基于蛋白组学探讨消癌解毒方通过IL-6/TNF-α/STAT3通路抑制肝癌小鼠肿瘤生长的作用机制  

Exploration on the Mechanism of Xiao'ai Jiedu Recipe for Inhibitingg Tumor Growth of Hepatoma Mice Through IL-6/TNF-α/STAT3 Pathway Based on Proteomics

作　　者：孙玉侠 吴勉华 孙莎莎[1,2] 谢美萍 姜泽群 李文婷[2,3] SUN Yuxia;WU Mianhua;SUN Shasha;XIE Meiping;JIANG Zequn;LI Wenting(School of Medicine&Holistic Integrative Medicine,Nanjing University of Chinese Medicine,Nanjing 210023 Jiangsu,China;Collaborative Innovation Canter of Traditional Chinese Medicine Prevention and Treatment of Tumor,Nanjing 210023 Jiangsu,China;The First Clinical Medical Shool of Nanjing University of Chinese Medicine,Nanjing 210029 Jiangsu,China)

机构地区：[1]南京中医药大学医学院·整合医学院,江苏南京210023 [2]江苏省中医药防治肿瘤协同创新中心,江苏南京210023 [3]南京中医药大学第一临床医学院,江苏南京210029

出　　处：《中药新药与临床药理》2023年第11期1505-1513,共9页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81804058);吴勉华全国名老中医药专家传承工作室{国中医药人教函[2022]75号};国家中医药管理局第七批全国老中医药专家学术经验继承工作项目{国中医药人教函[2022]76号};国家中医药管理局中医药传承与创新“百千万”人才工程(岐黄工程)岐黄学者项目{国中医药人教函[2018]284号};吴勉华江苏省名老中医药专家传承工作室{苏中医科教[2021]7号};江苏省大学生创新计划项目(202110315012)。

摘　　要：目的通过对H22肝癌荷瘤小鼠血清蛋白质组学检测,探讨消癌解毒方抑制肝癌小鼠肿瘤增殖促进凋亡的作用机制。方法将C57BL/6小鼠随机分为正常组、模型组、消癌解毒方组、顺铂组、消癌解毒方联合顺铂组。构建H22肝癌荷瘤小鼠模型,给药干预,连续给药11 d,次日剥取瘤体、胸腺和脾脏,计算抑瘤率和免疫系数;病理检测肿瘤光镜下差异表现;流式细胞仪检测各组凋亡情况;制备血清,以TMT肽段标记结合LC-MS/MS寻找差异蛋白表达谱,应用IPA软件进行分析;Elisa法检测血清中白细胞介素6(Interleukin6,IL-6)和血清肿瘤坏死因子α(TNF-α)表达;蛋白免疫印迹(Western Blot)法检测信号传导及转录激活因子3(STAT3)、磷酸化信号传导及转录激活因子3(p-STAT3)、c-Myc、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤2(Bcl-2)、裂解半胱氨酸蛋白酶3(Cleaved-Caspase-3)和裂解半胱氨酸蛋白酶9(Cleaved-Caspase-9)蛋白含量。结果与模型组比,用药后各组小鼠肿瘤质量均有不同程度的减小(P<0.01);消癌解毒方、顺铂和消癌解毒方联合顺铂组抑瘤率分别为40.59%、53.84%、67.32%;顺铂组免疫指数降低(P<0.05),消癌解毒方和消癌解毒方联合顺铂组免疫系数均较模型组明显上升(P<0.05,P<0.01);病理检测结果显示,与模型组比,各组瘤组织坏死程度加大,分裂增殖程度减轻;流式细胞仪检测结果显示各加药组凋亡率较模型组均明显上升(P<0.01)。蛋白组学检测显示,消癌解毒方与模型组比差异蛋白共132个,其中下调蛋白95个,上调蛋白37个,这些差异表达涉及脂质代谢、凋亡、炎症等方面。Elisa检测提示模型组IL-6和TNF-α的表达量均较正常组上调(P<0.01),各加药组与模型组比明显下降(P<0.05,P<0.01);Western Blot检测结果显示给药后瘤组织中p-STAT3蛋白表达明显降低(P<0.05,P<0.01),其下游基因c-Myc、Bcl-2蛋白含量明显降低(P<0.01),Bax、Cleaved-Caspase-3和Cleaved-Caspase-9蛋白Objective To investigate the mechanism of Xiao'ai Jiedu Recipe for inhibiting tumor proliferation and promoting apoptosis in mice with hepatocellular carcinoma through the detection of serum proteomics of H22 hepatoma-bearing mice.Methods C57BL/6 mice were randomly divided into normal group,model group,Xiao'ai Jiedu Recipe group,cisplatin group and Xiao'ai Jiedu Recipe combined with cisplatin group.After H22 hepatoma-bearing model was constructed,the rats were treated with drug for ll consecutive days.The tumors,thymus and spleen were taken on the day after the last administration,and the tumor suppression rate and immune index were calculated.Pathologyical examination for differential expression of tumors under light microscopy was conducted.Flow cytometry was used to detect apoptosis of cells in each group.Then,serum was prepared,and the dfferential protein expression profile was found by TMT peptide labeling coupled with LC-MS/MS,and bioinformatics analysis was performed by GO analysis and IPA software.Interleukin-6(IL-6)and serum tumor necrosis factor-α(TNF-α)expression were detected by ELISA.Western Blot was used to detect the protein contents of signal transduction and transcription activating factor 3(STAT3),phosphorylation of signal transducer and activator of transcription 3(p-STAT3),c-Myc,Bcl-2 related X protein(Bax),B cell lymphoma/leukemia-2 gene(Bcl-2),Cleaved-Caspase-3 and Cleaved-Caspase-9.Results Compared with the model group,the tumor mass in all groups decreased with different degree after drug administration(P<0.01).The tumor suppression rates were 40.59%,53.84%and 67.32%in the Xiao'ai Jiedu Recipe group,cisplatin group and Xiao'ai Jiedu Recipe combined with cisplatin group,respectively.The immune index in the cisplatin group was the lowest(P<0.05),but the immune index in both Xiao"ai Jiedu Recipe group and Xiao'ai Jiedu Recipe combined with cisplatin group showed a significant increase(P<0.05,P<0.01).The pathological results showed that tumor tissues necrosis in each group were increased,div

关 键 词：消癌解毒方 肝癌 蛋白组学 IL-6/TNF-α/STAT3信号通路 小鼠 

分 类 号：R285.5[医药卫生—中药学]