淫羊藿素抑制外泌体介导的黑色素瘤肺转移及机制初探

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...

Inhibition of exosomes-mediated melanoma metastasis by icaritin

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-induced tumor metastasis,and the inhibition is related to the inactivation of STING in metastatic foci.