Lactobacillus paracasei Expressing Porcine Trefoil Factor 3 and Epidermal Growth Factor: A Novel Approach for Superior Mucosal Repair
Description
Trefoil factor 3 (TFF3) and epidermal growth factor (EGF) are small bioactive proteins known for their significant roles in promoting intestinal epithelial cell proliferation, migration, and apoptosis inhibition, contributing to gastrointestinal mucosal protection and repair. Lactobacillus paracasei served as the host strain to expressing pig TFF3 (pTFF3), pig EGF (pEGF), and a fusion protein (pTE). Functional assays demonstrated that these recombinant strains significantly promoted IPEC-J2 cell proliferation and migration, with the fusion protein pTE showing a greater migratory effect compared to individual factors. In vivo, oral administration of pPG-SP-pTE/27-2 in DSS-induced colitis mice resulted in improved health, preserved colon length, and reduced colonic inflammation. The pPG-SP-pTE/27-2 treatment also led to increased expression of tight junction proteins (Claudin-2, Occludin, ZO-1) and elevated serum IL-10 levels, while reducing pro-inflammatory cytokines IL-6, IL-1β, and TNF-α. Histopathological examination showed reduced colonic lesions and improved mucosal integrity in treated animals. These findings strongly indicate that the recombinant Lactobacillus paracasei engineered to express porcine trefoil factor 3 (pTFF3) and porcine epidermal growth factor (pEGF) presents a highly promising strategy for augmenting intestinal epithelial cell proliferation, migration, and mucosal restitution. This approach holds significant potential for therapeutic applications in the prophylaxis and treatment of intestinal mucosal injury and IBD. Figure 1. DSS induced model Figure 2. The recombinant Lactobacillus expressing pTFF3 and pEGF were identified by PCR and Western blot Figure 3. Identification of proteins expressed in recombinant Lactobacillus by IFA Figure 4. The growth characteristics (a,b,c) and stability (e,f,g) of recombinant Lactobacillus were analyzed Figure 5. Analysis of the effect of pTFF3, pEGF and pTE on IPEC-J2 cell proliferation Note: *P < 0.05, **0.01 < P < 0.05, ***P < 0.01 vs 0 ng/mL Figure 6. The analysis of the migration effect about the target protein on IPEC-J2 Note: *P<0.05, **0.01<P<0.05, ***P<0.01 vs pPG; #P<0.05, ##0.01<P<0.05, ###P<0.01 vs pTE; ns, P>0.05 Figure 7. Effects of different treatments on disease activity index (a) Colonic H&E staining results of different groups. (b) Effects of different treatments on colonic MPO activity (c). Westren blot and Real-time PCR analysis of tight junction protein expression in colon of mice. (d). Note: *P < 0.05, **0.01 < P < 0.05, ***P < 0.01 vs pPG; #P < 0.05, ##0.01 < P < 0.05, ###P < 0.01 vs pTE; ns, P > 0.05 Figure 8. Effects of different treatments on colon length in colitis mice Figure 9. Determination of inflammatory secretion level