Casein hydrolysate promotes intestinal repair in adult mice with Salmonella enteritis through a lactate-GPR81-Wnt3A axis in intestinal stem cell niche.

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major enteric pathogen that causes severe intestinal damage, characterized by epithelial disruption, inflammation, and impaired regeneration. Nutritional intervention has emerged as a promising strategy to mitigate such injury and promote mucosal repair. Casein enzymatic hydrolysate (CEH) possesses well-documented anti-inflammatory and mucosa-protective properties. However, whether CEH exerts its protective effects against S. Typhimurium-induced intestinal injury by modulating the gut microbiota, and the underlying mechanisms, remains largely unknown. Twelve-month-old C57BL/6J mice were pretreated with streptomycin and challenged with S. Typhimurium. In the primary efficacy study, mice were assigned to four groups: control, control + CEH, model, and model + CEH (2% CEH in diet for 10 days before and throughout infection). Disease severity, intestinal histology (H&E and PAS staining), inflammation (TNF-α and IL-10 ELISA; CD45 + flow cytometry), barrier function (E-cadherin flow cytometry; PEPT1/SGLT1 immunofluorescence), stem cell activity (LGR5/Ki67 and CD24 + LGR5 + staining), Paneth cell niche factors (LYZ1/Wnt3A immunofluorescence and western blot), gut microbiota composition (16S rDNA sequencing), and luminal L-lactate levels (biochemical assay) were evaluated. To establish causality, additional mechanistic experiments were performed using exogenous L-lactate supplementation, the GPR81 inhibitor 2,5-DHBA, and the Wnt secretion inhibitor Wnt-C59, with the same panel of tests. CEH administration significantly improved survival, alleviated clinical disease severity, and attenuated systemic and intestinal inflammation. CEH preserved mucosal architecture, restored goblet cells and barrier proteins (PEPT1, SGLT1, E-cadherin), and promoted LGR5 + and CD24 + LGR5 + intestinal stem cell regeneration. CEH reshaped the gut microbiota, enriched lactate-producing genera, and increased luminal L-lactate levels, accompanied by restored Paneth cell LYZ1 expression and elevated Wnt3A in the crypt niche. Strikingly, lactate supplementation improved CEH's protective effects on Paneth cell function, ISC proliferation, barrier integrity, and inflammatory cytokines. LYZ1 + Paneth cells co-expressed the lactate-sensing receptor GPR81. Pharmacological blockade of GPR81 or Wnt abrogated the beneficial effects of both CEH and lactate, confirming that CEH acts through the lactate-GPR81-Wnt3A axis. Collectively, these findings demonstrate that CEH alleviates S. Typhimurium-induced enteritis in adult mice via a microbiota-lactate-GPR81-Wnt3A-ISC axis. CEH represents a promising nutritional strategy to counteract infection-induced intestinal injury and promote regeneration in adult hosts with diminished regenerative reserve.