摘要
目的探究脉络宁(MLN)对小鼠肝癌细胞Hepa1-6细胞异位移植肝癌模型肿瘤生长的抑制作用及其潜在机制。方法(1)C57BL/6J雄性小鼠腋下接种1×10~6个Hepa1-6细胞建立异位移植肝癌模型,随机分为:模型组、模型+MLN 5、15 g·kg^(-1)组和模型+索拉非尼(SRF)30 mg·kg^(-1)组。连续ig给予相应剂量药物14 d,每2天监测1次体重和瘤体积。给药结束后取瘤,通过肿瘤直观图、瘤重和瘤体积评价MLN抑制肝细胞癌生长的作用;通过生物信息学分析肝癌关键生物过程;天狼星红染色检测肿瘤组织胶原纤维含量;RT-qPCR检测肿瘤组织中细胞外基质成分Ⅰ型胶原α1链(Col1a1)、Col3a1和纤连蛋白,上皮-间充质转化标志物E-钙黏蛋白、N-钙黏蛋白、α-平滑肌肌动蛋白(α-SMA)和波形蛋白,及免疫检查点程序性死亡受体1(PD-1)、程序性死亡受体配体1(PD-L1)和B7同源物3(B7-H3)的mRNA表达水平;Western印迹法检测肿瘤组织中E-钙黏蛋白、α-SMA和PD-L1蛋白表达水平。(2)将人肝癌细胞HepG2细胞分为细胞对照组、β-蜕皮甾酮组、哈巴苷组、哈巴俄苷组、绿原酸组、异绿原酸A组、异绿原酸C组、木犀草苷组、灰毡毛忍冬皂苷乙组和川续断皂苷乙组,药物终浓度均为10μmol·L^(-1),孵育24 h,RT-qPCR检测细胞内Col1a1的mRNA表达水平。将HepG2细胞分为细胞对照组、β-蜕皮甾酮组、绿原酸组、木犀草苷组和灰毡毛忍冬皂苷乙组,药物终浓度均为10μmol·L^(-1),孵育24 h,Western印迹法检测细胞内COL1A1蛋白表达水平。结果(1)生物信息学分析显示,肝癌患者中细胞外基质与成纤维细胞增殖相关基因显著变化。与模型组相比,模型+MLN15 g·kg^(-1)组肿瘤重量和体积显著降低,天狼星红阳性面积显著减少,E-钙黏蛋白mRNA和蛋白表达水平均显著增加,Col1a1、Col3a1、纤连蛋白、N-钙黏蛋白、α-SMA、波形蛋白、PD-L1和B7-H3的mRNA表达水平显著降低;模型+MLN 5、15 g·kg^(-1)组α-SMA和PD-L1蛋白水平显著降低。(2)与细胞对照组相比,β-蜕皮甾酮、绿原酸、木犀草苷和灰毡毛忍冬皂苷乙组HepG2细胞内COL1A1的mRNA和蛋白表达水平均显著降低。结论MLN能抑制小鼠异位移植肝癌生长,其机制可能与抑制上皮-间充质转化,减少胶原生成,降低PD-L1蛋白表达水平,从而改善肿瘤微环境有关。
OBJECTIVE To explore the inhibitory effect of Mailuoning(MLN)on ectopic hepatocellular carcinoma(HCC)growth and the potential mechanism.METHODS①C57BL/6J male mice were inoculated with Hepa1-6 cells(1×106)in the axilla to establish ectopic HCC models and randomly divided into 4 groups:model group,model+MLN 5 g·kg^(-1) group,model+MLN 15 g·kg^(-1) group and model+sorafenib(SRF)30 mg·kg^(-1) group.The drug administration groups were intragastrically given corresponding doses of drugs for 14 consecutive days,and body mass and tumor volume were monitored every 2 days during this period.At the end of the animal experiment,mice were sacrificed and tumors were harvested.The anti-tumor effect of MLN was evaluated via tumor illustration,tumor mass and tumor size.Bioinformatic analyses were conducted to identify key biological processes in hepatocellular carcinoma.Sirius red staining was used to assess collage fiber content in tumors of mice.Quantitative realtime polymerase chain reaction(RT-qPCR)test was performed to detect mRNA expressions of extracellular matrix components including collagen typeⅠalpha 1 chain(Col1a1),Col3a1 and fibronectin,epithelial-mesenchymal transition markers like E-cadherin,N-cadherin,α-smooth muscle actin(α-SMA)and vimentin,and immune checkpoint-related molecules such as programmed death-1(PD-1),programmed death-ligand 1(PD-L1)and B7 homologue 3(B7-H3).Protein levels of E-cadherin,α-SMA,and PD-L1 were subsequently detected by Western blotting.②HepG2 cells were incubated with the vehicle,β-ecdysone,harpagide,harpagoside,chlorogenic acid,isochlorogenic acid A,isochlorogenic acid C,luteoloside,macranthoidin B and dipsacoside B(10μmol·L^(-1))for 24 h,respectively.After incubation,the expression of Col1a1 mRNA was measured to assess the impact of MLN active ingredients on COL1A1 production.Further more,HepG2 cells were incubated with the vehicle,β-ecdysone,chlorogenic acid,luteoloside or macranthoidin B(10μmol·L^(-1))for 24 h before the COL1A1 protein level was assessed.RESULTS①Bioinformatic analysis results showed that extracellular matrix and fibroblast proliferation related genes were notably changed in HCC patients.Compared with the model group,the mass and volume of tumors were significantly reduced in the model+MLN 15 g·kg^(-1) group,so was the positive area of sirius red staining in tumor tissues.In the model+MLN 15 g·kg^(-1) group,the mRNA expression of E-cadherin was upregulated while those of Col1a1,Col3a1,fibronectin,N-cadherin,α-SMA,vimentin,PD-L1 and B7-H3 were downregulated compared to the model group.Western blotting results revealed that the E-cadherin protein level in HCC tissues in the model+MLN 15 g·kg^(-1) group increased while the protein levels ofα-SMA and PD-L1 in HCC tissues in both the model+MLN 5 g·kg^(-1) and model+MLN 15 g·kg^(-1) group decreased compared to the model group.②MLN active ingredients-β-ecdysone,chlorogenic acid,luteoloside,macranthoidin B-were effective at decreasing the mRNA and protein expressions of Col1a1 in HepG2 cells compared with the control group.CONCLUSIN MLN can ameliorate ectopic HCC growth by inhibiting epithelial-mesenchymal transition,decreasing extracellular matrix deposition,reducing immune checkpoint-related molecule PD-L1 expressions and improving immune microenvironments.
作者
周思妍
吴泽奇
柯创
陆宾
黄镇林
季莉莉
ZHOU Siyan;WU Zeqi;KE Chuang;LU Bin;HUANG Zhenlin;JI Lili(Shanghai Key Laboratory of Compound Chinese Medicines,the MOE Key Laboratory for Standardiza-tion of Chinese Medicines,Institute of Chinese Materia Medica,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)
出处
《中国药理学与毒理学杂志》
北大核心
2025年第11期829-838,共10页
Chinese Journal of Pharmacology and Toxicology
基金
国家重点研发计划(2024YFC3506600)。
关键词
脉络宁
肝细胞癌
细胞外基质
上皮-间充质转化
免疫检查点
肿瘤微环境
Mailuoning
hepatocellular carcinoma
extracellular matrix
epithelial-mesenchymal transition
immune checkpoint
tumor microenvironment
