NOD1/RIP2信号通路对巨噬细胞炎性活化的作用

目的以人单核细胞株THP-1为基础,探讨NOD1/受体相互作用蛋白2(RIP2)信号通路对巨噬细胞炎性活化及表型的影响。方法用160 nmo L/L的佛波酯(PMA)将THP-1单核细胞诱导分化成巨噬细胞,分别用10、25和50 mg/L浓度的氧化型低密度脂蛋白(ox-LDL)刺激THP-1源性巨噬细胞24 h,以空白组为对照组。应用RT-PCR法检测NOD1和RIP2的mRNA表达情况;应用Western blot法检测NOD1和RIP2的蛋白表达情况;应用ELISA法检测细胞培养液中单核细胞趋化蛋白1(MCP-1)和巨噬细胞移动抑制因子(MIF)的分泌;应用流式细胞学检测巨噬细胞表面抗原CD16、CD68表达。结果 ox-LDL能以剂量依赖的方式激活THP-1源性巨噬细胞中NOD1/RIP2信号通路,随着ox-LDL刺激浓度的增加,NOD1、RIP2的mRNA和蛋白表达水平升高(P0.05)。NOD1/RIP2信号通路激活后能使细胞培养物上清液中炎症因子MIF和MCP-1的表达增加,随着ox-LDL刺激浓度的增加,MCP-1和MIF的分泌增多(P0.05)。NOD1/RIP2信号通路激活后能改变巨噬细胞表面抗原CD16、CD68的表达,随着ox-LDL刺激浓度的变化,巨噬细胞表面抗原CD16/CD68的平均荧光强度发生变化,其中50 mg/L组能显著下调CD16/CD68的表达(P0.01)。结论巨噬细胞中NOD1/RIP2信号通路能被ox-LDL以剂量依赖的方式激活,NOD1/RIP2信号通路激活后能导致巨噬细胞的炎性活化及其表型变化,这可能是其参与动脉粥样硬化形成和发展过程的主要机制。

Effects of NOD1/RIP2 Signal Pathway on Macrophage Inflammatory Activation

Aim To explore the effects of NOD1/receptor-interacting protein 2(RIP2) signal pathway on macrophage inflammatory activation and phenotype by human monocytic cell line THP-1. Methods Human THP-1 cells were differentiated into macrophages by addition of 160 nmol/l phorbol 12-myristate 13-acetate (PMA) for 24 h. Macrophages were incubated with different concentrations of ox-LDL(0,5, 50 mg/L) for 24 h. The expression of NOD1 and RIP2 was detected by RT-PCR and Western blot. ELISA was used to detect the secretion of monocyte chemotactic protein 1 (MCP-1) and macrophage migration inhibition factor (MIF). FACS was used to detect membrane molecule CDl6/CD68. Results ox-LDL could up-regulate the expression of NOD1/RIP2 signal pathway as a dose-dependent manner in THP-1 derived macrophages. The expression of NOD1, RIP2 mRNA and protein was up-regulated followed the increasing concentrations of ox-LDL. Activation of NOD1 /RIP2 signaling pathway increased the expression of MCP-1 and MIF from the cell culture supernatants. With the increasing concentrations of ox-LDL, the secretion of MCP-1 and MIF increased(P<0.05). The activation of NOD1/RIP2 signaling pathway could change the expression of membrane molecule CD16/CD68. With the different ox-LDL concentrations, the mean fluorescence intensity of CD16/CD68 varied. 50 mg/L group could significantly reduce the expression of CD16/CD68(P<0.01). Conclusion ox-LDL can up-regulate the expression of NOD1/RIP2 signal pathway in a dose-dependent manner in macrophages. NOD1/RIP2 signal pathway enables the macrophage inflammatory activation and polarity switch, which may be the main mechanism in the initiation and progression of atherosclerosis.