Exosomal miR-21-5p derived from typeⅡalveolar epithelial cells alleviates hyperoxia-induced acute lung injury by regulating epithelial sodium channels in mice
- Journal of Army Medical University Vol. 45, Issue 21, Pages: 2222-2230(2023)
- 作者机构：
1.563000 贵州 遵义，遵义市中医院重症医学科
2.563000 贵州 遵义，遵义医科大学附属医院重症医学科
3.563000 贵州 遵义，遵义医科大学附属医院儿内二科
- 作者简介：
- 基金信息：the Regional Science Fund Project of National Natural Science Foundation of China(81960024)
DOI：10.16016/j.2097-0927.202212027
CLC： R363.22;R394.1;R563.9Received：04 December 2022，
Revised：2023-01-28，
Published：15 November 2023
- 稿件说明：
移动端阅览
Banghai FENG, Yingcong REN, Ping YUAN, et al. Exosomal miR-21-5p derived from typeⅡalveolar epithelial cells alleviates hyperoxia-induced acute lung injury by regulating epithelial sodium channels in mice[J]. Journal of Army Medical University, 2023, 45(21): 2222-2230.
Banghai FENG, Yingcong REN, Ping YUAN, et al. Exosomal miR-21-5p derived from typeⅡalveolar epithelial cells alleviates hyperoxia-induced acute lung injury by regulating epithelial sodium channels in mice[J]. Journal of Army Medical University, 2023, 45(21): 2222-2230. DOI： 10.16016/j.2097-0927.202212027.
摘要
目的
2
探讨Ⅱ型肺泡上皮细胞(type Ⅱ alveolar epithelial cells，AEC-Ⅱ)来源外泌体(exosomes，Exos)-miR-21-5p(miR-21)调控上皮钠离子通道(epithelial sodium channel，ENaC)对高氧性急性肺损伤(hyperoxia-induced acute lung injury，HALI)的保护效应及可能机制。
方法
2
6~8周龄雄性C57BL/6J小鼠60只，其中20只使用差速贴壁离心法提取原代AEC-Ⅱ并培养，采用密度梯度离心法提取AEC-Ⅱ来源外泌体，透射电镜鉴定外泌体形态。余40只小鼠按随机数字表法分为(
n
=10)：常氧(Control)组、高氧(HALI)组、高氧+Exos-miR-21(HALI+miR-21)组和高氧+siPI3K+Exos-miR-21(HALI+siPI3K+miR-21)组。除Control组外，余3组小鼠暴露于95%O
2
的自制高氧饲养箱中构建HALI模型。72 h后RT-qPCR检测肺组织及外泌体miR-21表达；HE染色观察肺组织形态学变化并行肺损伤病理评分，计算肺组织湿/干质量比及肺泡液体清除率(alveolar fluid clearance，AFC)；可见分光光度法测定超氧化物歧化酶(superoxide dismutase，SOD)、丙二醛(malondialdehyde，MDA)及总抗氧化能力(total antioxidant capacity，T-AOC)水平；荧光分光光度法测定活性氧簇(reactive oxygen species，ROS)水平；Western blot检测PTEN、AKT、p-AKT、PI3K及α-ENaC、β-ENaC、γ-ENaC蛋白表达水平。双荧光素酶报告基因实验验证miR-21与PTEN靶向关系。
结果
2
原代AEC-Ⅱ提取的囊泡状物质经鉴定为外泌体，外泌体中miR-21表达显著增高(
P
＜0.05)，PTEN为miR-21靶基因。与Control组比较，HALI组HE染色可见肺组织肺间隔增厚、大量炎症细胞浸润及肺泡萎陷，HALI组、HALI+miR-21组及HALI+siPI3K+miR-21组肺损伤病理评分、肺湿/干质量比、MDA及PTEN蛋白表达升高(
P
＜0.05)；SOD、T-AOC、AFC、miR-21及p-Akt、α-ENaC、β-ENaC、γ-ENaC蛋白表达水平降低(
P
＜0.05)，以HALI+siPI3K+miR-21组最明显。与HALI组比较，HALI+miR-21组HE染色可见肺间隔增厚、炎症细胞浸润明显减少，肺泡萎陷显著减轻；肺损伤病理评分、肺湿/干质量比、MDA及PTEN蛋白表达显著降低(
P
＜0.05)；而SOD、T-AOC、AFC、miR-21及p-Akt、α-ENaC、β-ENaC、γ-ENaC蛋白表达显著升高(
P
＜0.05)。
结论
2
Ⅱ型肺泡上皮细胞来源外泌体miR-21-5p通过靶向PTEN激活PI3K/AKT信号通路调控上皮钠离子通道减轻高氧性急性肺损伤。
Abstract
Objective
2
To investigate the protective effect and possible mechanism of type Ⅱ alveolar epithelial cells (AEC-Ⅱ)-derived exosomal (Exos)-miR-21-5p (miR-21) on the regulation of epithelial sodium channels (ENaC) in hyperoxia-induced acute lung injury (HALI).
