Temporal Proteomic Analysis of Intestine Developing Necrotizing Enterocolitis following Enteral Formula Feeding to Preterm Pigs Pingping Jiang,† Per T. Sangild,§,# Wai-Hung Sit,† Heidi Hoi-Yee Ngai,† Ruojun Xu,† Jayda Lee Ann Siggers,§ and Jennifer Man-Fan Wan*,†,# Division of Agricultural, Food and Nutritional Science, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong S.A.R., P.R. China, Department of Human Nutrition, Faculty of Life Science, University of Copenhagen, 30 Rolighedsvej, DK-1958 Frederiksberg C, Denmark Received August 14, 2008
Necrotizing enterocolitis (NEC), a serious gastrointestinal inflammatory disease, frequently occurs in preterm neonates that fail to adapt to enteral nutrition. A temporal gel-based proteomics study was performed on porcine intestine with NEC lesions induced by enteral formula feeding. Functional assignment of the differentially expressed proteins revealed that important cellular functions, such as the heat shock response, protein processing; and purine, nitrogen, energy metabolism, were possible involved in the early progression of NEC. Keywords: premature neonates • enteral formula feeding • necrotizing enterocolitis • 2-DE • mass spectrometry • temporal study
Introduction Just after birth, neonates experience a dramatic nutritional change from obtaining parental nutrients via the placenta to uptake of nutrients from gastrointestinal tract (GIT). To cope with this change, the GIT of neonates has to undergo a fast maturation in a very short time period, which includes an increase in intestinal mass, DNA, gut motility, nutrient hydrolysis and absorption by enterocytes.1 Stimulated by enteral nutrition, the gut immune system also has to adapt to billions of colonizing microbes. This process is diet-dependent and affected by mode and route of nutrient administration.2 Feeding with total parenteral nutrition (TPN) after birth may be required to avoid digestive overload, but this may also be associated with mucosal atrophy and reduced digestive and absorptive capacity. Conversely, enteral feeding, especially with mother’s milk, is believed to be able to improve the maturation of intestine in newborns, even following preterm birth3,4 However, in preterm neonates, the intestine is relatively short and has a smaller absorptive area, lower hydrolase enzyme activities and absorption capacity as well as an immature gut immune system.2 Together, these factors may predispose to development of necrotizing enterocolitis (NEC) after receiving enteral feeds for certain periods.1 NEC is the most severe neonatal GIT emergency and is characterized by severe inflammatory-response bleeding and hemodynamic instability in the intestine. The incidence of NEC is 3-7% in preterm and low birth weight infants with surgical treatment required in 27-50%
of the cases and an overall mortality of 12-30%.5 Infants that recover from NEC may still suffer from complications, such as intestinal obstruction, liver failure, short bowel syndrome and associated nutritional deficiencies.6 After several decades of research, the pathophysiology of NEC remains unclear. The proposed mechanisms involve the breakdown of intestinal mucosal barrier, translocation of bacteria and endotoxins, excessive production of pro-inflammatory mediators and increased apoptosis that together lead to necrosis of enterocytes. Among the epidemiologic risk factors, prematurity and enteral formula feeding are found most consistently.6 NEC may be initiated with a rapid advancement of feeding that exceed the digestive capacity, leaving unfavorable metabolites to disturb the microflora balance.6,7 Our previous study has shown that 24 h of enteral formula feeding with formula diet to premature piglets can trigger multiple proteome responses leading to NEC.8 To further understand the dynamic cellular responses of the premature GIT to enteral formula feeding (FF), a temporal proteomic study was performed in this study aiming to identify proteins in NEC with differential expression over time. We believe that the synergistic influence of these proteins is a key part of the overall phenotypic response to FF and development of NEC. Functional assignment of proteins into clusters revealed that the stress response and detoxification, protein processing, and metabolism pathways were highly affected over time of NEC development.
Materials and Methods * To whom correspondence should be addressed. Dr. Jennifer Man-Fan Wan, School of Biological Sciences, Kardoorie Biological Science Building, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, P.R. China. Tel: 852-22990838. Fax: 852-25599114. E-mail:
[email protected]. † The University of Hong Kong. § University of Copenhagen. # These authors contributed equally to this work.
