Luo C, Ji X, Fan J, Hou Z, Wang T, Wu B, Ni C
Department of Occupational Medicine and Environmental Health, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China.
Department of Occupational Medicine and Environmental Health, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China
Toxicol Ind Health. 2016 Sep;32(9):1628-38. doi: 10.1177/0748233715572744. Epub 2015 Mar 10.
To investigate the contributions and underlying molecular mechanisms of annexin A5 toward silica-induced pulmonary fibrosis.
Male C57BL/6 mice were randomly divided into three groups and instilled intratracheally with silica, saline, or air. Mice were euthanized at 3, 7, 14, or 28 days following treatment. Annexin A5 levels in serum and lung tissues were detected by enzyme-linked immunosorbant assay (ELISA) assays or Western blots. The association of annexin A5 levels with silica-induced lung fibrosis was further investigated in the macrophage cell line, RAW264.7. Following exposure of these cells to silica at a concentration of 200 μg/ml for 6 or 12 h, the expression levels of transforming growth factor β1 (TGF-β1), interleukin 1α (IL-1α), Fas ligand (FasL), and their downstream targets were evaluated by Western blots. Furthermore, annexin A5 and FasL were knocked down by small interfering ribonucleic acid (siRNA) and TGF-β1 secretion into the cell culture medium was measured by ELISA assays or Western blots.
Mice treated with silica demonstrated lung fibrosis at 28 days following exposure, whereas, in controls, only mild and transient inflammation was evident at day 3 and day 7 postinstillation and was not present at day 14. Furthermore, silica-exposed mice exhibited significantly (p < 0.05) elevated levels of annexin A5 in serum and lung tissues, relative to control groups. Consistent with these findings, silica exposure of RAW264.7 cells for 6 or 12 h, led to an annexin A5-dependent increase in the expression levels of TGF-β1, IL-1α, FasL, and their downstream target molecules. These silica-induced changes were reversed by siRNA-mediated knockdown of annexin A5, but downregulation of FasL led to increased annexin A5 expression and reduced levels of TGF-β1, IL-1α, and FasL downstream target molecules.
These findings define a role of annexin A5 in promoting macrophage activation via Fas/FasL pathways in silica-induced lung fibrosis.
探讨膜联蛋白A5在二氧化硅诱导的肺纤维化中的作用及潜在分子机制。
将雄性C57BL/6小鼠随机分为三组，分别经气管内滴注二氧化硅、生理盐水或空气。在处理后3、7、14或28天对小鼠实施安乐死。采用酶联免疫吸附测定（ELISA）法或蛋白质印迹法检测血清和肺组织中膜联蛋白A5的水平。在巨噬细胞系RAW264.7中进一步研究膜联蛋白A5水平与二氧化硅诱导的肺纤维化的关系。将这些细胞暴露于浓度为200μg/ml的二氧化硅中6或12小时后，通过蛋白质印迹法评估转化生长因子β1（TGF-β1）、白细胞介素1α（IL-1α）、Fas配体（FasL）及其下游靶点的表达水平。此外，通过小干扰核糖核酸（siRNA）敲低膜联蛋白A5和FasL，并采用ELISA法或蛋白质印迹法测定细胞培养基中TGF-β1的分泌量。
二氧化硅处理的小鼠在暴露后28天出现肺纤维化，而对照组在滴注后第3天和第7天仅出现轻度短暂炎症，第14天则无炎症。此外，与对照组相比，暴露于二氧化硅的小鼠血清和肺组织中膜联蛋白A5水平显著升高（p<0.05）。与这些发现一致，RAW264.7细胞暴露于二氧化硅6或12小时后，导致TGF-β1、IL-1α、FasL及其下游靶分子的表达水平依赖于膜联蛋白A5增加。siRNA介导的膜联蛋白A5敲低可逆转这些二氧化硅诱导的变化，但FasL的下调导致膜联蛋白A5表达增加，TGF-β1、IL-1α和FasL下游靶分子水平降低。
这些发现确定了膜联蛋白A5在二氧化硅诱导的肺纤维化中通过Fas/FasL途径促进巨噬细胞活化的作用。
