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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

张冰¹, 孟超², 康继宇¹, 周华成^2,*

¹哈尔滨医科大学附属第四医院疼痛科，哈尔滨 150001；²青岛大学附属医院疼痛诊疗科，青岛 266000

摘要

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-reperfusion injury, IRI)的影响及miR-335在其中的作用及机制。通过左肺夹闭60 min、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，Western blot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、cleaved-caspase-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。

关键词： 骨髓间充质干细胞; 外泌体; 肺缺血再灌注损伤; 线粒体; miR-335; NF-κB

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

ZHANG Bing¹, MENG Chao², KANG Ji-Yu¹, ZHOU Hua-Cheng^2,*

¹Department of Pain, Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China；²Department of Pain, Affiliated Hospital of Qingdao University, Qingdao 266000, China

Abstract

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 treatment. 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 factor α (TNF-α), myeloperoxidase (MPO), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured at the end of reperfusion. Mitochondria 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 oxygenation 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.

Key words: bone marrow mesenchymal stem cells; exosomes; lung ischemia-reperfusion injury; mitochondria; miR-335; NF-κB

收稿日期：　　录用日期：

通讯作者：周华成　　E-mail: zhouhuacheng@163.com

DOI: 10.13294/j.aps.2024.0024

引用本文：

张冰, 孟超, 康继宇, 周华成. 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[J]. 生理学报 2024; 76 (2): 247-256.

ZHANG Bing, MENG Chao, KANG Ji-Yu, ZHOU Hua-Cheng. Exosomes derived from bone marrow mesenchymal stem cells regulate NF-κB pathway and reduce lung ischemia-reperfusion injury in rats by miR-335. Acta Physiol Sin 2024; 76 (2): 247-256 (in Chinese with English abstract).