Effect Of Intrauterine Growth Restriction And Nutrition On Gut Development And Necrotizing Enterocolitis In Neonatal Pigs

EXPERIMENT 1:GUT ADAPTIVE RESPONSE TO ENTERAL NUTRITION AND INCIDENCE OF NECROTIZING ENTEROCOLITIS IN INTRAUTERINE GROWTH RESTRICTION PIGLETSThe current study includes three sections to investigate the effect of intrauterine growth restriction (IUGR) on the gut development and NEC incidence of both term and preterm piglets.Firstly, we determined the effect of IUGR on internal visera and gut development of term piglets. Piglets with birth weight 2SD below the mean birth weight of experimental population were allocated to IUGR, whereas piglets with birth weight near the mean birth weight (±0.5SD) were allocated to normal birth weight(NBW).According to this selective criteria,12 IUGR piglets and 12 NBW piglets were selected from 18 term litters. The results indicated there were no significant differences for relative internal visera weight.gut growth and digestive enzyme activities between IUGR and NBW piglets at the time of birth. After 2 days of suckling,however,IUGR piglets had impaired gut morphology (villi/crypt, ileum density) and ileum sucrase activity (P<0.05),further more.cecum and ileum mucosal bacteria adhesion appeared to be higher in IUGR piglets(P<0.05) but no NEC was observed.Secondly, the gut responses to enteral nutrition (Colostrum or Formula) between IUGR and NBW piglets were investigated.Preterm piglets were delivered by caecerean section at 92%gestation (105d). Piglets with birth weight 1.5 SD below the mean birth weight of experimental population were allocated to IUGR (n=25),while piglets with the birth weight near the mean birth weight (±SD) were defined as NBW(n=63). In order to study the gut respone of IUGR or NBW piglets to total enteral nutrition (TEN), colostrum vs.formula, IUGR piglets were allocated to colostrum (n=8) or formula feeding group(n=17), NBW piglets were allocated to colostrum (n=21) and formula feeding group(n=42),respectively. All piglets were fixed orogastric tubes for TEN and reared in infant incubators.The results showed IUGR piglets had higher mortality rate than NBW piglets(24%vs.18%,P=0.18) within 24 hours of feeding.Regardless of enteral nutrition type, strikingly.the survival IUGR piglets had higher relative small intestine weight than NBW piglets (+25-30%, P< 0.05, table),and tissue-specific ApA and ApN enzyme activities were higher than NBW piglets (P<0.05),but no differences were observed for mucosa percentage and gut morphology (villi/crypt) between IUGR and NBW piglets. Correspondingly, mucosal bacteria adhesion from FISH(fluorence in situ hybridization) was not different between IUGR and NBW piglets.eventually no difference was observed on NEC incidence between groups. Interestingly, analysed across birth weight (IUGR or NBW), colostrum improved mucosa percentage, villous height and digestive enzyme activities (such as ApA,ApN,DPPIV and maltase)(P<0.05). Next, we observed the effect of IUGR on gut responses to enteral nutrition when total parenteral nutrition (TPN) was introduced to preterm piglets for the first 2 days after brith. The selective criteria for IUGR or NBW piglets is same as section 2, totally 21 IUGR piglets were allocated to colostrum (n=12) and formula feeding group(n=9),84 NBW piglets were allocated to colostrum (n=65) and formula feeding group (n=19). All piglets were fitted with orogastric tubes and arterial catheters insered into the dorsal aorta via the transected umbilical cord for TEN and TPN.respectively.All piglets were reared in infant incubators.The results indicated the mortality rate of IUGR was higher than NBW piglets (31%vs.8%, P<0.01), consistently, the rectal temperature of IUGR piglets was lower than NBW piglets(35.7±0.3 vs.37.0±0.3, P<0.01). Adrenal weight was increased under colostrum or formula feeding (+40-50%,P<0.05). Similar to piglets without TPN introduction, regardless of enteral nutrition type, IUGR piglets had higher relative small intestine weight and intestinal length than that of NBW piglets(+50-80%, P< 0.01 and+30-40%, P < 0.05), however, the intestinal morphology between IUGR and NBW piglets was not significantly different. Digestive enzyme activities appeared consistent with the piglets without TPN,most of digestive enzyme activities were dependent on enteral nutrition types (colostrum vs.formula,P<0.05)but not birth weight(IUGR or NBW). Likely.no differences were observed for bacteria adhesion and NEC incidence between IUGR and NBW piglets. In addition, regardless of birth weight, NEC incidence with TPN introduction was higher than piglets without TPN (colostrum feeding group:61%vs.34%, P<0.01; formula feeding group: 22%vs.5%, P=0.095).Analysed across feeding