目的 探讨M2-样巨噬细胞源性外泌体(M2 macrophage-derived exosomes,M2-Exos)对小鼠急性肝衰竭(acute liver failure,ALF)的保护作用及其分子机制.方法 选取健康雄性Balbc(6～8周龄)小鼠20只,采用D-氨基半乳糖/脂多糖(D-galactosamine/lipopolysaccharide,D-GalN/LPS)处理,构建ALF动物模型.将小鼠分为正常对照组、M0-Exos+急性攻击组(M0-Exos+D-GalN/LPS组)、M1-Exos+急性攻击组(M1-Exos+D-GalN/LPS组)、M2-Exos+急性攻击组(M2-Exos+D-GalN/LPS组),每组5只.分别收集M0-、M1-、M2-样巨噬细胞培养上清,采用超速离心法提取外泌体(Exosomes,Exos)并鉴定;分别将M0-Exos、M1-Exos、M2-Exos回输给正常小鼠,然后给予D-GalN/LPS攻击.比较各组小鼠的肝损害程度.采用定量聚合酶链反应(quantitative polymerase chain reaction,qPCR)及Western blot检测分析各组小鼠肝组织中自噬信号分子的表达水平.结果 Exos呈典型的杯状结构,囊泡直径约130 nm,Exos标志物CD63、TSG101、HSP70、Syntenin达呈阳性.病理结果显示,M2-Exos+D-GalN/LPS组小鼠的肝组织损害较M0-Exos+D-GalN/LPS组、M1-Exos+D-GalN/LPS组小鼠明显减轻,其相应病理评分明显降低.Western blot结果显示,M2-Exos+D-GalN/LPS组小鼠肝组织中自噬标志物自噬相关蛋白16样1(autophagy related 16 like 1,ATG16L1)、磷酸化的ATG16L1(p-ATG16L1)及微管相关蛋白轻链3B(microtubule-associated protein light chain 3B,LC3B)的表达较M0-Exos+D-GalN/LPS组、M1-Exos+D-GalN/LPS组小鼠明显增强.qPCR结果显示,M2-Exos+D-GalN/LPS组小鼠肝组织中ATG5 和ATG16L1的mRNA水平明显升高.结论 巨噬细胞源性Exos分离成功,M2-Exos可能通过促进自噬保护小鼠抵抗ALF.
Study on the protective effect and mechanism of M2 macrophage-derived exosomes on acute liver failure in mice
Objective To explore the protective effect of M2 macrophage-derived exosomes(M2-Exos)on acute liver failure(ALF)and its molecular mechanism.Methods A total of 20 healthy male Balbc mice(6-8 weeks)were selected,and were treated with D-galactosamine/lipopolysaccharide(D-GalN/LPS)to construct an ALF animal model.Mice were divided into the normal control group,M0-Exos+acute attack group(M0-Exos+D-GalN/LPS group),M1-Exos+acute attack group(M1-Exos+D-GalN/LPS group)and M2-Exos+acute attack group(M2-Exos+D-GalN/LPS group),with five mice in each group.The culture supernatants of M0-,M1-and M2-macrophages were collected respectively,and exosomes(Exos)were extracted by ultracentrifugation and identified.M0-Exos,M1-Exos and M2-Exos were transfused to normal mice respectively,and then mice were challenged with D-GalN/LPS.The degree of liver damage of mice in each group was compared.The expression level of autophagy signal molecules in liver tissue of mice in each group were analyzed by quantitative polymerase chain reaction(qPCR)and Western blot.Results Exos was a typical cup-shaped structure with a vesicle diameter of about 130 nm,and Exos markers the tetraspanin protein family members(CD63),tumor susceptibility gene 101(TSG101),heat shock protein 70(HSP70)and Syntenin were positive.The pathological results showed that the damage of liver tissue in M2-Exos+D-GalN/LPS group was significantly less than those in M0-Exos+D-GalN/LPS group and M1-Exos+D-GalN/LPS group,and its corresponding pathological score was significantly reduced.Western blot results showed that the expression of autophagy markers autophagy related 16 like 1(ATG16L1),phosphorylated-ATG16L1 and microtubule-associated protein light chain 3B(LC3B)in the liver tissue of M2-Exos+D-GalN/LPS group was significantly higher than those of M0-Exos+D-GalN/LPS group and M1-Exos+D-GalN/LPS group.The qPCR results showed that the mRNA levels of autophagy markers autophagy related 5(ATH5)and ATG16L1 in the liver tissue of M2-Exos+D-GalN/LPS group were significantly increased.Conclusions Exos derived from macrophages are successfully isolated,and M2-Exos can protect mice from ALF induced by promoting autophagy.
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