Trefoil factor 3 (TFF3) and epidermal growth factor (EGF) exert a promotive effect on the functions of intestinal epithelial cells and offer protection to the intestinal mucosa. Lactobacillus paracasei can ameliorate intestinal mucosal damage. In this study, pPG-pTFF3/27-2, pPG-pEGF/27-2, and pPG-pTE/27-2 were constructed to express porcine TFF3, EGF, and a fusion protein (pTE). Functional assays showed they promoted Immortalized Porcine Enterocyte Cell line J2 (IPEC-J2) proliferation and migration, with pTE having a greater migratory effect. In dextran sulfate sodium (DSS)-induced colitis mice, oral administration of pPG-pTE/27-2 reduced colitis, improved mucosal integrity, increased the expression of tight-junction proteins and the serum level of Interleukin-10 (IL-10), and decreased the levels of pro-inflammatory Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), and Interleukin-1β (IL-1β). These results imply that recombinant L. paracasei 27-2 strains engineered to express pTFF3 and pEGF represent a promising approach for augmenting intestinal epithelial cell function and facilitating mucosal restitution, and they possess significant potential in the treatment of intestinal mucosal injury and inflammatory bowel disease (IBD).

Porcine β-defensin 2 (pBD2) possesses broad-spectrum antimicrobial properties and is crucial for gastrointestinal mucosal repair. Lactic acid bacteria (LAB) serve as optimal vectors for exogenous protein delivery due to their high biosafety, intestinal colonization capacity, and ability to modulate gut microecology. In this study, we engineered a recombinant Lactobacillus paracasei strain (pPG-N1-pBD2/27-2) that efficiently secretes pBD2. In vitro, this recombinant strain significantly enhanced the proliferation and migration of porcine intestinal epithelial cells (IPEC-J2). In vivo, oral administration of pPG-N1-pBD2/27-2 markedly alleviated dextran sulfate sodium (DSS)-induced colitis in mice. This protective effect was evidenced by reduced Disease Activity Index (DAI) scores, prevention of colon shortening, and decreased colonic activities of myeloperoxidase (MPO) and eosinophil peroxidase (EPO), alongside normalized N-acetyl-β-D-glucosaminidase (NAG) levels. Histopathological analysis revealed that the treatment preserved mucosal architecture, including goblet cells and crypts, and fortified the physical barrier by upregulating tight junction proteins. Mechanistically, the recombinant strain suppressed the colonic iNOS/COX-2 inflammatory axis, decreased serum pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α), and elevated the anti-inflammatory cytokine IL-10. Furthermore, it restored systemic immune homeostasis by normalizing the proportions of splenic macrophages, T/B lymphocytes, and natural killer (NK) cells. In conclusion, pPG-N1-pBD2/27-2 mitigates colitis through a dual mechanism: reinforcing the intestinal physical barrier and rebalancing the innate–adaptive immune axis. These findings highlight the potential of pBD2-engineered probiotics as novel biological therapeutics for intestinal inflammatory diseases.