Studies Of The Intervention Of RhBD3in Necrotizing Enterocolitis Via Rho-ROCK Signaling Pathway

Objective: The aim of this study was to investigate the effects of recombinant humanbeta-defensin-3(rhBD3) on intestinal wound healing and to test the hypothesis thatthe rhBD3might have beneficial effects on a neonatal rat necrotizing enterocolitis(NEC) model.Methods: The antimicrobial activity of rhBD3against clinical isolates Escherichiacoli (E. coli) and Staphylococcus aureus (S. aureus) was determined using a liquidmicrodilution assay. Cytotoxicity against human and rat intestinal epithelial cell linesCaco-2and IEC-6was first measured by MTT assay. Enterocyte migration (woundclosure assay, real-time electrical-impedance based detection analyzer, and BrdUimmunostaining) and proliferation (CCK8, BrdU incorporation assay, cell cycleanalysis, and PCNA immunostaining) was detected in vitro and in vivo. Intracellularcalcium mobilization was measured using the Fluo-4NW assay. The CCR6expression in Caco-2and IEC-6cells and in rat small intestine and colon wasevaluated by western blot analysis and immunostaining. To determine the role ofCCR6in rhBD3-stimulated cell migration, neutralizing anti-CCR6antibody andspecific CCR6siRNA were used to block CCR6activation. Then the effect of rhBD3on the Rho-ROCK signaling pathway was assessed. All experimental protocols wereapproved by the Animal Care Committee of the Children’s Hospital of Shanghai.Sixty-eight newborn Sprague-Dawley rats were randomly divided into four groups:Control+NS (n=12), Control+hBD3(n=12), NEC+NS (n=20), and NEC+hBD3(n=24). Rats in the former two groups were mother-fed and gavaged with0.1mlnormal saline daily. Experimental NEC was induced by exposure to hypertonicfeeding, asphyxia (60seconds exposure to100%nitrogen) and hypothermia (4℃for10minutes). Pups received recombinant hBD3(100μg/kg with a volume of0.1ml,once a day) via gastric tube or the same dose of normal saline. Body weights wererecorded daily. Animals were sacrificed via cervical dislocation upon the developmentof NEC clinical signs (abdominal distension, discolored abdominal walls, or bloody stools) or at the end of the experiment on day4. After the killing, the intestine wasremoved and hematoxylin and eosin (H&E) stained. Pathological changes werescored blindly by an experienced pathologist using a previously published NECscoring system. Survival time, cytokines expression (tumor necrosis factor-(TNF-),interleukin6(IL-6), and IL-10), mucosal integrity (the DAO level in serum, and theexpression of ZO-1in the ileum), enterocyte proliferation and migration wereevaluated.Results: For both tested microorganisms, rhBD3activity was maintained throughouta broad pH range. The bactericidal effect of hBD3was slightly inhibited at pH8.5forE. coli, and at pH5.8for S. aureus. No significant cytotoxicity against IEC-6andCaco-2was observed over a broad range of concentrations and incubation periods.Recombinant hBD3could stimulate enterocyte migration, but not proliferation, bothin cultured enterocytes and in the ileum of necrotizing enterocolitis rat. The migratoryeffect was slightly dose-dependent. Calcium mobilization was involved inrhBD3-mediated migration of wounded epithelial cells. Neutralizing antibody andsiRNA confirmed this stimulatory effect was mediated by CCR6. Epithelial cells fromileum and colon expressed CCR6both in newborn and adult rats. However, areduction of CCR6expression in neonatal rat was observed when compared withadult rat. Furthermore, rhBD3treatment induced Rho activation (GTP-bound form ofRho), myosin light chain2phosphorylation (at serine19), and F-actin accumulation.ROCK inhibition resulted in strikingly reduction of F-actin accumulation andenterocyte migration. Strikingly, neonatal rats in group NEC+hBD3could maintainthe body weight during the experiment. And rhBD3administration decreased theincidence of NEC from80%in group NEC+NS to50%in group NEC+hBD3(P=0.039), increased the survival rate from25%in group NEC+NS to62.5%in groupNEC+hBD3(P<0.001), and reduced the severity of NEC (mean NEC scores in thesetwo groups were2.50and1.67, respectively, P=0.01). Moreover, rhBD3reduced thepro-inflammatory cytokines (TNF-alpha, IL-6) including the anti-inflammatorycytokine IL-10expression in ileum and serum by real-time PCR and ELISA,respectively. There were no differences of all the three cytokines expression between the two mother-fed groups. IEC-6cells were incubated with various concentrations ofrhBD3, and rhBD3treatment did not induce the mRNA and protein expression ofTNF-. In addition, the serum level of diamine oxidase (DAO) and the expression ofZO-1were also evaluated. Decreased levels of DAO activity were detected in theNEC group. And rhBD3treatment increased the concentration of DAO in the serumand ZO-1expression in the ileum.Conclusion: This study, for the first time, provided evidence that rhBD3couldpromote enterocyte migration via the action on CCR6and regulation of itsdownstream Rho-ROCK signaling pathway. Calcium mobilization was also involvedin rhBD3-mediated migration of enterocyte. In vivo results demonstrated that rhBD3might protect newborn rats from NEC due to reducing inflammatory mediators,preserving mucosal integrity, and promoting intestinal restitution.