<scp> <i>Lactobacillus rhamnosus</i> </scp> ‐Derived Extracellular Vesicles Mitigate Nonalcoholic Fatty Liver Disease Progression Through Activation of the Apelin Signaling Pathway

Non-alcoholic fatty liver disease (NAFLD), a chronic metabolic disorder characterized by ectopic lipid deposition in hepatocytes, currently lacks evidence-based pharmacotherapies, rendering probiotic-based strategies-particularly those utilizing Lactobacillus rhamnosus (LR)-a promising therapeutic avenue. Bacterial extracellular vesicles from Lactobacillus rhamnosus modulate NAFLD progression, but their molecular mechanisms remain unelucidated. Lactobacillus rhamnosus (LR-BEVs) and Escherichia coli (E-BEVs) were isolated via sequential ultracentrifugation, validated for morphology/size by TEM and NTA, and their uptake into THLE-2 hepatocytes was traced with DIL fluorescent dye. In vitro, oleic acid (OA)-induced steatosis in THLE-2 cells was used to assess BEV effects via CCK-8, ALT/AST/TG, IL-1β/IL-6, ROS, and Oil Red O. In vivo, high-fat diet-induced murine NAFLD evaluated BEV therapeutic potential. Mechanistically, transcriptomics, Apelin receptor inhibition (ML221), and Western blotting explored the Apelin pathway in LR-BEV-mediated NAFLD regulation. LR-BEVs (~110 nm) were isolated and internalized by THLE-2 hepatocytes. At 100 ng/mL, LR-BEVs significantly suppressed IL-1β and IL-6 release in THLE-2 cells, contrasting with the pro-inflammatory effects of E-BEVs. In vitro, LR-BEVs ameliorated OA-induced steatosis, reversing suppressed proliferation, elevated ALT/AST levels, inflammatory cytokine secretion, ROS accumulation, and lipid droplet deposition. In a murine model of high-fat diet-induced hepatic steatosis, LR-BEV administration markedly attenuated hepatic lipid accumulation and concomitantly reduced serum levels of ALT, AST, TC, and TG, along with ameliorating hepatic inflammation and oxidative stress. Transcriptomic analysis implicated Apelin signaling as a key pathway modulated by LR-BEVs, with enrichment of target genes (APLNR, SERPINE1, EGR1, ACTA2). Functional validation demonstrated that LR-BEVs exert their anti-steatotic effects-promoting proliferation and inhibiting lipid accumulation, ALT/AST elevation, TC, and TG increase-specifically via Apelin pathway activation, as evidenced by the abrogation of these benefits by the Apelin receptor antagonist ML221. ML221 also reversed LR-BEV-induced upregulation of key Apelin pathway targets. LR-BEVs exert anti-steatotic efficacy via the Apelin signaling pathway activation in vitro and in vivo. These findings establish LR-BEVs as novel NAFLD therapeutics and highlight their translational potential for metabolic liver diseases.