Exosomal miR-21-5p derived from typeⅡalveolar epithelial cells alleviates hyperoxia-induced acute lung injury by regulating epithelial sodium channels in mice

Abstract

Objective 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 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%O2 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 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 atrophy 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 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. [Key words] type Ⅱ alveolar epithelial cells , exosomes , microRNA-21-5p, epithelial sodium channels , hyperoxic acute lung injury , PI3K/Akt signaling pathway ,

Keywords

• type ⅱ alveolar epithelial cells
• exosomes
• microrna-21-5p
• epithelial sodium channels
• hyperoxic acute lung injury
• pi3k/akt signaling pathway