Study On The Intervention Of Lactoferrin In A Neonatal Rat Necrotizing Enterocolitis Model Through Inhibiting Epithelial-mesenchymal Transition

ObjectiveThe aim of this study was to investigate the interventional effect of lactoferrin(LF)on neonatal rats with necrotizing enterocolitis(NEC)by inhibiting intestinal epithelialmesenchymal transition(EMT)and its possible molecular mechanism.Methods1.To evaluate the effect of LF on protecting neonatal rats from NEC by inhibiting EMTNEC model of newborn Sprague-Dawley rats was established by formula feeding combined with asphyxia and cold stimulation.Neonatal rats were randomly divided into four groups: Control(n=12),Control + LF(n=13),NEC(n=33)and NEC + LF(n=20).Both pups in Control and Control + LF group were breastfed.We administered LF(200μg/time)or the same dose of normal saline to pups via the gastric tube 30 min before asphyxia stimulation,twice a day for three consecutive days.Body weight,survival rate,and intestinal pathology scores were compared between groups.The terminal ileum tissues were collected to measure the expression of EMT-related proteins(E-cadherin,N-cadherin and vimentin)through immunohistochemistry,immunofluorescence staining and western blot.2.To evaluate the effect of LF on LPS-induced IEC-6 cell EMT model in vitroIn vitro,rat-derived intestinal epithelial cell line IEC-6 cells were divided into four groups: Control,Control + LF(200μg/ml),LPS(50μg/ml)and LF + LPS.The cell morphology of each group was observed under an inverted microscope.The expression of EMT-related proteins was detected by western blot.Pull down assay was performed to detect the activated level of ADP-ribosylation factor 6(Arf6)in each group.Finally,to verify whether LF regulates EMT through Arf6 signaling,cells were treated with Arf6 activation inhibitor Secin H3(20μM)before LPS stimulation and EMT markers were assessed.Results1.The body weight of rats in the Control and Control + LF group increased steadily,while the NEC group showed continuous weight loss throughout the NEC modeling experiment.Both pups in the Control and Control + LF group survived the entire 96 h protocol.In contrast,the NEC group showed a lower survival rate and a higher pathological score of intestinal injury.LF supplementation would significantly decrease NEC morbidity and mortality,and alleviate intestinal damage in experimental rat NEC models.Compared with the Control group,the protein expression of epithelial marker E-cadherin was suppressed in the NEC group,whereas the expression of mesenchymal markers N-cadherin and vimentin was higher,indicating the occurrence of EMT in the intestine of those experimental NEC rats.Conversely,EMT was dramatically inhibited in the NEC + LF group.2.In vitro,treatment with 50μg/ml LPS for 4 days can successfully establish the EMT model of IEC-6 cells.LF pretreatment appeared to significantly inhibit LPSinduced EMT changes in IEC-6 cells.Compared with the Control group,LPS stimulation also enhanced the activation of Arf6(elevated levels of Arf6-GTP).LF preincubation could obviously downregulate Arf6-GTP expression.Pull down assays revealed that the levels of activated Arf6 was clearly augmented upon LPS exposure,and Secin H3 pretreatment displayed a compromised effect.Interestingly,Secin H3 particularly ameliorated the reduced expression of E-cadherin by LPS and further downregulated N-cadherin and vimentin expression notably.ConclusionLF could significantly alleviate the severity of intestinal injury and improve the survival rate in experimental NEC rats,and its mechanism might be partially related to inhibiting the development of EMT by suppressing Arf6 activation.