姜黄素阻断DAMPs-TLR4信号通路抑制黑色素瘤肺转移的效应及机制

Inhibitory Effect of Curcumin on Lung Metastasis of Melanoma B16 Cells via Blocking DAMPs-TLR4 Signal Pathway

目的探讨姜黄素抑制黑色素瘤细胞DAMPs-TLR4信号通路的激活,从而抑制黑色素瘤肺转移的效应及机制。方法体外培养小鼠黑色素瘤B16细胞,制备肿瘤损伤分子(DTC-Ms)模拟肿瘤微环境中DAMPs分子混合培养,并加入最佳工作浓度姜黄素处理,CCK8细胞活性试剂盒检测细胞活性;CFSE标记B16细胞,尾静脉注射接种,分别在接种后6h和24h取肺组织做冰冻切片,观察各组细胞在肺部聚集和对肺组织侵袭情况;接种后15d解剖小鼠取肺,观察B16细胞在肺表面形成转移结节数量差异;Zymography法检测肿瘤细胞分泌MMP-9水平;Western blot检测肿瘤细胞TLR4信号通路的关键分子表达。结果姜黄素能有效逆转DTC-Ms刺激所致的细胞活性增强及体内转移能力增强的效应,抑制DTC-Ms刺激所致的肿瘤细胞MMP-9和Pro-MMP-9分泌增多的生物学效应,并能明显抑制DTC-Ms刺激下肿瘤细胞TLR4信号通路的激活。结论姜黄素能阻断DAMPs对黑色素瘤细胞TLR4信号通路的激活,抑制实验性黑色素瘤肺转移。

Objective To investigate the inhibitory effect of curcumin on the lung metastasis of melanoma by suppressing the activation of DAMPs-Toll-like receptor 4（TLR4）signal in melanoma cells and the underlying mechanisms.Methods Mela-noma B1 6 cells were treated with DTC-Ms in vitro to simulate the in vivo microenvironment of cells cultured with DAMPs.The tumor cells were then treated or not with curcumin at the best working concentration.Cell viability was measured by CCK-8 cytotoxicity assay kit.The accumulation of B1 6 cells in lung and their invasion to lung tissue were observed after in-jection of B16 cells labeled with CFSE into mice via the tail vein,removal of the lungs 6 and 24 h later and preparation of frozen lung sections.The metastatic loci formed by B1 6 cells in lung were observed 1 5 days after inoculation.Zymography was used to analyze the activity of MMP-9 and Western blotting to detect the key molecules in the TLR4 signal pathway.Results Curcumin could significantly inhibit the DTC-Ms-induced increase of the invasion ability of tumor cells,down-regulate the activities of MMP-9 and Pro-MMP-9 and suppress the activation of TLR4 signal pathway in the presence of DTC-Ms.Conclusion Curcu-min may inhibit the lung metastasis of melanoma cells by abrogating DAMPs-induced activation of TLR4 signal pathway.