Background The pathogenesis of acute lung injury (ALI) induced by high-altitude hypobaric hypoxia remains incompletely understood, necessitating reliable animal models to investigate its underlying mechanisms. Objective To establish a novel murine model of ALI by combining hypobaric hypoxia with exercise stress. Methods A hypobaric hypoxia chamber was

used to simulate a high-altitude low-oxygen environment, and an animal treadmill was employed to mimic the exercise state of mice. A total of 100 C57BL/6J mice were randomly divided into control group, and 12 h, 24 h, 48 h, and 72 h groups, with 20 mice in each group. Mice were placed on the animal treadmill inside the hypobaric hypoxia chamber, where the simulated altitude was set to 8 000 meters. The treadmill speed was adjusted as follows: low speed at 4 m/min and high speed at 6 m/min, alternating in a high-low speed cycle mode, with an acceleration time of 10 seconds. After 1 hour of exercise, mice rested for 20 minutes. The 12 h group underwent a total of 6 hours of exercise followed by rest, while other experimental groups performed 6 hours of daily exercise with the remaining time allocated for rest. At 12 h, 24 h, 48 h, and 72 h post-treatment, mice were removed from the chamber to observe behavioral states, physiological manifestations, and survival outcomes under simulated high-altitude exercise conditions. After euthanasia, lung tissue pathological changes and water content were examined. Bronchoalveolar lavage fluid (BALF) protein content, pulmonary oxidative stress markers (malondialdehyde, MDA; superoxide dismutase, SOD), and serum inflammatory cytokines (interleukin-1 IL-1, interleukin-6 IL-6, tumor necrosis factor-α TNF-α) were also analyzed. Results The survival rates of the control group, 12 h, 24 h, 48 h, and 72 h groups were 100%, 100%, 90%, 80%, and 50%, respectively. Hematoxylineosin (HE) staining of lung tissues revealed normal alveolar septa in the control group, with no evidence of widened alveolar septa, inflammatory cell infiltration, alveolar collapse, or alveolar hemorrhage. Mild alveolar septal thickening and mild inflammatory cell infiltration were observed in the 12 h and 24 h groups, without alveolar hemorrhage. In contrast, the 48 h and 72 h groups exhibited significant alveolar septal thickening, extensive inflammatory cell infiltration, and alveolar hemorrhage. The lung injury scores (Smith scores) in the 24 h, 48 h, and 72 h groups were significantly higher than that in the control group (all P < 0.001). MDA levels in the 24 h, 48 h, and 72 h groups were elevated compared to the control group (P < 0.05), with the highest level in the 72 h group (P < 0.001). SOD content in the 24 h, 48 h, and 72 h groups was significantly lower than that in the control group (all P < 0.05), reaching its lowest in the 72 h group (P < 0.001). Bronchoalveolar lavage fluid (BALF) protein concentrations in the 24 h, 48 h, and 72 h groups were increased compared to the blank control group (all P < 0.001), with the highest concentration in the 72 h group. Serum inflammatory cytokine IL-1 levels in the 24 h, 48 h, and 72 h groups were significantly higher than those in the control group (all P < 0.001). Similarly, sera IL-6 levels in these groups were markedly elevated compared to the control group (all P < 0.001). TNF- α levels in the 24 h, 48 h, and 72 h groups were also significantly increased relative to the control group (all P < 0.001). Conclusion By placing mice in a simulated hypobaric hypoxia environment at an altitude of 8 000 meters combined with intermittent exercise stimulation, a stable high-altitude acute lung injury model in mice can be successfully established within 48 hours.