strategy and birth weight.colostrum decreased NEC incidence (P<0.05). The conclusion could be the gut responses to enteral nutrition in term IUGR piglets were different with that in preterm IUGR piglets. Preterm IUGR piglets survived in postnatal period have adaptive gut responses to enteral nutrition. TPN introduction immediately after birh couldn't improve mortality rate of piglets, conversely predispose neonates to higher risk of NEC.In addition,colostrum improves gut adaptation and reduces NEC incidence in neonates.EXPERIMENT 2:NUTRIENTS UPTAKE AND CYTOKINES GENE RESPONSES OF PRIMARY PIG INTESTINAL EPITHELIAL CELLS TO ENTEROTOXIN ARE DEVELOPMENTALLY DEPENDENTTo investigate if the response of intestinal epithelial cells(IECs) to enterotoxin is developmentally dependent, caesarean-delivered preterm piglets(105 days gestation) and term piglets (114 days gestation) were employed for isolation of primary IECs at day 0 (n=6) and after 2 days colostrum enteral feeding(n=6). Four treatments were applied to the short-term cultured cells including control group(DMEM-F12), LPS group (100μg LPS/ml DMEM) TNF-a group (50μg TNF-α/ml DMEM) and NEC-MIX group (200μl NEC-MlX/ml DMEM). NEC-MIX was the isolated supernatant from the intestinal content of piglets suffering necrotizing enterocolitis (NEC). After 4 hours culture.cells were collected for nutrients uptake(glucose and leucine) and cytokines gene expression (TNF-a,IL-6 and INF-y).The results indicated 1) the leucine uptake of IECs from both preterm and term piglets were significantly decreased in NEC-MIX group and TNF-a group at birth (-25-30%, P<0.05),but only IECs of preterm piglets had lower glucose uptake (-28%and-30%, P<0.05) in LPS and NEC-MIX group. After 2 days of enteral feeding, only leucing uptake in IECs of preterm piglets was significantly reduced by NEC-MIX (P<0.05),no other enterotoxin reduced the nutrients uptake of IECs.2) Relative to mature IECs from term piglets, premature IECs from preterm piglets had obviously lower leucing uptake at birth after cultured with NEC-MIX and TNF-a, similarly glucose uptake was lower than that of mature IECs when it came to LPS or NEC-MIX (P<0.05). After 2 days of enteral feeding.the developmental regulation of nutrients uptake (Leucing and Glucose) in IECs was only observed in NEC-MIX group.3)Correspondingly,cytokine genes expression were stimulated by enterotoxin, TNF-a gene expression was increased by NEC-MIX in both premature and mature IECs (P<0.05), whereas LPS or NEC-MIX-stimulated IL-6 and INF-γgenes over-expression were only occurred in premature IECs(P<0.05).4)Thus, TNF-a and IL-6 genes expression of IECs were developmentally dependent when suffering NEC-MIX stimulus (P<0.05). LPS-stimulated IL-6 gene expression tended to increase in premature IECs (P=0.09). The conlusion is the nutrients uptake and cytokines expression of IECs under stimulation of proinflammatory factors are developmentally dependent, and this results imply to us the possibility that cytokines play a role to regulate nutrients uptake in IECs.which together play a crucial role in NEC.EXPERIMENT 3:PROTEOMIC STUDY OF INTESTINAL NECROTIZING ENTEROCOLITIS IN NEONATAL PIGLETSTo further study the molecular mechansim of necrotizing enterocolitis (NEC), gel-based differential proteome was employed to investigate the proteins expression differences between NEC and healthy tissue, which were collected from preterm piglets and term piglets fed formula for 48 hours.respectively. According to the results from tissue morphology,2-Dimension electrophoresis was used to scatter proteins that later identified via mass spectrum,the scattered proteins on the SDS-PAGE gel were dyed by SYBRORuby and laser-scanned to obtain pictures for analysis of PDQuest(8.0) which was used to match protein spots, standardized spots and analysis management.The results indicated NEC tissues showed necrosis.dislocation of mucosal villi to baserlateral and lamonia, proteomic study demonstrated the significant changes of 14 proteins expression along with NEC, among them signal transduction proteins (G protein,GDP dissociation inhibitor) were increased (+160-200%, P<0.05). DNA synthesis inhibitor PHB and iron transferring protein (Serotransferrin) were 2.1 and 3.3 times more than health tissues (P<0.05), respectively. In addition to these upregulated proteins in NEC tissue, the obveriously down-regulated proteins including cell structure-related proteins (Actin,Keratin 10,), metabolism-related proteins (FBCA, LDH, PCK.GIyRS and ETF-QO) and serum albumin precursor were observed (-200-300%, P<0.01 or 0.05). The protein changes in NEC tissue imply to us the dysfunction of energy metabolism,cellular skeleton and over-oxidation in gut tissue is responsible for NEC.