Antimicrobial peptides produced by Clostridium butyricum alleviate LPS-induced intestinal injury in piglet by modulating gut microbiota, bile acid, and GPR43-NLRP3 pathway

Abstract Weaning stress impacts piglet performance, prompting antimicrobial resistance concerns from antibiotic overuse. Clostridium butyricum -derived antimicrobial peptides (CBP) show potentials as a safe, effective antibiotic alternative. We initially characterized novel antimicrobial peptides within the CBP fraction, synthesizing and confirming their potent activity. This study evaluated CBP’s effects on intestinal health and growth performance of weaned piglets using a lipopolysaccharide (LPS)-induced inflammation model. Fifty weaned Jinhua piglets (45 days, 9.95 ± 2.03 kg) were randomly allocated to control group (CON) and CBP group ( n = 25), with five replicates each. Piglets in the CBP group were orally administered 3 mL of CBP daily (145.59 mg of total peptide) for 21 days, while the CON group received sterile water. During this period, CBP significantly improved growth performance, evidenced by increased average daily gain ( P = 0.047) and reduced feed conversion ratio ( P = 0.015), alongside a decrease in diarrhea incidence ( P < 0.05). To further investigate the mechanism, a subset of animals from each group was challenged with LPS on day 21 to induce intestinal inflammation. Mechanistically, CBP enhanced intestinal barrier functions by optimizing crypt architecture and upregulating tight junction proteins expression ( P < 0.05). CBP also exerted a potent anti-inflammatory effect, substantially reducing pro-inflammatory cytokines ( P < 0.05) and suppressing NLRP3 inflammasome activation. Integrated microbiome and metabolomic analyses revealed CBP modulated the gut microbiota by increasing beneficial bacteria Lactobacillus and Coprococcus ( P < 0.05) and elevating protective metabolites, including butyrate and hyocholic acid ( P < 0.05). In conclusion, our findings demonstrate that CBP supplementation effectively promotes piglet growth and alleviates intestinal injury by regulating the gut microbiota and associated metabolic profiles. These effects are mediated through enhanced intestinal barrier functions and suppressed inflammation via the GPR43-NLRP3 pathway. This study provides strong evidence for CBP as a promising, safe alternative to antibiotics.