脐带间充质干细胞外泌体对慢性阻塞性肺疾病大鼠肺部炎症的作用机制探讨

The effect and mechanism of exosomes from umbilical cord mesenchymal stem cells on pulmonary inflammation in chronic obstructive pulmonary disease rats

目的探讨人脐带间充质干细胞外泌体对慢性阻塞性肺疾病(COPD)大鼠肺部炎症的作用及机制。方法将18只SD雄性大鼠随机分为Control组、COPD组、COPD+Exo组,每组6只;COPD组、COPD+Exo组大鼠采用香烟烟熏+脂多糖(LPS)雾化吸入的方法建立COPD大鼠模型,COPD+Exo组经尾静脉注射100μL人脐带间充质干细胞外泌体稀释液(含2×107个外泌体),Control组和COPD组注射等量的磷酸盐缓冲液。干预后1周处死大鼠,收集外周血、肺泡灌洗液(BALF)和肺组织。HE染色观察大鼠肺组织病理改变,全自动血细胞分析仪计数外周血炎性细胞数目,酶联免疫吸附试验(ELISA)检测BALF和血清中白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α水平,qRTPCR检测肺组织中IL-1β、IL-6、TNF-αmRNA的表达,Western blot检测肺组织中Toll样受体4(TLR4)、磷酸化核因子κB(p-NF-κB p65)和核因子抑制蛋白(IκBα)的表达。结果Control组大鼠肺泡结构完整、形态规则;COPD组大鼠肺泡结构紊乱,部分肺泡不规则扩张、融合成肺大疱甚至破裂,肺泡间隔增宽且可见较多炎性细胞浸润。与COPD组比较,COPD+Exo组大鼠肺泡结构较完整,肺泡间隔炎性细胞浸润减少,外周血中炎性细胞减少,BALF、血清及肺组织中IL-1β、IL-6、TNF-α表达水平降低(P<0.05);肺组织TLR4、IκBα蛋白表达水平升高,p-NF-κB p65表达水平降低(P<0.05)。结论人脐带间充质干细胞外泌体通过调控TLR4/核因子κB(NF-κB)信号通路减轻COPD大鼠的肺部炎症,从而发挥肺保护作用。

Objective To investigate the effect and mechanism of human umbilical cord mesenchymal stem cell exosomes(Exo)on pulmonary inflammation in chronic obstructive pulmonary disease(COPD)rats.Methods A total of 18 male SD rats were randomly divided into the control group,the COPD group and the COPD+Exo group with 6 rats in each group.COPD rat model was estalished by giving cigarette smoke and lipopolysaccharide(LPS)inhalation in the COPD group and the COPD+Exo group.Rats in the COPD+Exo group were injected with 100μL of human umbilical mesenchymal stem cell exosome diluent(containing 2×107 exosomes)via tail vein.The control group and the COPD group were given equal amounts of phosphate buffered solution.One week after intervention,rats were killed and the peripheral blood,alveolar lavage fluid(BALF)and lung tissue samples were collected.The histopathological changes in lung tissue of each group were observed using Hematoxylin and Eosin(HE)staining.The number of inflammatory cells in peripheral blood was counted by fully automated hematology analyzer.The serum levels of IL-1β,IL-6 and TNF-αwere detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of IL-1β,IL-6 and TNF-αmRNA in lung tissue were detected by qRT-PCR.Western blot assay was used to detect the protein expression levels of TLR4,p-NF-κB p65 and IκBαin lung tissue.Results In the control group,the structure of pulmonary alveolus was intact and regular,while in the COPD group,the structure of alveolus was disordered,some alveolus expanded irregularly,fused into bullae and even ruptured.The alveolar septa were widened and filled with a large number of inflammatory cells.Compared with the COPD group,the COPD+Exo group had more intact alveolar structure and less infiltration of inflammatory cells in the alveolar septum.The inflammatory cells in peripheral blood decreased.The expression levels of IL-1β,IL-6 and TNF-αin serum and lung tissue were decreased(P<0.05).The expression of TLR4 and IκBαprotein in lung tissue were increased,while the expression of p-NF-κb p65 protein decreased(P<0.05).Conclusion Human umbilical cord mesenchymal stem cell exosomes can alleviate pulmonary inflammation in COPD rats by modulating TLR4/NF-κB signaling pathway,thereby playing a lungprotective effect.