Lactobacillus mucosae extracellular vesicles improve metabolic dysfunction-associated fatty liver disease by activating the AMPK pathway via the gut-liver axis

Metabolic dysfunction-associated fatty liver disease (MAFLD) is the most prevalent chronic liver disease worldwide but lacks effective therapies. Gut microbiota play a key role in MAFLD via the gut-liver axis, yet targeted interventions remain unclear. This study investigates the therapeutic effects and mechanisms of Lactobacillus mucosae ( L. mucosae ) and its extracellular vesicles (EVs) in MAFLD. At the clinical level, 16S rRNA sequencing was performed to analyze differences in gut microbiota composition between MAFLD patients and healthy controls, followed by the identification of a core probiotic strain, L. mucosae , using culture-based approaches. A high-fat diet-induced MAFLD mouse model was subsequently established to evaluate the therapeutic effects of L. mucosae , while free fatty acid-induced hepatocyte models were used to identify L. mucosae -EVs as the key active component. Mechanistic investigations and validation were further conducted in vivo and in vitro using RNA sequencing, qPCR, Western blot analyses, DIA-based EV proteomics, and Proteinase K-mediated EV protein functional assays. MAFLD patients showed reduced intestinal Lactobacillus abundance, negatively correlated with hepatic lipid parameters. L. mucosae administration alleviated steatosis, metabolic dysfunction, inflammation, and oxidative stress in mice. In vitro, L. mucosae -EVs were confirmed as the main active component, and Proteinase K-pretreated EVs showed attenuated protective effects on hepatocytes. Mechanistically, L. mucosae -EVs activated hepatic AMPK signaling via the gut-liver axis, remodeled gut microbiota, and restored intestinal barrier integrity. This study systematically demonstrates for the first time that L. mucosae -EVs ameliorate MAFLD through multiple mechanisms, including gut microbiota remodeling, intestinal barrier restoration, and activation of hepatic AMPK signaling via the gut-liver axis. Proteinase K-mediated functional assays suggest that EV-associated protein cargo contributes, at least in part, to these protective effects. These findings provide a solid experimental foundation and theoretical basis for the development of precision microbiome-based therapeutic strategies for MAFLD based on L. mucosae and its EVs. L. mucosae -EVs improve MAFLD by activating the AMPK pathway through the gut-liver axis. Orally administered L. mucosae -EVs act through the gut-liver axis and activate hepatic AMPK signaling, resulting in synchronized metabolic improvements, including enhanced fatty acid oxidation, suppressed lipogenesis, improved insulin sensitivity, and attenuated oxidative stress, inflammation, and fibrosis. Restoration of gut microbiota homeostasis and reinforcement of intestinal barrier integrity further contribute to this systemic amelioration. EVs, extracellular vesicles; UC, ultracentrifugation