Supplementary Material for: Akkermansia muciniphila and Its Extracellular Vesicles affect endocannabinoid system in in vitro model

Introduction: Recent studies indicate that the gut microbiota controls the host's immune system. Probiotics use different signaling pathways to regulate intestinal permeability, barrier integrity, and energy balance. Methods: This research examined how Akkermansia muciniphila and its extracellular vesicles (EVs) impact inflammation and genes related to the endocannabinoid system in the STC-1 cell line through RT-PCR and ELISA assays. Results: The study's results indicated that EVs had a significant impact on GLP-1 expression compared to the multiplicity of infections (MOI) ratio. Notably, there was a substantial increase in the expression of PYY and GLP-1 genes across all treatments (P<0.05). Conversely, the expression of CB-1, CB-2, and FAAH genes notably decreased in the STC-1 cell line when treated with MOI 50 of A. muciniphila and an EV concentration of 100 μg/mL (P<0.05). Both MOI 50 of A. muciniphila and an EV concentration of 100 μg/mL significantly enhanced the expression of the TLR-2 gene. In contrast, EVs at a concentration of 100 μg/mL substantially reduced TLR-4 gene expression. A. muciniphila-derived EVs notably decreased the levels of inflammatory cytokines (TNF-α and IL-6), while increasing IL-10 expression at MOI 100 and an EV concentration of 100 μg/mL. These findings suggest that A. muciniphila and its EVs could regulate the expression of specific genes, serving as targets for maintaining host energy balance. Conclusions: In summary, this study illustrates that A. muciniphila-derived EVs exhibit anti-inflammatory properties and have the potential to modulate gene expression in cases of obesity and gastrointestinal tract inflammation.