Inhibitory effects of Levilactobacillus brevis IBRC-M10790 on apoptosis and inflammation induced by Clostridioides difficile culture supernatant in vitro

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated diarrhea that contributes significantly to global morbidity and mortality. Bacterial virulence factors, mostly toxins, play key roles in CDI pathogenesis. Probiotic supplementation is a potential strategy to reduce the adverse effects of C. difficile and support intestinal homeostasis. This study aimed to investigate the inhibitory effects of live Levilactobacillus brevis IBRC-M10790 (LLB) and its membrane vesicles (LBMVs) on apoptosis and inflammation induced by released C. difficile virulence factors in vitro. We employed human colorectal adenocarcinoma Caco-2 and HT-29 cell lines, which are widely used as in vitro models against CDI. Viability and apoptosis of both cell lines were assessed using MTT and Annexin V/PI flow cytometry assays. Anti-inflammatory and anti-apoptotic effects of LLB and LBMVs were investigated following treatment with cell-free supernatants of toxigenic C. difficile RT001 (Tox-S), as well as the culture filtrates of non-toxigenic C. difficile RT084 and ATCC 700057 strains. The expression of apoptosis-related genes (BAX, BCL-2, Caspase-3, Caspase-9) and inflammatory markers (IL-6, IL-8, IL-1β, TNF-α) was measured by RT-qPCR, and cytokine production was analyzed by ELISA. C. difficile Tox-S and culture filtrate significantly reduced cell viability and increased the expression of apoptotic and proinflammatory markers in Caco-2 and HT-29 cells. LLB and LBMVs effectively modulated cell viability, reduced apoptosis, and downregulated the expression and production of inflammatory cytokines in both cell lines after exposure to C. difficile culture supernatants. These findings suggest that LLB and LBMVs could be exploited as potential supplement to the current treatment strategies against C. difficile-induced cellular injury and inflammation.