Methods
2
Sixty male C57BL/6J mice (6-8 weeks old) were subjected
and 20 of them were used to isolate AEC-Ⅱ using differential wall centrifugation
which were further cultured extract derived exosomes with density gradient centrifugation. Transmission electron microscopy was employed to identify the morphology of obtained exosomes. The 40 remaining mice were randomly divided into (
n
=10): normoxia (Control)
hyperoxia (HALI)
hyperoxia+Exo-miR-21 (HALI+miR-21)
and hyperoxia+siPI3K+miR-21 groups. Except for the Control group
the other 3 groups were exposed to self-made hyperoxia chamber containing 95%O
2
to establish rat model of HALI. In 72 h after exposure
RT-qPCR was performed to detect the miR-21 expression in lung tissue and in AEC-Ⅱ-derived exosomes. HE staining was performed to observe morphological changes in lung tissue and to score the pathology of lung injury
and the wet/dry mass ratio of lung tissue and alveolar fluid clearance (AFC) were calculated. Superoxide dismutase (SOD)
malondialdehyde (MDA) and total antioxidant capacity (T-AOC) were measured by visible spectrophotometry. The levels of reactive oxygen species (ROS) were measured by fluorescence spectrophotometry. The protein levels PTEN
AKT
p-AKT
PI3K and α-ENaC
β-ENaC
γ-ENaC were detected by Western blotting. Dual luciferase reporter gene assay was conducted to verify the relationship between miR-21 and PTEN targeting.
Results
2
The vesicular material extracted from primary cultured AEC-Ⅱ was identified as exosomes
the expression level of miR-21 was significantly increased in the exosomes (
P
<
0.05)
and PTEN was identified as the target gene of miR-21. Compared with the mice of the Control group
thickening pulmonary septum
massive infiltration of inflammatory cells and alveolar atrop
hy were observed
and pathological score of lung injury
lung wet/dry mass ratio
and protein levels of MDA and PTEN were elevated in the the HALI group
HALI+miR-21 group and HALI+siPI3K+miR-21 group (
P
<
0.05). The protein expression levels of SOD
T-AOC
AFC
miR-21 and p-Akt
α-ENaC
β-ENaC
γ-ENaC were reduced in the above 3 groups (
P
<
0.05)
significantly in the HALI+siPI3K+miR-21 group. HE staining showed that no significant differences in thickening of pulmonary septum and inflammatory cell infiltration were observed between the HALI+miR-21 group and the HALI group
but milder alveolar atrophy was seen in the former group. What's more
the former group also got obviously reduced pathological score of lung injury
lower lung wet/dry mass ratio
and decreased MDA and PTEN expression (
P
<
0.05)
but increased protein levels of SOD
T-AOC
AFC
miR-21 and p-Akt
α-ENaC
β-ENaC
γ-ENaC (
P
<
0.05) when compared with the latter group.
Conclusion
2
Exosomal miR-21 derived from AEC-Ⅱ targets PTEN to activate PI3K/AKT signaling pathway
and attenuates hyperoxic acute lung injury by regulating epithelial sodium channels.
关键词
Keywords
references
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