72 Journal of Proteome Research 2009, 8, 72–81 Published on Web 11/05/2008
Piglet Model of NEC. The development of the FF inducedNEC premature piglet model has been described previously.7 Preterm piglets from three sows were delivered by caesarean section at 107-108 day (92%) gestation. All delivered piglets were housed individually in the infant incubators (Air-Shields) 10.1021/pr800638w CCC: $40.75
2009 American Chemical Society
Enteral Formula Feeding Induced NEC in Premature Pigs kept at 34-37 °C and 80-100% humidity and supplied with extra oxygen (0.5-2 L/min). Immediately after birth, a vascular catheter (infant feeding tube 4F; Portex, Kent, U.K.) was inserted into the dorsal aorta of the anesthetized newborn pigs. After recovery from surgery, all animals were given TPN for 2 days as described previously.3,4 After TPN feeding, one group of piglets (n ) 5) was killed and termed as group 0 h. Other piglets were changed to enteral formula feeding (15 mL/kg body weight) for 8, 17, or 24 h before being killed (n ) 5 each group). The formula diet for enteral feeding, which contained Peptide 2-0 (SHS, Liverpool, U.K.) 80 g, Maxipro (SHS,Liverpool, U.K.) 70 g, and 75 mL of Liquigen-MCT (SHS, Liverpool, U.K.) per 1 L water, was designed to match the composition of sow’s milk during lactation as previously described.7,9 The protein source in the formula diet was a mixture of low-molecular-weight peptides of both vegetable origin and from cow’s milk whey. It is considered an ideal protein source adapted to low luminal proteolytic activity and does not result in the normal formation of a solid curd in the stomach of preterm pigs.7,9 Tissue Collection. The piglets were killed after 2-3 h following the last feeding using sodium pentobarbital (200 mg/ kg) intra-arterially. The GIT were immediately removed, and approximately 6 cm sections of the middle small intestine were cut with half saved for histological analysis and the other half saved for proteomic analysis. Tissue sections for histological analysis were fixed in 4% paraformaldehyde for 48 h before storage in 70% ethanol at 4 °C. The intestine samples saved for proteomics analysis were frozen in liquid nitrogen and kept at -80 °C. The National Committee on Animal Experimentation in Denmark approved all procedures. Histology Assessment of NEC. The paraformaldehyde fixed samples were embedded in paraffin and sectioned at 3 µm before they were mounted on the slides for staining with hematoxylin and eosin. All slides were checked with a light microscope (Orthoplane, Leitz) and imaginized by NIH Image J software (version 1.22c, NIH). The NEC scoring system for intestinal tissue was based on a system developed earlier1 according to the following criteria: 0 ) normal histological appearance, 1 ) mild local disruption of villus tips and no changes in the submucosal layer, 2 ) local villus disruption and separation of the submucosal layer, 3 ) regional villus disruption, blood congestion and separation of the submucosa and lamina propria, 4 ) extensive and complete destruction of villus architecture, presence of blood congestion and transmural necrosis. With the use of this histopathological scoring system, piglets scoring 2 or greater in any region of the gastrointestinal tract were deemed NEC. Protein Extraction. Protein extraction has been previously described.8 Briefly, the tissue samples were disrupted with a tissue teaser (Biospec Products, OK) in a cocktail buffer (1% Triton X-100, 25 mmol/L Hepes, 150 mmol/L NaCl, 1 mmol/L EDTA disodium salt, 1 mmol/L DTT) containing also Protease Inhibitor Cocktail Set III (Bio-Rad, CA). The resulting ice-kept tissue homogenate was centrifuged at 15 800g for 30 min at 4 °C. The superfluous salt in the supernatant extract was removed with trichloroacetic acid (TCA)-acetone solution (20% TCA, 20 mmol/L DTT in acetone). Excess TCA was removed by washing twice with acetone containing 20 mmol/L DTT. The protein pellet was resuspended in a buffer containing 7 mol/L urea, 2 mol/L thiourea, 4% Chaps, 100 mmol/L DTT, and 5% glycerol. The final protein solution was stored at -80 °C until further analysis of 2-DE. Protein concentration of the samples was determined by Bio-Rad Protein Assay (Bio-Rad, CA,).
