012,China;School of Integrated Chinese and Western Medicine,Anhui University of Chinese Medicine,Hefei 230012,China;Anhui Province Key Laboratory of Translational Cancer Research,Bengbu Medical College,Bengbu 233030,China)

机构地区：[1]安徽中医药大学研究生院,安徽合肥230012 [2]安徽中医药大学中西医结合学院,安徽合肥230012 [3]蚌埠医学院安徽省转化性肿瘤研究重点实验室,安徽蚌埠233030

出　　处：《中国中药杂志》2024年第15期4158-4166,共9页China Journal of Chinese Materia Medica

基　　金：安徽省自然科学基金项目(2108085Y29);癌症转化医学安徽省重点实验室(蚌埠医学院)项目(KFZZ202205);安徽省教育厅重点项目(2023AH050870)。

摘　　要：基于蛋白组学探讨灵芝酸X(ganoderic acid X,GAX)对人肝母细胞瘤HepG2、HuH6细胞模型和非肥胖型糖尿病/重症联合免疫缺陷(nonobese diabetic-severe combined immune deficient,NOD-SCID)小鼠皮下移植瘤模型的作用机制,为灵芝酸X的临床应用提供依据。采用CCK-8法检测灵芝酸X对HepG2、HuH6细胞活力的影响;EdU实验检测灵芝酸X对细胞增殖的影响;划痕实验检测灵芝酸X对细胞迁移能力的影响;Hoechst 33258染色检测灵芝酸X对细胞凋亡的影响;建立NOD-SCID小鼠皮下移植瘤模型,分析对照组及灵芝酸X低、中、高剂量组(5、10、20 mg·kg^(-1))肿瘤体积和质量;苏木素-伊红(HE)染色评估灵芝酸X的药物毒性。此外,收集HepG2细胞对照组和灵芝酸X高剂量组的细胞,分别进行laber-free蛋白组学分析,筛选差异蛋白并富集相关信号通路,CYTO-ID?染色检测细胞自噬情况,Western blot实验检测相关蛋白的表达量。体外结果显示,灵芝酸X呈剂量依赖性抑制HepG2、HuH6细胞的增殖、迁移、诱导凋亡;体内研究显示灵芝酸X显著抑制肿瘤体积和质量,且对小鼠主要脏器(心、肝、脾、肺、肾)无明显损害;laber-free蛋白组学结果显示灵芝酸X在肝母细胞瘤治疗过程中参与多种信号通路,其中自噬途径高度富集。CYTO-ID?染色及Western blot结果显示灵芝酸X诱导细胞自噬并上调苄氯素1(Beclin-1)、自噬相关基因5(ATG5)、微管相关蛋白1A/1B-轻链3(LC3)-Ⅱ蛋白表达量,下调螯合体1(p62)蛋白表达量。该研究表明,灵芝酸X通过诱导自噬进而抑制肝母细胞瘤细胞的增殖、迁移和诱导凋亡,并显著抑制肿瘤生长,是一种有希望的癌症辅助治疗药物。This research explored the mechanism of ganoderic acid X(GAX)on human hepatocellular carcinoma cell models(HepG2,HuH6)and nonobese diabetic-severe combined immune deficient(NOD-SCID)mouse subcutaneous tumor models using proteomics,aiming to provide a basis for the clinical application of GAX.CCK-8 assay was employed to evaluate the effect of GAX on the viability of HepG2 and HuH6 cells.EdU assay was used to assess the effect of GAX on cell proliferation.Scratch assay was used to examine the effect of GAX on cell migration ability.Hoechst 33258 staining was used to investigate the effect of GAX on cell apoptosis.Moreover,a NOD-SCID mouse subcutaneous tumor model was established to analyze the tumor volume and weight in control group and GAX low-,medium-,and high-dose groups(5,10,and 20 mg·kg^(-1)).HE staining was conducted to evaluate the drug toxicity of GAX.Additionally,HepG2 cells in the control group and the GAX high-dose group were subjected to label-free proteomics analysis to identify differential proteins and enrich relevant signaling pathways.CYTO-ID?staining was performed to detect autophagy,and Western blot was conducted to measure the expression levels of relevant proteins.In vitro results demonstrated that GAX dose-depen-dently inhibited proliferation,migration,and induced apoptosis in HepG2 and HuH6 cells.In vivo studies showed that GAX significantly inhibited tumor volume and weight without causing significant damage to major organs(heart,liver,spleen,lung,and kidney)in mice.Label-free proteomics analysis revealed that GAX participated in multiple signaling pathways during the treatment of hepatocellular carcinoma,with a high enrichment in the autophagy pathway.CYTO-ID?staining and Western blot results showed that GAX induced autophagy,upregulated the expression of Beclin-1,ATG5,and LC3-Ⅱproteins,and downregulated the expression of p62 protein.This study suggests that GAX inhibits the proliferation,migration,and induces apoptosis of hepatocellular carcinoma cells by inducing autophagy,thereby sig

关 键 词：灵芝酸X 肝母细胞瘤 蛋白组学 自噬 

分 类 号：R285.5[医药卫生—中药学]