Lactobacillus helveticus R0052-derived membrane vesicles ameliorate DSS-induced inflammatory bowel disease by modulating the gut microbiota and activating the cholinergic anti-inflammatory pathway.

Inflammatory bowel disease (IBD) involves persistent immune dysregulation in the gut, accompanied by changes in microbiota and metabolite profiles. Lactobacillus helveticus R0052, a probiotic with known anti-inflammatory effects, has been shown to provide health benefits; however, its precise mechanisms in the treatment of IBD remain unclear. This study investigated the ability of L. helveticus R0052 to produce membrane vesicles (MVs), their therapeutic effects in a murine model of dextran sulfate sodium (DSS)-induced colitis, and the underlying mechanisms. The results revealed that L. helveticus R0052 secretes anti-inflammatory MVs (052-MVs), which modulate cytokine expression in RAW 264.7 mouse macrophage cells. In DSS-induced colitis mice, 052-MVs alleviated colitis symptoms, suppressed pro-inflammatory cytokines (IL-6, IL-1β, TNF-α), increased the expression of anti-inflammatory cytokine (IL-10), enhanced the expression of tight junction proteins in colon tissues, and reduced macrophage polarization toward the M1 phenotype. Additionally, 16S rDNA and UHPLC/Orbitrap mass spectrometry analyses showed that 052-MVs improved the gut microbiota structure and diversity and mitigated DSS-induced metabolite dysregulation. Mechanistically, 052-MVs activated the cholinergic anti-inflammatory pathway (CAP) by upregulating α7 nAChR, acetylcholine (ACh), and the ACh-related enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE). Notably, pre-treatment with the α7 nAChR antagonist methyllycaconitine (MLA) abolished 052-MV-induced α7 nAChR upregulation and reversed TNF-α suppression, confirming that the anti-inflammatory effects of 052-MVs are mediated, at least in part, through α7 nAChR-dependent CAP activation. These findings indicate that 052-MVs ameliorate DSS-induced colitis by modulating the gut microbiota and activating the CAP, highlighting the gut microbiota-CAP axis as a potential therapeutic target for IBD.