晚期糖基化终末产物受体(receptor for advanced glycation end product, RAGE)是免疫球蛋白受体,能够与多种配体结合,研究发现RAGE参与了非专职吞噬细胞对组蛋白修饰的微米颗粒的吞噬过程,但目前没有研究证明组蛋白是RAGE的配体。该研究对组蛋白分子能否直接激活RAGE及其下游信号通路进行探究。使用共聚焦显微镜和流式细胞仪检测哺乳动物细胞对可溶性组蛋白分子的内化;采用钙黄绿素乙酰氧基甲酯/碘化丙啶染色法检测细胞活性;通过荧光探针2′,7′-二氯二氢荧光素二乙酸酯检测胞内活性氧水平;利用酶联免疫吸附剂测定法(enzyme-linked immunosorbent assay, ELISA)测定炎症因子的释放。结果表明,组蛋白分子能够被野生型HEK293T细胞内化并于胞内溶酶体中聚集,RAGE-敲除细胞则无法内化组蛋白;培养基组蛋白质量浓度在100 μg/mL时对细胞无明显毒性,细胞活性正常;组蛋白能够通过RAGE刺激细胞活性氧水平升高,并释放炎症因子肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)和白细胞介素-6(interleukin-6, IL-6);组蛋白与DNA结合能够增强对RAGE的刺激。以上结果表明,组蛋白能够激活RAGE及其下游炎症通路。
The receptor for advanced glycation end products (RAGE) is an immunoglobulin-like receptor and can bind to multiple ligands. Previous study showed that histone-bound macromolecules were phagocytosed by non-professional phagocytes in a RAGE dependent manner. However, histone has not been reported as a ligand for RAGE. Thus, the present study aims at assessing whether histone molecules can activate RAGE and its downstream signaling pathway. Microscopy and flow cytometry analyses were used to assess internalization of histone in mammalian cells. Cell viability was analyzed by Calcein-AM/propidium iodide staining assay. Cellular reactive oxygen species (ROS) levels were assayed by using a 2′,7′-dichlorodihydrofluorescein diacetate fluorescent probe. The secretion of inflammatory cytokines was evaluated by enzyme-linked immunosorbent assay. Compared to RAGE knock out cells, wild-type mammalian cells efficiently internalize soluble histone molecules in lysosomes, and induce more cellular ROS production and secretion of proinflammatory cytokines including tumor necrosis factor-TNF and interleukin-6 (IL-6). Notably, these effects were enhanced when histone molecules were attached to DNA. Taken together, histone can activate RAGE and its downstream proinflammation pathway.
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