| Antibiotics has been used in stockbreeding for a long time, but the problem of antibiotic resistance is growing. Consequently, it is urgent to search for new antimicrobial agents. PR39 is a proline-arginine-rich antimicrobial peptide that has been shown to exert multiple and diverse activities, including wound healing regulation, chemoattraction of neutrophils and several other biological processes. Recent study has shown that bacteria challenge could significantly up-regulate PR39 mRNA expression in the ileum, liver and spleen of pigs, leading us to explore the role of PR39 in bacterial infection. Therefore, we have analyzed the antimicrobial activity and stability of PR39 in vivo first, and evaluated the effects of PR39 on modulating the bacterial infection in two mouse models, and further determined the mechanisms of PR39 in regulating epithelial barrier function in epithelial cells. The main results were as below:1. Antibacterial activity, stability and biodistribution of PR39The antimicrobial activity and stability of PR39 in the presence of simulated gastrointestinal fluids and serum were analyzed. PR39 significantly retarded bacterial growth in serum, but when incubated with SGF or SEF, PR39 failed to inhibit bacterial growth. RP-HPLC analysis revealed that PR39 was completely and quickly degraded in SBF within 5 min, and was also degraded entirely in SGF within 1 h, but approximately 70% of PR39 remained intact after incubated with serum for 1 h, the half-life of PR39 in serum was approximately 2 h, indicating that PR39 is highly unstable in gastrointestinal tract, but remains substantially intact and active in serum. The in vivo imaging results showed that PR39 could be absorbed into the systemic circulation, and was expelled out of the body within 24 h.2. Effects of PR39 on bacterial infection in miceEffects of PR39 on Salmonella typhimurium infection in mice:PR39 ameliorated the clinical symptoms such as less vitality, hunched over and ruffled fur during the pathological process of Salmonella-induced disease, and bacteria translocation to both spleen and liver was significantly reduced (P<0.05) by Salmonella+PR39 group compared with the Salmonella group. The results of inflammatory responses of mice showed that colon MPO levels of mice in Salmonella group dramatically increased compared with control mice, while administration of PR39 significantly reduced colon MPO activity (P<0.05). But the gene expression and secretion of TNF-α, IL-8 and IL-10 revealed that PR39 had no significant impact on the gene expression and secretion of inflammatory cytokines. A significant reduction in villus height and altered mucosal architecture were observed in mice inoculated with Salmonella typhimurium, however, PR39 treatment significantly decreased these damage (P<0.05). Electrical resistance of Salmonella-infected mice was reduced significantly compared with the values obtained from control mice, however, Salmonella-infected mice given PR39 showed a significant recovery in TER (P<0.05). Consistent with this, PR39 significantly suppressed the increase in FITC-dextran permeability induced by Salmonella infection, indicating that PR39 could restore intestinal tight junction destruction caused by Salmonella infection, and PR39 alone may enhanced the intestinal barrier function in mice.Effects of PR39 on EHEC infection in mice:PR39 ameliorated the clinical symptoms such as less vitality, hunched over and ruffled fur during EHEC infection, reduced the MPO levels in colon tissues (P<0.05), and bacteria translocation to both spleen and liver was also reduced. H&E stained images of kidney showed that treatment of PR39 alleviated renal glomeruli and kidney interstitial tissue damage such as severe congestion and membrane blebbing were, and restored the intestinal architecture damage such as atrophy and edema of mucosa, reduction in villus height and increases in crypt depth induced by EHEC infection. TEM images of colon showed that treatment of PR39 attenuated the extensive distortion and effacement of microvilli observed in mice inoculated with EHEC. PR39 significantly prevented the TER and FITC-dextran permeability changes, indicating that PR39 ameliorated intestinal epithelial barrier destruction caused by EHEC infection. Besides, PR39 also enhanced tight junction protein ZO-1 and Occludin expression in intestine impaired by EHEC (P<0.05), and the expression of ZO-2 and Claudin-1 were not altered in all groups.3. Mechanisms of PR39 in regulating epithelial barrier functionBased on the results above, a series of analysis were conducted to explore the effects of PR39 in vitro in IPEC-1 cells model. The cytotoxicity tests showed that PR39 exhibited little cytotoxic effects towards IPEC-1 cells. The Western blot results confirmed that PR39 could significantly inhibit the EHEC-induced reduction of occluding (P<0.05). Immunofluorescence images showed that pretreatment with PR39 for 24 h obviously prevented the EHEC-induced redistribution of Occludin and F-actin. We performed a study that cells were incubated with PR39 for 24 h and washed out before EHEC infection, which means PR39 was not contact with the bacteria. The results showed that PR39 significantly reduced EHEC adhesion and attenuated EHEC-induced decreases in TER in epithelial cells, and we also found that PR39 could not inhibit EHEC growth at concentration of 2 μg/mL in DMEM&F 12 medium. Therefore, we speculated that PR39 protect host from EHEC infection by affecting barrier functions of epithelial cells.Calcium switch assay, which is a well-validated model for studying the assembly of tight junction, was conducted to evaluating the effect of PR39 alone on IPEC-1 monolayers. During the recovery period, monolayers treated with PR39 (≥2μg/mL) recovered TER values within 12 h significantly higher compared to the control monolayer. Western blot analysis revealed that PR39 (≥0.5μg/mL) increased the protein expression of Occludin in 12 h after calcium repletion. These data suggested that PR39 promoted the assembly of Occludin and regulated the barrier permeability of epithelial cells.To further examine the mechanism by which PR39 regulates TJ barrier function, we investigated the involvement of Rho GTPase family, which controls TJ assembly and regulates epithelial barrier function. PR39 induced the activation of the Rac 1, and specific inhibition of Rac 1 blocked the PR39-mediated promotion of occludin, and reduction of E.coli adhesion. These data revealed that, in addition to its antibacterial and other immunoregulatory functions, PR39 could protect the intestinal barrier from bacterial infection by modulating tight junction via Rac 1 activation.In summary, this is the first report to our knowledge showing the role of PR39 in modulating intestinal epithelial barrier function and preventing EHEC-induced barrier damage. Our results provide a first indication for the potential of PR39 as a new therapeutic option for bacterial infection in both human and farm animals. |
