Establishing rat models of MASLD and MASLD + OSA

Male clean-grade Sprague–Dawley rats (n = 48; 200 ± 20 g) were purchased from Shanghai Slack Experimental Animal Co., Ltd. (Shanghai, China). The rats were housed in an environment with a relative humidity of 45–55% at 18–22 °C and allowed free access to water and food for one week. 12 rats were fed a standard chow diet (53% carbohydrates, 23% proteins and 5% fats; El-Nasr Company, Abu Zaabal, Egypt) during the second week and included in the healthy Control group. The remaining 36 rats were fed a high-fat diet consisting of 30% sucrose, 4% palmitic acid, 2% cholesterol and 0.5% cholic acid (Sigma-Aldrich, St Louis, MO, USA) and 30% lard stearin (El-Nasr Company, Abu Zaabal, Egypt) and included in the MASLD group. Rats in both groups were euthanised using 1% pentobarbital sodium after eight weeks, and their liver tissues were isolated, immersed in 10% buffered formalin and embedded in paraffin. The formalin-fixed liver tissues were stained with haematoxylin and eosin (H&E; Sigma-Aldrich) for histologic analysis. The tissue samples were initially incubated with haematoxylin for 10 min at 25 °C and then with 0.5% eosin for 3 min. Oil red O staining was performed to observe lipid droplets in the liver and verify the establishment of MASLD models.

Nonalcoholic fatty liver disease (NAFLD) activity score (NAS), which originally evaluated 14 histologic features, was calculated to analyse histologic changes⁴⁴. Five features are independently associated with MASLD diagnosis, namely steatosis, hepatocellular ballooning, lobular inflammation, fibrosis and the absence of lipogranuloma. NAS (0–8) is determined by calculating the sum of three equal-weighted features: steatosis (0–3), lobular inflammation (0–3) and hepatocellular ballooning (0–2). NAS ≥ 5 correlates with MASLD, whereas NAS < 3 is not considered MASLD.

Chronic intermittent hypoxia (CIH) was used to construct an OSA model in this study. The induction principle of OSA-related diseases including MASLD was caused by chronic hypoxia caused by OSA^41–43. OSA could directly cause a decrease in oxygen inhalation. Therefore, the hypoxia caused by OSA was simulated by directly controlling the inhaled concentration of oxygen, that was, CIH was used to simulate OSA. We kept 6 rats with MASLD in a glass chamber (30 × 20 × 20 in; BioSpherix, Redfield, NY, USA) to control the concentration of inhaled O₂. The intermittent low O₂ concentration was provided by keeping the average O₂ concentration at 10 ± 1% for 60 s (hypoxia phase) and then raised to 20–21% for 30 s (normal phase). OSA models were constructed by maintaining the rats under intermittent low O₂ concentration for 8 h/day (9:00 am to 5:00 pm) for 6 weeks^41–43. The 24 rats of MASLD model were randomly divided into four groups, namely MASLD, MASLD + OSA-Exo, MASLD + miR-455-3p and MASLD + miR-455-3p + OSA-Exo groups.

All animal experiments were performed in accordance with the guidelines of the Animal Experiment Committee of Yan’an Hospital affiliated with Kunming Medical University. We have complied with all relevant ethical regulations for animal use.