AECII-derived miR-21a-5p exosomes alleviate HALI via targeting and regulating PGAM5-mediated necroptosis

Hyperoxic Acute Lung Injury (HALI) is a serious complication of prolonged high-concentration oxygen therapy, primarily leading to Acute Respiratory Distress Syndrome (ARDS), which primarily affects alveolar epithelial cells (AECs). Exosomes (Exos) derived from type II alveolar epithelial cells (AEC IIs) play a crucial role in lung protection through their contained microRNAs (miRNAs). Previous research has established miR-21a-5p as a key player in pulmonary defense mechanisms. In this study, we utilized a C57BL/6 mouse model of HALI established by hyperoxic conditions (FiO₂ > 90 %) to demonstrate a gradual decrease in miR-21a-5p levels concomitant with an increase in PGAM5 levels with prolonged hyperoxia exposure. Exosomal transcriptome sequencing suggested significant downregulation of miR-21a-5p expression in hyperoxia-stimulated AECII exosomes. We employed dual-luciferase reporter assays and Chromatin Isolation by RNA Purification (ChIRP) to confirm the direct interaction between miR-21a-5p and PGAM5. AECII-derived exosomal miR-21a-5p effectively attenuated lung injury and inhibited the expression of proteins associated with PGAM5-mediated necroptosis (RIPK1, RIPK3, p-MLKL). Furthermore, in vitro assays using MLE-12 cells confirmed that AECII-derived exosomal miR-21a-5p intervention reversed the elevated levels of necroptotic apoptotic proteins in hyperoxia-stimulated MLE-12 cells. These findings collectively demonstrate that AECII-derived exosomal miR-21a-5p inhibits necroptosis pathway activity by modulating PGAM5, thereby exerting lung-protective effects. Therefore, exosomal miR-21a-5p may serve as a novel therapeutic target for attenuating HALI via modulation of the PGAM5-mediated necroptotic pathway.