Table_1_Immunomodulatory Effects of Colistin on Macrophages in Rats by Activating the p38/MAPK Pathway.xlsx

Objectives: Colistin has been identified in a Caenorhabditis elegans chemical screening as an immunostimulatory agent that activates the conserved p38/PMK-1 pathway and provides protection against pathogens. Here we aimed to extend those findings to a mammalian model and evaluate the immunomodulatory effects of colistin on rat macrophages.Methods: Macrophages were isolated from Sprague-Dawley (SD) rat. The effects of colistin on the cytokine secretion, phagocytic activity and protein expression were determined by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blotting analysis, respectively. The relative microRNA expression was determined by microarray, and Kyoto Encylopedia of Genes and Genomes (KEGG) was used to identify potential signaling pathways.Results: Our data showed that 5, 10, and 20 µg/ml colistin significantly increased the secretion of TNF-α, while 20 and 5 µg/ml colistin significantly increased the levels of IL-1β and IL-6, respectively. Flow cytometry results showed that the relative mean fluorescence intensity and percentage of phagocytosis in colistin treatment groups were significantly higher compared with the control group, while the increased phagocytosis phenomenon can be blocked by p38 inhibitor. The phagocytic ability of macrophages against Staphylococcus aureus was significantly increased after colistin treatment. Microarray and KEGG pathway analyses revealed that mitogen-activated protein kinase (MAPK), mammalian target of rapamycin (mTOR), chemokine, and B cell receptor were the main pathways involved in the colistin stimulation process. Western blotting analysis demonstrated that the phosphorylated p38 protein level of colistin treatment groups was increased in a dose dependent manner.Conclusions: Present study is the first to demonstrate that colistin had immunomodulatory effects on macrophages in mammals, and the p38/MAPK pathway was involved in such colistin-induced immunomodulatory effect.

研究目标：在本研究中，我们已将粘菌素在秀丽线虫化学筛选中鉴定为一种免疫刺激剂，该剂能够激活保守的p38/PMK-1通路并对抗病原体提供保护。本研究旨在将此发现扩展至哺乳动物模型，并评估粘菌素对大鼠巨噬细胞的免疫调节作用。研究方法：从Sprague-Dawley（SD）大鼠中分离巨噬细胞。通过酶联免疫吸附测定（ELISA）、流式细胞术和蛋白质印迹分析，分别确定粘菌素对细胞因子分泌、吞噬活性和蛋白质表达的影响。通过微阵列确定相对微RNA表达，并使用京都基因与基因组百科全书（KEGG）识别潜在的信号通路。研究结果：我们的数据显示，5、10和20 µg/ml的粘菌素显著增加了TNF-α的分泌，而20和5 µg/ml的粘菌素分别显著增加了IL-1β和IL-6的水平。流式细胞术结果表明，与对照组相比，粘菌素处理组的相对平均荧光强度和吞噬百分比显著升高，而p38抑制剂可以阻断这种增加的吞噬现象。粘菌素处理后，巨噬细胞对金黄色葡萄球菌的吞噬能力显著增强。微阵列和KEGG通路分析揭示了丝裂原活化蛋白激酶（MAPK）、哺乳动物雷帕霉素靶点（mTOR）、趋化因子和B细胞受体是粘菌素刺激过程中的主要通路。蛋白质印迹分析表明，粘菌素处理组的磷酸化p38蛋白水平以剂量依赖性方式增加。研究结论：本研究首次证实了粘菌素对哺乳动物巨噬细胞的免疫调节作用，p38/MAPK通路参与了粘菌素诱导的免疫调节效应。