Type Ⅱ alveolar epithelial cell-derived exosomal miR-21-5p targeting SKP2 alleviate bronchopulmonary dysplasia
10.3969/j.issn.1006-5725.2024.23.004
- VernacularTitle: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
- Author:
Yan JIANG
1
;
Xiaoqin WANG
;
Hong MEI
;
Xinxin LIU
;
Zhenliang LIAO
;
Kun YU
;
Banghai FENG
;
Song QIN
Author Information
1. 遵义医科大学附属医院小儿内二科(贵州遵义 563000)
- Publication Type:Journal Article
- Keywords:
type Ⅱ alveolar epithelial cells;
exosomes;
micro RNA-21-5p;
bronchopulmonary dys-plasia;
SKP2
- From:
The Journal of Practical Medicine
2024;40(23):3298-3305
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the impact of exosomal(Exos)-miR-21-5p(miR-21)targeting S-phase kinase associated protein 2(SKP2)derived from Type Ⅱ alveolar 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-Ⅱ derived exosomes,while vesicles from AEC-Ⅱ me-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-Ⅱ cells 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 VEGF-A 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-Ⅱ may 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.
