Study Of The Protective Effect And Mechanism Of HBD3 In Neonatal Necrotizing Enterocolitis Via Regulating Autophagy

Objective: The aim of this study was to investigate the effects of human ?-defensin-3(h BD3)-mediated autophagy regulation on intestinal epithelial cell migration and on the neonatal rat model of necrotizing enterocolitis(NEC).Methods: Intestinal epithelial cell lines were induced by rapamycin for 24 h to establish the autophagy model in vitro.The effect of h BD3 on m TOR autophagy signaling pathway in intestinal epithelial cells was detected by transmission electron microscope(TEM),western blotting and immunofluorescent techniques.AMD3100(CXCR4 inhibitor)and CXCR4 small interfering RNA were used to testify that CXCR4 signaling pathway is essential for the above process.Wound healing assay and transwell migration assay were measured to clarify the relationship between h BD3-mediated autophagy regulation and intestinal epithelial cell migration,which was verified by the Atg-7-deficient enterocyte generated by the transfection of ATG7 si RNA.In addition,Rho activation and its downstream effectors such as myosin light chain 2(MLC2)and F-actin were assessed.Newborn Sprague-Dawley rats were induced by formula feeding,asphyxia-cold stimulation exposure to establish a rat model of NEC.Rat pups were divided randomly into four groups: control+NS(n=11),control+rapamycin(n=11),NEC+NS(n=12)and NEC+h BD3(n=8).Rats in group control+NS and group control+rapamycin were mother-fed.Administration of rapamycin(5mg/kg/d),h BD3(100ug/kg/d)or the same dose of normal saline was conducted respectively 1h before asphyxia stimulation.Body weight and survival time were recorded in detail.18 h before the end of the experiment,Brd U(50mg/kg)was intra-peritoneal injected to assess the migration of intestinal epithelial cells.Hematoxylin and eosin(H&E)staining was used for pathologists to score the pathological changes blindly according to the published NEC scoring system.Intestinal tissues and serum were collected to measure the expression of autophagy related genes and inflammatory cytokines,enterocyte migration and intestinal mucosal integrity were assessed.Results: According to the expression of autophagy-related proteins,the autophagy flux in Caco2 increased significantly after being incubated with 50 n M rapamycin for 24 h.Accordingly,the autophagy in IEC-6 was effectively induced by rapamycin at the concentration of 200 n M for 24 h.h BD3 could inhibit the intestinal epithelial cell autophagy induced by rapamycin via the phosphorylation of AKT and m TOR(P < 0.05).After being pretreated with 10?M AMD3100 or CXCR4 si RNA(The knockdown efficiency of CXCR4 si RNA was approximately 60% and resulted in a remarkable reduction in CXCR4 expression compared with control si RNA(P < 0.05)),the autophagy-associated proteins Beclin1 and LC3-II/LC3-I ratio increased,while protein p62,p-AKT and p-m TOR were showed in decline expression.The migration distance of IEC-6 cells was significantly decreased in the group incubated with rapamycin compared with the unstimulated control(P < 0.05).However,pretreatment of h BD3 could noticeably reverse the inhibition effect of rapamycin(P < 0.05).IEC-6 cells were transfected with ATG7 si RNA to confirm the effect of h BD3-mediated autophagy suppression on the migration of intestinal epithelial cells.Knockdown efficiency of ATG7 si RNA was more than 60% and markedly down-regulated the protein expression of ATG7 compared with control si RNA(P < 0.05).The migration distance of IEC-6 cells transfected with ATG7 si RNA increased obviously compared with autophagy induction group no matter whether h BD3 incubation or not(P < 0.05),which indicated that h BD3 did regulate intestinal epithelial cell migration by inhibiting autophagy.Autophagy induced by rapamycin down-regulated the activation of Rho,phosphorylation of Myosin Light Chain 2(MLC2)and the accumulation of F-actin in IEC-6 and Caco2 cells,h BD3 could reverse the inhibition effect of rapamycin to some extent.Rats in group NEC+NS gained obvious weight loss,and the survival rate was significantly decreased(42%)when compared with rats in group control+NS(100%),however,the body weight and survival rate increased to some extent after h BD3 intervention and the difference was statistically significant(P < 0.05).The degree of bowel damage,evaluated by the histological scores of the ileum,was reduced in group NEC+h BD3,compared with the group NEC+NS,with a mean NEC score of 1.33 and 2.52,respectively(P < 0.05).what's more,in group NEC+NS,inflammatory cytokines including IL-6,IL-10 and TNF-a were noticeably increased in the ileum and serum,but the intervention of h BD3 could relatively down-regulated the indicated cytokines(P < 0.05)and then attenuated the inflammatory injury of rat NEC model.The expression of autophagy related genes(Beclin1 and LC3)in group NEC+NS was significantly increased,which was noticeably reduced by h BD3 administration(P < 0.05).The intestinal epithelial cells migration ability was markedly decreased in group NEC+NS,which was effectively up-regulated after h BD3 administration(P < 0.05).ZO-1 staining loss was observed in group NEC+NS,which was consistent with occludin staining.The inhibition of ZO-1 and occludin expression could be prevented to some extent after h BD3 intervention.Conclusion: This study unveiled that h BD3 could specifically bind to CXCR4 and down-regulate autophagy via the AKT-m TOR signaling pathway,and then significantly mitigate the inhibition effect on intestinal epithelial cell migration induced by autophagy via the promotion of Rho activation,MLC2 phosphorylation and F-actin accumulation.h BD3 adminstration could attenuate the intestinal damage,reduce the excessive autophagy and promote intestinal epithelial cells migration and mucosal integrity in NEC rats.