Transcriptomic profile of trametinib on LPS-mediated gene expression in murine peritoneal macrophages

To determine the underlying mechanisms by which trametinib affected LPS-induced inflammatory response in macrophages, we performed microarrays to define the global gene expression in murine peritoneal macrophages treating with trametinib or vehicle followed by LPS stimulation. We injected three mice with thioglycollate broth intraperitoneally. Three days later, the mice were injected with 5 ml of cold PBS into the peritoneal cavity and gently massaged. Then, the peritoneal fluid was collected, centrifuged, and the cell pellet was resuspended in DMEM supplemented with 10% FBS and cultured in 6-well-plate at 37℃. After adhering for 1h, the cells were gently washed with warm PBS for 3 times to remove non-adherent cells and then cultured with fresh medium. After additional 24h of culture, these cells were treated with trametinib (100nM) or vehicle (DMSO) for 2h, and then challenged by LPS (100ng/ml) for 12h.

为明确曲美替尼（trametinib）调控巨噬细胞脂多糖（LPS）诱导炎症反应的潜在分子机制，我们采用基因芯片（microarrays）技术分析经曲美替尼或溶剂处理后再经脂多糖刺激的小鼠腹腔巨噬细胞的全基因表达谱。我们向3只小鼠腹腔注射硫代乙醇酸钠肉汤（thioglycollate broth），3天后向小鼠腹腔注入5mL预冷磷酸盐缓冲液（PBS）并轻轻按摩腹腔。随后收集腹腔灌洗液，经离心后将细胞沉淀重悬于添加10%胎牛血清（FBS）的达尔伯克改良伊格尔培养基（DMEM）中，接种于6孔板并置于37℃培养箱培养。细胞贴壁1小时后，用预热PBS轻柔洗涤3次以去除未贴壁细胞，更换新鲜培养基继续培养。继续培养24小时后，用100nM曲美替尼或溶剂二甲基亚砜（DMSO）处理细胞2小时，随后以100ng/mL脂多糖（LPS）刺激细胞12小时。