淫羊藿素抑制外泌体介导的黑色素瘤肺转移及机制初探  

Inhibition of exosomes-mediated melanoma metastasis by icaritin

作　　者：屠书梅 刘玉萍[2,3] 陈彦[2,3] TU Shu-mei;LIU Yu-ping;CHEN Yan(Jiangsu University,Zhenjiang 212013,China;Affiliated Hospital of Integrated Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 210028,China;Multi-component of Traditional Chinese Medicine and Microecology Research Center,Jiangsu Provincial Academy of Chinese Medicine,Nanjing 210028,China)

机构地区：[1]江苏大学,江苏镇江212013 [2]南京中医药大学附属中西医结合医院,江苏南京210028 [3]江苏省中医药研究院,中药组分与微生态研究中心,江苏南京210028

出　　处：《药学学报》2021年第3期778-785,共8页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81903858,81873016);江苏省科教强卫医学重点人才项目(ZDRCA2016036);江苏省卫生健康委科研项目面上项目(H2019097)。

摘　　要：本研究从体内外考察淫羊藿素(icaritin,ICT)对外泌体(exosomes,Exo)诱导的小鼠黑色素瘤B16BL6细胞肺转移的影响,并探讨其潜在分子机制。收集B16BL6细胞培养上清液,超速离心法提取Exo,使用透射电镜和Western blotting对Exo进行表征,BCA法对Exo总蛋白进行定量分析。细胞划痕实验考察ICT对Exo诱导的B16BL6细胞迁移能力的影响。建立C57BL/6小鼠黑色素瘤实验性转移模型,动物实验获得南京中医药大学伦理委员会的同意,H&E染色考察ICT体内抑制Exo介导的黑色素瘤转移作用。ELISA及免疫荧光检测转移组织中促炎因子白细胞介素6(interleukin-6,IL-6)、S100钙结合蛋白A8/A9复合物(S100 calcium-binding protein A8/A9 complex,S100A8/A9)、血清淀粉样蛋白A(serum amyloid A,SAA)及纤连蛋白(fibronectin)的表达。免疫组化及Western blotting检测ICT对Exo诱导的转移组织相关蛋白干扰素基因刺激因子(stimulator of interferon gene,STING)、磷酸化STING(p-STING)、TANK结合激酶1(TANK-binding kinase 1,TBK1)和磷酸化的TBK1(p-TBK1)的表达情况。结果表明,所提取的外泌体粒径为(149.33±2.68)nm、多分散系数为(0.192±0.02)、zeta电位为(-32.22±0.50)mV,电镜下呈现茶托样双层膜结构,测得蛋白浓度为(838.66±62.14)μg·mL^(-1)。ICT浓度为5、10和20μmol·L^(-1)时,能够抑制Exo诱导的B16BL6细胞迁移。ICT能够抑制Exo介导的黑色素瘤肺转移,抑制肺组织中促炎因子S100A8/A9、SAA和IL-6的表达,能够抑制转移肺组织的p-STING和p-TBK1的表达。本研究结果表明,ICT能够明显抑制Exo诱导的肿瘤转移,且与STING通路的失活有关。This study investigated the mechanism by which icaritin(ICT)inhibits exosomes-induced lung metastasis of B16 BL6 mouse melanoma cells.The culture supernatant of B16 BL6 cells was collected for extraction of exosomes by ultracentrifugation and their characterization by transmission electron microscopy and Western blotting.Exosomal protein was quantified by BCA.A wound-healing assay was used to determine the effect of ICT on the migratory ability of B16 BL6 cells induced by exosomes.After establishing an experimental melanoma lung metastasis model in C57 BL/6 mice,we used H&E staining to study the ability of ICT to inhibit exosomes-induced melanoma metastasis.Animal experiments were approved by the Ethics Committee of Nanjing University of Chinese Medicine.ELISA and immunofluorescence were used to detect pro-inflammatory factors interleukin 6(IL-6),S100 calcium-binding protein A8/A9 complex(S100 A8/A9),serum amyloid A(SAA)and fibronectin in metastatic tumors.The expression of metastatic tissue-related proteins stimulator of interferon gene(STING),phospho-STING(p-STING),TANK-binding kinase 1(TBK1)and phospho-TBK1(p-TBK1)was detected by immunohistochemistry or Western blotting.The results showed that the particle size of exosomes was 149.33±2.68 nm,the polydispersity index(PDI)was 0.192±0.02,the zeta potential was-32.22±0.50 mV,and the particles had classic tea tray-like membrane structure under TEM.The protein concentration of exosomes was measured to be 838.66±62.14μg·mL^(-1).The results of the cell scratch test showed that ICT can inhibit exosomes-induced migration of B16 BL6 cells at a concentration of 5,10,and 20μmol·L^(-1).In vivo experimental results also showed that ICT can inhibit exosomes-induced metastasis of melanoma to the lungs and can significantly inhibit the expression of pro-inflammatory factors S100 A8/A9,SAA and IL-6 in lung tissue,and inhibit the expression of p-STING and p-TBK1 in metastatic lung tissue.Taken together,these results indicated that ICT can significantly inhibit exosomes-in

关 键 词：淫羊藿素 外泌体 黑色素瘤 肿瘤转移 STING 

分 类 号：R966[医药卫生—药理学]