铁过载通过 ROS/乙酰化-p53 通路使巨噬细胞极化向促炎表型。
Iron overloaded polarizes macrophage to proinflammation phenotype through ROS/acetyl-p53 pathway.
机构信息
Chongqing Medical University, Chongqing, China.
出版信息
Cancer Med. 2018 Aug;7(8):4012-4022. doi: 10.1002/cam4.1670. Epub 2018 Jul 10.
PURPOSE
Macrophages play critical roles in inflammation and wound healing and can be divided into two subtypes: classically activated (M1) and alternatively activated (M2) macrophages. Macrophages also play important roles in regulating iron homeostasis, and intracellular iron accumulation induces M1-type macrophage polarization which provides a potential approach to tumor immunotherapy through M2 tumor-associated macrophage repolarization. However, the mechanisms underlying iron-induced M1 polarization remain unclear.
METHODS
Western blotting, qRT-PCR, and flow cytometry were used to detect the polarization indexes in RAW 264.7 murine macrophages treated with iron, and Western bloting and qRT-PCR were used to detect p21 expression. The compound 2,7-dichlorofluorescein diacetate was used to measure reactive oxygen species (ROS) levels in macrophages after iron or N-acetyl-l-cysteine (NAC) treatment. The p300/CREB-binding protein (CBP) inhibitor C646 was used to inhibit p53 acetylation, and Western bloting, qRT-PCR, and immunofluorescence were used to detect p53 expression and acetylation. BALB/c mice were subcutaneously injected with H22 hepatoma cells, and macrophage polarization status was investigated after tail intravenous injection of iron. Immunohistochemical staining was used to evaluate the protein expression of cluster of differentiation 86 (CD86) and EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80) in the subcutaneous tumors.
RESULTS
Iron overload induced M1 polarization by increasing ROS production and inducing p53 acetylation in RAW cells, and reduction in ROS levels by NAC repressed M1 polarization and p53 acetylation. Inhibition of acetyl-p53 by a p300/CBP inhibitor prevented M1 polarization and inhibited p21 expression. These results showed that high ROS levels induced by iron overload polarized macrophages to the M1 subtype by enhancing p300/CBP acetyltransferase activity and promoting p53 acetylation.
目的
巨噬细胞在炎症和伤口愈合中发挥关键作用，可分为两种亚型：经典激活（M1）和交替激活（M2）巨噬细胞。巨噬细胞在调节铁平衡方面也起着重要作用，细胞内铁积累诱导 M1 型巨噬细胞极化，这为通过 M2 肿瘤相关巨噬细胞再极化进行肿瘤免疫治疗提供了一种潜在方法。然而，铁诱导 M1 极化的机制尚不清楚。
方法
用铁处理 RAW 264.7 鼠巨噬细胞后，采用 Western blot、qRT-PCR 和流式细胞术检测极化指数，Western blot 和 qRT-PCR 检测 p21 表达。用 2,7-二氯二氢荧光素二乙酸酯检测铁或 N-乙酰-L-半胱氨酸（NAC）处理后巨噬细胞内的活性氧（ROS）水平。用 p300/CREB 结合蛋白（CBP）抑制剂 C646 抑制 p53 乙酰化，用 Western blot、qRT-PCR 和免疫荧光法检测 p53 表达和乙酰化。用 H22 肝癌细胞皮下注射 BALB/c 小鼠，尾静脉注射铁后观察巨噬细胞极化状态。用免疫组化染色法检测皮下肿瘤中 CD86 和 EGF 样模块包含粘蛋白样激素受体样 1（F4/80）的蛋白表达。
结果
铁过载通过增加 ROS 产生和诱导 RAW 细胞中 p53 乙酰化诱导 M1 极化，NAC 降低 ROS 水平抑制 M1 极化和 p53 乙酰化。p300/CBP 抑制剂抑制乙酰化 p53 可防止 M1 极化并抑制 p21 表达。这些结果表明，铁过载引起的高 ROS 水平通过增强 p300/CBP 乙酰转移酶活性和促进 p53 乙酰化，将巨噬细胞极化为 M1 亚型。
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