BACKGROUNDS: While lung-protective ventilation strategies are standard care, effective adjunctive pharmacotherapies for ventilator-induced lung injury (VILI) remain lacking. Disruption of circadian (diurnal) rhythms is implicated in various lung pathologies, and the exploration of circadian regulation in VILI has emerged as a clinically relevant research direction for optimizing mechanical ventilation strategies. We hypothesized that the pulmonary Bmal1/Per2 axis confers protection against VILI by activating the Nrf2 antioxidant pathway.
METHODS: A murine model of VILI was established via high-tidal volume ventilation. Lung epithelial cell-specific Bmal1 knockout mice (Bmal1; SPC-CreERT2) and wild-type littermates were used to define the role of the Bmal1/Per2 axis. Lung injury was assessed histologically, by bronchoalveolar lavage fluid protein, and wet/dry weight ratio. Fibrosis was evaluated after a recovery period using hydroxyproline assay and Masson's trichrome staining. Molecular mechanisms were analyzed by qPCR, western blot, and immunohistochemistry. The specific Nrf2 inhibitor ML385 was employed to validate pathway involvement. fl/fl
RESULTS: VILI significantly disrupted pulmonary circadian rhythms, suppressing rhythmic Bmal1 and Per2 expression. Bmal1 deficiency markedly exacerbated VILI, increasing lung injury scores by approximately 2-fold, alveolar permeability by 1.8-fold, and edema. These mice subsequently developed more severe pulmonary fibrosis. This aggravated phenotype was associated with a blunted activation of the Nrf2-mediated antioxidant response, demonstrated by reduced expression of Nrf2 and its downstream targets HO-1 and NQO1. Pharmacological inhibition of Nrf2 with ML385 in wild-type mice abolished the protective effect, resulting in injury and fibrosis severity comparable to Bmal1-deficient animals.
CONCLUSION: This study establishes a crucial functional link between the circadian clock and oxidative stress in mechanical lung injury, identifying the Bmal1/Per2-Nrf2 axis as a potential target for chronotherapeutic intervention. Notably, this work is the first to define the specific Bmal1/Per2 regulatory module and its direct causal connection with the Nrf2 pathway in mediating VILI-induced pulmonary fibrosis, extending prior observations of general circadian rhythm disruption in lung injury to a mechanistically actionable signaling axis.
