骨髓间充质干细胞来源的外泌体通过miR-335调控NF-κB通路并减轻大鼠肺缺血再灌注损伤

Exosomes derived from bone marrow mesenchymal stem cells regulate NF-κB pathway and reduce lung ischemia-reperfusion injury in rats by miR-335

本文旨在探讨骨髓间充质干细胞来源的外泌体(exosomes derived from bone marrow mesenchymal stem cells,BMSCs-EXO)对大鼠肺缺血再灌注损伤(ischemia-reperfusioninjury,IRI)的影响及miR-335在其中的作用及机制。通过左肺夹闭60min、开放180 min建立大鼠肺IRI模型。Sprague-Dawley大鼠40只随机分为假手术组(sham)、IRI组、IRI+PBS组、IRI+EXO组和IRI+inhibitor-EXO组(n=8)。Sham组大鼠仅开胸,但不建立肺IRI模型;IRI组建立肺IRI模型,无其他任何处理;其它3组建立肺IRI模型后,于再灌注前分别给予PBS、无任何处理的BMSCs-EXO和经miR-335抑制剂处理的BMSCs-EXO。实验中进行血气分析;再灌注结束后检测肺组织湿/干重比(wet/dry ratio,W/D),以及白介素1β(interleukin-1β,IL-1β)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、髓过氧化物酶(myeloperoxidase,MPO)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)含量;电镜下观察线粒体并记录Flameng评分;光镜下观察肺组织病理学,记录肺损伤评分(lung injury score,LIS);TUNEL法检测细胞凋亡情况,并计算细胞凋亡指数(apoptosis index,AI);RT-qPCR检测miR-335表达,Westernblot检测caspase-3、cleaved-caspase-3、caspase-9、cleaved-caspase-9、NF-κB蛋白表达。结果显示,再灌注后,与sham组相比,IRI组和IRI+PBS组氧合指数、pH、碱剩余(base excess,BE)显著降低,而与IRI+PBS组相比,IRI+EXO组氧合指数、pH、BE显著升高,与IRI+EXO组相比,IRI+inhibitor-EXO组氧合指数、pH、BE显著降低(P<0.05)。与sham组相比,IRI组W/D、IL-1β、TNF-α、MPO、MDA、LIS、AI、Flameng评分及caspase-3、cleaved-caspase-3、caspase-9、cleavedcaspase-9蛋白表达显著升高,NF-κB蛋白表达降低,SOD和miR-335表达显著降低(P<0.05),但IRI组和IRI+PBS组相比无显著差异。与IRI+PBS组相比,IRI+EXO组W/D、IL-1β、TNF-α、MPO、MDA、LIS、AI、Flameng评分以及caspase-3、cleaved-caspase-3、caspase-9、cleaved-caspase-9蛋白表达显著降低,NF-κB蛋白表达升高,SOD和miR-335表达显著升高(P<0.05)。与IRI+EXO组相比,IRI+inhibitor-EXO组中上述指标的变化均反转,但仍优于IRI+PBS组(P<0.05)。上述结果表明,BMSCs-EXO能够上调miR-335,维持线粒体结构稳定,激活NF-κB通路并减轻大鼠肺IRI。

This study aimed to investigate the effect of exosomes derived from bone marrow mesenchymal stem cells(BMSCs-EXO)on lung ischemia-reperfusion injury(IRI)in rats and to explore the role of miR-335.The model of rat lung IRI was established by clipping the hilum of left lung for 60 min and opening for 180 min.Forty Sprague-Dawley rats were randomly divided into sham group,IRI group,IRI+PBS group,IRI+EXO group,and IRI+miR-335 inhibitor EXO(IRI+inhibitor-EXO)group(n=8).Rats in the sham group underwent thoracotomies without IRI.Rats in the IRI group were used to establish IRI model without any additional treat-ment.In the IRI+PBS,IRI+EXO,and IRI+inhibitor-EXO groups,the rats were used to establish IRI model and given PBS,EXO from BMSCs without any treatment,and EXO from BMSCs with miR-335 inhibitor treatment before reperfusion,respectively.Blood gases were analyzed during the experiment.Lung tissue wet/dry ratio(W/D),interleukin 1β(IL-1β),tumor necrosis factorα(TNF-α),myeloperoxidase(MPO),malondialdehyde(MDA),and superoxide dismutase(SOD)were measured at the end of reperfusion.Mito-chondria were observed by electron microscopy and the Flameng scores were counted.Lung histopathology and apoptosis(TUNEL staining)were observed by light microscopy,and the lung injury scores(LIS)and apoptosis index(AI)were detected.The miR-335 expression was detected by RT-qPCR,and the expression of caspase-3,cleaved-caspase-3,caspase-9,cleaved-caspase-9,and NF-κB proteins were detected by Western blot at the end of reperfusion.The results showed that compared with the sham group,the oxygen-ation index,pH,and base excess(BE)were significantly lower in the IRI group and IRI+PBS group after reperfusion,whereas those indices were significantly higher in the IRI+EXO group than those in the IRI+PBS group(P<0.05).Compared with the sham group,there were significant increases in W/D,IL-1β,TNF-α,MPO,MDA,LIS,AI,Flameng score,caspase-3,cleaved-caspase-3,caspase-9,and cleaved-caspase-9,however significant decreases in the SOD,miR-335 and NF-κB in the IRI group(P<0.05).These indices in the IRI and IRI+PBS groups showed no significant differences.Compared with the IRI+PBS group,there were significant decreases in W/D,IL-1β,TNF-α,MPO,MDA,LIS,AI,Flameng score,caspase-3,cleaved-caspase-3,caspase-9,and cleaved-caspase-9,however significant increases in the SOD,miR-335 and NF-κB in the IRI+EXO group(P<0.05).While,the changes of the above mentioned indices were reversed in the IRI+inhibitor-EXO group compared with IRI+EXO group,which were still better than those in the IRI+PBS group(P<0.05).The results suggest that BMSCs-EXO could attenuate lung IRI in rats,activate NF-κB pathway,and maintain mitochondrial stability by up-regulating miR-335.