Lactobacillus rhamnosus-Derived Extracellular Vesicles Mitigate Nonalcoholic Fatty Liver Disease Progression Through Activation of the Apelin Signaling Pathway.

Lactobacillus rhamnosus-derived bacterial extracellular vesicles (LR-BEVs) were successfully isolated and characterized at approximately 110 nm and were internalized by THLE-2 hepatocytes.

• LR-BEVs were isolated via sequential ultracentrifugation.
• Morphology and size were validated by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA).
• Uptake into THLE-2 hepatocytes was traced using DIL fluorescent dye.
• LR-BEVs measured approximately 110 nm in size.

At 100 ng/mL, LR-BEVs significantly suppressed IL-1β and IL-6 release in THLE-2 cells, in contrast to the pro-inflammatory effects of E. coli-derived BEVs (E-BEVs).

• LR-BEVs at 100 ng/mL were used for in vitro treatment of THLE-2 hepatocytes.
• E-BEVs exhibited pro-inflammatory effects, increasing IL-1β and IL-6, while LR-BEVs suppressed these cytokines.
• Inflammatory cytokine secretion was assessed in oleic acid (OA)-induced steatosis model in THLE-2 cells.

LR-BEVs ameliorated oleic acid-induced steatosis in THLE-2 cells in vitro by reversing suppressed proliferation, elevated ALT/AST levels, inflammatory cytokine secretion, ROS accumulation, and lipid droplet deposition.

• Steatosis was induced in THLE-2 cells using oleic acid (OA).
• Cell viability/proliferation was assessed via CCK-8 assay.
• Lipid accumulation was evaluated by Oil Red O staining.
• ALT, AST, TG, IL-1β, IL-6, and ROS levels were measured as outcome parameters.

In a murine high-fat diet-induced NAFLD model, LR-BEV administration markedly attenuated hepatic lipid accumulation and reduced serum levels of ALT, AST, TC, and TG, along with ameliorating hepatic inflammation and oxidative stress.

• NAFLD was induced in mice using a high-fat diet.
• LR-BEV treatment reduced serum ALT, AST, total cholesterol (TC), and triglycerides (TG).
• Hepatic lipid accumulation was markedly attenuated.
• Hepatic inflammation and oxidative stress were also ameliorated by LR-BEV administration.

Transcriptomic analysis identified Apelin signaling as a key pathway modulated by LR-BEVs, with enrichment of target genes including APLNR, SERPINE1, EGR1, and ACTA2.

• Mechanistic investigation employed transcriptomics, Apelin receptor inhibition with ML221, and Western blotting.
• Key enriched Apelin pathway target genes identified were APLNR, SERPINE1, EGR1, and ACTA2.
• Transcriptomic analysis was used to implicate the Apelin signaling pathway as the key mechanism.

The anti-steatotic effects of LR-BEVs were specifically mediated through Apelin pathway activation, as demonstrated by abrogation of these benefits using the Apelin receptor antagonist ML221.

• ML221 is an Apelin receptor (APLNR) antagonist used for functional validation.
• ML221 reversed LR-BEV-promoted proliferation and inhibition of lipid accumulation.
• ML221 also reversed LR-BEV-induced reductions in ALT/AST elevation, TC, and TG increase.
• ML221 reversed LR-BEV-induced upregulation of key Apelin pathway targets.
• These results confirmed that LR-BEV anti-steatotic effects operate specifically via Apelin pathway activation.