巨噬细胞LSD1抑制H3K9Me2水平增强动脉粥样硬化中血清炎症因子的表达

Down-regulation of H3K9Me2 Level by LSD1 in Macrophage Enhances the Cytokines Expressions in Atherosclerosis

目的:检测动脉粥样硬化患者巨噬细胞中组蛋白第9位赖氨酸的二甲基化(histone H3 lysine 9 dimethylation,H3K9Me2)的表达并探究其对炎症因子表达的影响及其机制。方法:选择厦门大学附属东南医院收治的动脉粥样硬化患者(n=20)与健康对照人群(n=22)作为研究对象,采用蛋白免疫印迹方法检测巨噬细胞中H3K9Me2的表达,并通过酶联免疫吸附的方法检测其血清炎症因子表达。随后,通过小干扰RNA(small interfering RNA,siRNA)敲减赖氨酸特异去甲基化酶1A(lysine-specific demethylase 1A,LSD1)后,在氧化低密度脂蛋白(oxidized low-density lipoproteins,ox-LDL)诱导的巨噬细胞中结合白免疫印迹和染色质免疫共沉淀的方法检测细胞整体H3K9Me2的表达及炎症因子启动子区H3K9Me2水平。同时,分析巨噬细胞中上清中炎症因子表达变化。结果:动脉粥样硬化病人巨噬细胞中H3K9Me2水平显著下调,并与血清中炎症因子表达呈负相关。在敲减LSD1后,ox-LDL诱导的巨噬细胞中H3K9Me2整体水平以及炎症因子启动子区H3K9Me2水平下调均被抑制。同时,核因子-κB(NF-κB)结合启动子区介导的炎症因子表达增强也被抑制。结论:巨噬细胞中H3K9Me2的表达与动脉粥样硬化病人血清中炎症因子的表达成负相关,而组蛋白去甲基化酶LSD1很可能在其中参与H3K9Me2水平调控,并通过影响NF-κB与启动子区结合调节炎症因子表达。

Objective： This study is to measure the expression of H3K9Me2 in the macrophages of the atherosclerosis patients and investigate the effect and mechanism of H3K9Me2 level on macrophage tumor necrosis factor-α, interleukin-1 β, and interleukin-6 production in the serum of atherosclerosis patients. Methods： The blood samples were obtained from the atherosclerosis patients（n=20）and healthy controls（n=22） in the Affiliated Dongnan Hospital of Xiamen University（PLA 175 th Hospital）.The expression of H3K9Me2 in the macrophages of the atherosclerosis patients was analyzed by western blotting and the tumor necrosis factor-α, interleukin-1β, and interleukin-6 production in the serum was measured by enzyme-linked immunosorbent assay. Sequentially, the expression of H3K9Me2 in the macrophages and in the promoters of tumor necrosis factor-α, interleukin-1β, and interleukin-6 was analyzed by western blotting or chromatin immunoprecipitation in ox-LDL treated macrophages after knocking down LSD1 by siRNA transfection. Additionally, the production of tumor necrosis factor-α, interleukin-1 β, and interleukin-6 was also detected in supernatant of LSD1 knock-down macrophages. Results： the expression of H3K9Me2 in the macrophages of the atherosclerosis patients was greatly down-regulated and inversely correlated with the tumor necrosis factor-α, interleukin-1β, and interleukin-6 production in the serum. Moreover, the reduction of H3K9Me2 levels in the macrophages and in the promoters of tumor necrosis factor-α, interleukin-1β, and interleukin-6 was prevented by LSD1 knock-down in ox-LDL treated macrophages. Meanwhile, the binding of NF-κB in the tumor promoters of necrosis factor-α,interleukin-1β, and interleukin-6 and their production induced by ox-LDL were both inhibited in LSD1 knock-down macrophages.Conclusion： The expression of H3K9Me2 in the macrophages is inversely correlated with the tumor necrosis factor-α, interleukin-1β, and interleukin-6 production in the serum of the atherosclerosis patients and LSD1 probably contributes to the regulation of H3K9Me2 level and further enhances the tumor necrosis factor-α, interleukin-1β, and interleukin-6 production via inhibition of the binding between NF-κB and the promoters.