Ⅱ型肺泡上皮细胞来源外泌体miR-21-5p靶向SKP2缓解支气管肺发育不良

TypeⅡalveolar epithelial cell⁃derived exosomal miR⁃21⁃5p targeting SKP2 alleviate bronchopulmonary dysplasia

目的探讨来源于Ⅱ型肺泡上皮细胞(typeⅡalveolar epithelial cells,AEC-Ⅱ)外泌体(exosomal,Exos)-miR-21-5p(简称miR-21)靶向S期激酶相关蛋白2(S-phase kinase associated protein 2,SKP2)对支气管肺泡发育不良(bronchopulmonary dysplasia,BPD)的保护效应及作用机制。方法使用60只6~8周龄的SD大鼠,其中30只通过差速贴壁离心法提取原代AEC-Ⅱ并进行培养。密度梯度离心法用于获得AEC-Ⅱ来源的外泌体,密度梯度离心法用于提取AEC-Ⅱ培养基中囊泡,并通过透射电镜及粒径分析对其验证,双荧光素报告基因实验以确认miR-21与SKP2的靶向关系。剩余的30只大鼠按照3∶1的雌雄比例混合,以便于进行怀孕检测。将这些新生小鼠随机分为4个不同的实验组:空气对照组(Con组)、高氧处理组(BPD+PBS组)、接受高氧和外泌体治疗的小鼠(BPD+Exos-miR-21组),以及高氧联合外泌体miR-21抑制剂处理组(BPD+Exos-AV-miR-21组)。新生SD大鼠将暴露于85%的氧气环境中,以此建立BPD模型。经过14 d高氧处理后,使用RTq-PCR技术检测肺组织和外泌体中miR-21的表达水平。肺组织经HE染色观察其病理学变化,并计算了平均肺泡线性截距(MLI)和放射状肺泡计数(RAC)。分光光度法测定超氧化物歧化酶(SOD)、丙二醛(MDA)和总抗氧化能力(T-AOC)的水平,荧光分光光度法测定活性氧簇(ROS)水平。此外,利用Western blot技术检测了SKP2、NR2F2和VEGF-A蛋白的表达水平。结果电镜及粒径分析结果显示AEC-Ⅱ细胞提取的小泡结构物质属于外泌体,miR-21在外泌体中表达显著上调(P<0.01),双荧光素酶基因报告实验证实SKP2为miR-21的作用靶标。与Con组比较,BPD+PBS组及BPD+Exos-AVmiR-21组肺组织HE可见肺组织结构紊乱,肺泡增大、简化,ROS、MDA、MLI增高及SKP2蛋白表达升高(P<0.01);而RAC、SOD、T-AOC、miR-21下调及NR2F2、VEGF-A蛋白表达降低(P<0.01);与BPD+PBS组比较,BPD+Exos-miR-21组肺组织HE可见无肺泡数目增加,肺泡简化程度好转,同时MLI、ROS、MDA降低及SKP2蛋白表达降低(P<0.01);而ROC、SOD、T-AOC、miR-21上调及NR2F2、VEGF-A蛋白表达升高(P<0.01)。结论AEC-Ⅱ来源的Exos-miR-21可能靶向SKP2通过促进NR2F2、VEGF-A蛋白表达抑制氧化应激促进肺泡发育改善BPD。

Objective To investigate the impact of exosomal(Exos)-miR-21-5p(miR-21)targeting S-phase kinase associated protein 2(SKP2)derived from TypeⅡalveolar epithelial cells(AEC⁃Ⅱ)on the patho⁃genesis of bronchopulmonary dysplasia(BPD).Methods A total of 60 SD rats aged 6~8 weeks were utilized in this study,with 30 of them subjected to extraction and culture through differential adherent centrifugation.Density gradient centrifugation was employed for the isolation of AEC⁃Ⅱderived exosomes,while vesicles from AEC⁃Ⅱme⁃dium were extracted using density gradient centrifugation.These isolates were subsequently confirmed by transmission electron microscopy and particle size analysis,and the targeting relationship between miR-21 and SKP2 was validated through dual-fluorescein reporter gene assay.The remaining 30 mice were combined in a male-to-female ratio of 3∶1 to facilitate pregnancy testing.These neonatal mice were randomly assigned into four experimental groups:air control group(Con group),hyperoxia group(BPD+PBS group),hyperoxia-treated mice receiving exosomes(BPD+Exos-miR-21 group),and hyperoxia combined with exosome miR-21 inhibitor treatment group(BPD+Exos-AV-miR-21 group).Neonatal SD rats will be exposed to 85%oxygen to establish a BPD model.Follow⁃ing 14 days of high oxygen treatment,the expression levels of miR-21 in lung tissues and exosomes will be assessed using RT-qPCR.HE staining will be employed to observe pathological changes in lung tissue,while mean alveolar linear intercept(MLI)and radial alveolar count(RAC)will be calculated.Superoxide dismutase(SOD),malondialdehyde(MDA),and total antioxidant capacity(T-AOC)levels will be determined spectrophoto⁃metrically,whereas reactive oxygen species(ROS)levels will be measured via fluorescence spectrophotometry.Additionally,Western blot analysis will assess the expression levels of SKP2,NR2F2,and VEGF-A proteins.Results The results obtained from electron microscopy and particle size analysis demonstrated that the vesicle structure isolated from AEC⁃Ⅱcells corresponded to exosomes.Moreover,there was a significant upregulation of miR-21 expression in exosomes(P<0.01).Subsequently,the dual luciferase gene reporter assay con⁃firmed SKP2 as the target of miR-21.Comparative analysis revealed that compared to the Con group,both BPD+PBS and BPD+Exos-AV-miR-21 groups exhibited disordered lung tissue structure with enlarged and simpli⁃fied alveoli,increased levels of ROS,MDA,and MLI along with elevated expression of SKP2 protein(P<0.01).Conversely,RAC,SOD,T-AOC levels were downregulated alongside miR-21 expression while NR2F2 and VEGFA protein expressions decreased significantly(P<0.01).In contrast to the BPD+PBS group,the number of alveoli without alveoli increased in the BPD+Exos-miR-21 group leading to improved degree of alveolar simplification accom⁃panied by reduced MLI,ROS,MDA levels as well as decreased SKP2 protein expression(P<0.01).Addition⁃ally ROC,SOD,T-AOC,and miR-21 expressions were upregulated while NR2F2and VEGF-A expressions were increased(P<0.01).Conclusions The exosomal miR-21 derived from AEC⁃Ⅱmay potentially target SKP2,thereby inhibiting oxidative stress and promoting alveolar development.Consequently,it can improve BPD by enhancing the protein expression of NR2F2 and VEGF-A.