research articles Two-Dimensional Gel Electrophoresis (2-DE). For the running of the 2-DE, a total of 20 gels were run, with one gel per animal from each of the four time points (0, 8, 17 and 24 h). The conditions for setting up the 2-DE follows the modified method as described before.8 Protein samples (100 µg) were applied onto an 18 cm ReadyStrip IPG Strips (pI 3-10 NL, BioRad, CA), and the strips were rehydrated for 7 h with rehydration buffer (9.5 mol/L urea, 2% CHAPS, 0.28% DTT, 0.5% IPG Buffer PI 3-10, 0.002% Bromophenol blue) in a Ettan IPGphor III IEF System (GE Healthcare). After an active rehydration step for 7 h, isoelectric focusing (IEF) was carried out on an Ettan IPGphor IEF system following a five-step program. Step 1 of the program was performed linearly up to 500 V in 1 h, held at 500 V for 3 h, followed by a linear increase up to 10 000 V in 3 h, followed by a linear increase up to 10 000 V in 3 h and finally, held at 10 000 V to reach a total of 90 000 Vh. Focused IPG gel strips were equilibrated for 15 min in a solution (50 mmol/L, pH 8.8, Tris-HCl, 6 mol/L urea, 30% glycerol, 2% SDS) containing 20 mmol/L DTT) followed by incubation with the same buffer containing 20 mmol/L iodoacetamide (IAA) for another 15 min. Equilibrated gel strips were then placed onto a 1.0 mm-thick 12.5% polyacrylamide gels and SDS-PAGE was carried out at a constant current in a PROTEAN xi II cell (BioRad, CA). After electrophoresis, gels were fixed in fixation solution (10% ethanol, 7% acetic acid in water) for at least 30 min before stained with SYPRO Ruby Protein Stain (Bio-Rad, CA) according to the manufacturer’s guide. Image Analysis and Protein Identification. The stained 2-DE maps were obtained by scanning the gels using the Molecular Imager PharosFX Plus System (Bio-Rad). Analysis of the 2-DE maps was undertaken using the PDQuest 8.0 for Windows (BioRad). The analysis of protein spots by the software included background subtraction, spots detection and volume normalization. A total of 20 gels (5 from each time point group) were normalized and each spot on the gel was assigned a spot number by the software with reference to the reference gel automatically. Any underdetected spots were manually assigned with the number according to the reference gel by the researcher. Expression data of protein spots, as Quantity, was exported to SPSS 11.5 for Windows for statistical analysis. Protein Identification by MS. All 2-DE gels were visualized with silver nitrite for spots picking. Spots were cut out manually and saved in microtiter plate for mass spectrometry (MS) analysis. The in-gel trypsin digestion and other pre-MS process were performed as described previously.10 MALDI-TOF/TOF MS (Matrix Assisted Laser Desorption Ionization-Time Of Flight/Time Of Flight MS) for protein identification was carried out on a 4800 MALDI TOF/TOF Analyzer (AppliedBiosystem, CA) with MS setting as MS reflector positive and scan range 900-400 Da and MS/MS setting as 2KV positive, CID on and 5 monoisotopic precursors selected. The identity searching is a combination of Peptide Mass Fingerprint (PMF) and MS/MS search on GPS Explorer Workstation (AppliedBiosystem, CA) with in-house searching engine, Mascot, with searching taxonomy limited as Mammalia (mammals) against NCBInr database. The search parameters allowed modification as carbamidomethyl (C) and Oxidation (M) and a mass tolerance 75 ppm and up to 1 missed enzymatic cleavage. Basic information of identified proteins were obtained from MS platform, which included Protein name, GenInfo Identifier, Protein score and Searched pI and relative molar mass (Mr). The apparent pI and Mr are calculated according to their position of the gels Journal of Proteome Research • Vol. 8, No. 1, 2009 73
research articles
Figure 1. Representative histopathological pictures of Hematoxylin and eosin stained intestine from preterm piglets fed with enteral formula for 0 h (A), showed normal villus architecture (score ) 0), for 8 h (B) showed mild local disruption of villus tips with no changes in the submucosal layer (score ) 1), 17 h (C) showed local villus disruption and separation of the submucosal layer (score ) 2) and for 24 h (D), showed extensive and destruction of villus architecture with presence of transmural necrosis (score ) 4).
and confirmed with the search data. In this study, a protein match with a Protein score >71 was considered significant. Western Blot Analysis. Briefly, 25 µg of protein extracted was mixed with sample buffer (62.5 mmol/L Tris, pH 6.8, 25% glycerol, 2% SDS, 350 mmol/L DTT, 0.01% Bromophenol blue) with a ratio 1:1 (v/v) and was resolved by electrophoresis on a 12.5% SDS-PAGE gel. Anti-GDI 1, anti-GDI 2, anti-PHB and anti-GRP78 (ABCAM, U.K.) were used to detect the expression of GDI 1, GDI 2, PHB and GRP78 with dilution of 1:2000, 1:2000, 1:500 and 1:1000, respectively. The protein bands were visualized by Immun-Star HRP Peroxide Buffer plus Immun-Star HRP Luminol/Enhancer (Bio-Rad, CA) onto a blue film. The density of the protein bands was detected using Quantity One (a 1-D analyzing software, Bio-Rad, CA) and expressed in %Adj.Vol. The data were exported into SPSS 11.5 for Windows for statistical analysis. Statistical Analysis. All data are expressed as mean ( SEM. Differences between time points were analyzed for statistical significance by One-Way Analysis of Variance (ANOVA) and the Levene’s test for testing between any two time points using SPSS software (version 11.5). A p value of
