M2型巨噬细胞外泌体对小鼠自身免疫性肝炎的保护作用

The protective effect of M2 macrophage-derived exosomes on mouse model of autoimmune hepatitis

目的探究M2型巨噬细胞来源的外泌体(M2 Exos)对刀豆蛋白A(Con A)诱导的小鼠自身免疫性肝炎(AIH)的保护作用。方法采用IL-4(20μg/L)刺激RAW264.7巨噬细胞24 h后提取M2 Exos并用透射电子显微镜、纳米颗粒跟踪分析技术和蛋白质免疫印迹对其进行鉴定;免疫荧光观察RAW264.7巨噬细胞对M2 Exos的摄取情况。将12只C57BL/6J小鼠随机分为PBS组、DiR-M0 Exos组、DiR-M2 Exos组和DiR染料对照组。Con A(15 mg/kg)注射完成1 h后通过尾静脉分组注射PBS溶液、DiR-M0 Exos(100μg)、DiR-M2 Exos(100μg)和DiR染料,活体成像技术观察Exos在AIH小鼠肝、脾、心、肺、肾、肠的分布情况。将20只C57BL/6J小鼠随机分为Control组(PBS溶液)、Con A组(15 mg/kg Con A),M0 Exos组(15 mg/kg Con A+200μg M0 Exos)和M2 Exos组(15 mg/kg Con A+200μg M2 Exos),每组5只。Con A注射完成12 h后处死小鼠,收集外周血和肝组织,全自动生化仪测定血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)水平;苏木精-伊红(HE)染色观察肝脏病理形态变化;实时荧光定量PCR(qPCR)检测肝组织肿瘤坏死因子(TNF)-α和白细胞介素(IL)-6的mRNA表达水平;流式细胞术检测肝脏巨噬细胞亚群变化情况。结果成功诱导并分离了M2 Exos,可被RAW264.7巨噬细胞摄取;经尾静脉注射后,M2 Exos主要在小鼠肝脏和脾脏中蓄积。与Control组相比,Con A组小鼠血清ALT和AST明显升高(P<0.05),肝脏结构紊乱,肝细胞大片坏死,肝组织TNF-α和IL-6 mRNA表达升高(P<0.05),肝脏单核细胞来源巨噬细胞(MoMFs)的浸润增多(P<0.05)。与Con A组相比,M2 Exos组小鼠血清ALT和AST水平显著下降(P<0.05),肝脏坏死明显减轻,肝组织TNF-α和IL-6 mRNA表达降低(P<0.05),肝脏MoMFs的浸润减少(P<0.05)。结论M2 Exos可对小鼠AIH起到保护作用,其作用机制可能与降低肝脏炎性细胞因子的表达及减少对MoMFs的招募有关。

Objective To explore the protective effect of exosomes derived from M2 macrophages(M2 Exos)on concanavalin(Con)A induced autoimmune hepatitis in mice.Methods RAW264.7 macrophages were treated with IL-4(20μg/L)for 24 h to obtain cell culture supernatant containing M2 Exos.M2 Exos were separated by ultracentrifugation and identified by transmission electron microscope,nanoparticle tracking analysis and Western blot assay.Immunofluorescence was used to detect whether M2 Exos were uptaken by RAW264.7 macrophages.Twelve C57BL/6J mice were randomly divided into PBS group,DiR-M0 Exos group,DiR-M2 Exos group and DiR dye control group.PBS solution,DiR-M0 Exos(100μg),DiR-M2 Exos(100μg)and DiR dye were given to corresponding groups through tail veins after the injection of Con A(15 mg/kg)for 1h.The distribution of Exos in liver,spleen,heart,lung,kidney and intestine of mice with autoimmune hepatitis was observed by vivo imaging system.Twenty C57BL/6J mice were randomly divided into control group(PBS solution),Con A group(15 mg/kg Con A),M0 Exos group(15 mg/kg Con A+200μg M0 Exos)and M2 Exos group(15 mg/kg Con A+200μg M0 Exos),5 mice in each group.Peripheral blood and liver tissues were collected 12 h after Con A injection.The serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were determined by automated biochemistry analyzer.The histological profiles of liver tissue were observed by hematoxylin-eosin(HE)staining.The mRNA expression levels of liver TNF-αand IL-6 were detected with quantitative real-time PCR(qPCR).The changes of liver macrophage subpopulations were detected with flow cytometric analysis.Results M2 Exos were successfully induced and isolated,which can be taken up by RAW264.7 macrophages in large quantities.M2 Exos were mainly absorbed by liver and spleen of mice through tail vein injection.Compared with Control group,the serum levels of ALT and AST were significantly increased in Con A group(both P<0.05).The disordered liver structure and increased necrosis areas were found in Con A group.Meanwhile,the mRNA expression of TNF-αand IL-6 in liver increased(both P<0.05).The infiltration of liver monocyte-derived macrophages(MoMFs)increased(P<0.05).Compared with Con A group,the serum levels of ALT and AST were significantly decreased in M2 Exos group(both P<0.05).M2 Exos group also showed lower liver TNF-α,IL-6 mRNA expression levels(both P<0.05)and lower MoMFs infiltration.HE staining showed that the necrosis areas of liver cells were relieved in M2 Exos group.Conclusion M2 Exos plays a protective role in autoimmune hepatitis in model mice,and its mechanism may be related to the decreased production of liver inflammatory cytokines and decreased recruitment of MoMFs.