M2型巨噬细胞来源的外泌体对类风湿关节炎小鼠的作用及其机制研究

Effect and mechanism study of M2 macrophages-derived exosomes in treatment of collagen induced rheumatoid arthritis mouse

摘要:

目的 探讨M2型巨噬细胞来源的外泌体（M2-Exo）对胶原诱导的类风湿关节炎（RA）小鼠的作用及可能机制。方法 分离提取C57BL/6J小鼠骨髓来源的巨噬细胞（BMDMs），收集白细胞介素-4（IL-4）诱导的M2型巨噬细胞培养的上清液，用超速离心法分离出M2-Exo。使用透射电子显微镜（TEM）和动态光散射（DLS）观察M2-Exo的形态结构及粒径分布；采用细胞免疫荧光染色及Western blotting法检测M2-Exo处理的M1型巨噬细胞的表面标志物诱导型一氧化氮合酶（iNOS）、精氨酸酶（Arginase）的表达。复制胶原诱导的DBA/1小鼠RA模型，随机分为模型组和M2-Exo组，以正常DBA/1小鼠为对照组。M2-Exo组在首次免疫后第16天及第26天关节腔注射M2-Exo（100 μg/只），发病全过程监测关节炎病变并评分，于第42天安乐处死小鼠。对3组小鼠左后足拍照并测定厚度；采用酶联免疫吸附试验（ELISA）检测小鼠关节中促炎症细胞因子白细胞介素-1β（IL-1β）、肿瘤坏死因子ɑ（TNF-ɑ）和白细胞介素-6（IL-6）的表达；采用RT-PCR检测3组小鼠关节中M1型和M2型巨噬细胞的表面标志物iNOS2、CD86、Arginase、CD206 mRNA的相对表达量。结果 M2-Exo为类圆形、有完整的包膜的囊状结构，粒径均值为44.1 nm。M2-Exo处理24 h后的M1型巨噬细胞表达Arginase，不表达iNOS。在RA发病过程中，M2-Exo组小鼠平均关节炎指数（AI）低于模型组（P <0.05）。与模型组比较，模型复制第42天M2-Exo组小鼠的足趾关节红肿不明显，活动障碍程度不高，差异无统计学意义（P >0.05）；M2-Exo组小鼠关节IL-1β、TNF-ɑ和IL-6水平均降低（P <0.05）。M2-Exo组小鼠关节组织中iNOS和CD86 mRNA的相对表达量低于模型组（P <0.05），而CD206和Arginase mRNA的相对表达量高于模型组（P <0.05）。结论 M2-Exo可能通过重编程炎症M1型巨噬细胞转换为M2型抗炎的巨噬细胞，以缓解RA病情。

Abstract:

Objective To explore the physiological therapeutic actions and its mechanism of M2-derived exosome on the collagen-induced rheumatoid arthritis (RA) mouse.Methods The C57BL/6J mouse bone marrow-derived macrophages (BMDMs) were separated. The supernatant of IL-4 induced M2 macrophage were collected, and the M2-Exo were collected by ultracentrifugation. The transmission electron microscope (TEM) and dynamic light scattering (DLS) were used to observe the morphological structure and particle size distribution of M2-Exo. The cell immunofluorescence and Western blotting were used to detect the transformation of M1-type macrophages to M2-type macrophages after M2-Exo treatment. The collagen-induced IA model of DBA/1 mice were randomly divide into the model group and M2-Exo group, normal DBA/1 mice were as the control group. M2-Exo (100 μg/mouse) was injected into the joint cavity of the M2-Exo group on d16 and d26 after the first immunization. The arthritis lesions were monitored and scored during the whole course of the onset. The mice were euthanized on d42. The left hind feet of each group of mice were photographed and the thickness was measured. The pro-inflammatory cytokines interleukin 1β (IL-1β), tumor necrosis factor ɑ (TNF-ɑ) and interleukin 6 (IL-6) expression in the arthritis lesions were defined by ELISA. The relative expression of the surface markers iNOS, CD86, Arginase, and CD206 mRNA of M1 and M2 macrophages in the joints was detected by RT-PCR.Results M2-Exo was a round-like, cyst-like structure with a complete envelope, with an average particle size of 44.1 nm. M1 macrophages was treated with M2-Exo for 24 h, arginase was expressed but not iNOS. During the onset of RA, the average arthritis index (AI) of the M2-Exo group was significantly lower than that in the model group (P < 0.05). Compared with the model group, the toe joints of the model M2-Exo group on d42 were not swollen and the toe joints were not high, and the difference was not statistically significant (P > 0.05). The mice in the M2-Exo group had no significant difference (P > 0.05). The levels of TNF-ɑ and IL-6 were significantly reduced (P < 0.05). The relative expression levels of M1 marker iNOS and CD86 in the joint tissues of the M2-Exo group were significantly lower than those in the model group (P < 0.05), while the relative expression levels of CD206 and arginase were significantly higher than those in the model group (P < 0.05).Conclusion M2-Exo may reprogram inflammatory M1 macrophages into M2 anti-inflammatory macrophages to alleviate the condition of RA.