Introduction: The progression to pulmonary fibrosis in late-stage acute respiratory distress syndrome (ARDS) represents a critical clinical challenge, with dysregulation of the mTORC1/autophagy axis implicated in its pathogenesis. Emerging evidence suggests that biomolecular phase separation dynamically modulates signaling pathways in fibrotic diseases. However, whether nutritional metabolites exert protective effects through this mechanism in ARDS remains unknown. This study investigates whether alanyl-glutamine (Ala-Gln) regulates the mTORC1/autophagy axis via phase separation, thereby providing biphasic protection during ARDS injury-fibrosis transition. Methods: A bleomycin-induced mouse model of ARDS-pulmonary fibrosis transition was established, with interventions administered during acute and fibrotic phases. Phase separation events were assessed by transmission electron microscopy and fluorescence labeling. mTORC1 activity was analyzed via co-immunoprecipitation, while autophagic flux was evaluated through LC3-II/lysosome colocalization. In vitro experiments utilized human alveolar epithelial cells (A549) and lung fibroblasts (MRC-5), with HNRNPA1 knockdown to validate phase separation mechanisms. Results: Our findings demonstrate that Ala-Gln intervention exhibits distinct protective effects across ARDS progression stages. During the acute phase, Ala-Gln treatment significantly promoted stress granule (SG) formation, suppressed mTORC1 overactivation, and reduced levels of IL-6 and TNFα. As the disease progressed to the fibrotic phase, Ala-Gln effectively dissolved aberrant protein aggregates, enhanced autophagosome-lysosome fusion efficiency, and decreased expression of α-SMA and COL1A1. Mechanistic studies revealed that Ala-Gln precisely regulates mTORC1/autophagy activity by maintaining HNRNPA1-dependent phase separation boundary stability. Conclusions: Ala-Gln orchestrates biphasic protection in ARDS by fine-tuning the mTORC1/autophagy axis through phase separation - attenuating inflammation during acute injury and promoting degradation of aberrant proteins during fibrotic transition. This study unveils a novel mechanism by which nutritional metabolites regulate cellular homeostasis via phase separation, providing a theoretical framework for stage-specific targeted therapy in ARDS.
Chen et al. (Sun,) studied this question.