ABSTRACT
Recent reports indicate that epidermal growth factor (EGF) plays a crucial role for graft adaptation in rat model of small bowel transplantation (SBT). The administration of EGF enhances intestinal cell proliferating rate and the recovery of mucosal structure. However, the effect of EGF on biological functions including glucose absorption in intestinal graft remains to be elucidated. SBT was performed in the two-step procedure. On the first step, intestinal graft (30-cm jejunum) from Brown Norway rats was exteriorized through abdominal wall as a Thiry-Vella loop in recipient Lewis rats for one week. On the second surgery (POD 7), recipient jejunum was replaced orthotopically by the graft, and transplanted rats were treated intraperitoneally with EGF or its vehicle for 3 days. Analyses of histology and biological functions in the graft were done at POD 14. EGF increased both levels of villus height and crypt depth in the graft of transplanted groups. EGF enhanced the glucose absorption as well as the induction of sodium glucose cotransporter 2- to 3-fold in transplanted groups. Further, EGF stimulated the activities of disaccharidase (maltase and sucrase) and the induction of dipeptide cotransporter. These results demonstrate that EGF enhances the structural and functional adaptation of intestinal grafts after SBT. EGF may be useful therapy for patients following intestinal transplantation.
Subject(s)
Adaptation, Physiological/drug effects , Epidermal Growth Factor/pharmacology , Graft Survival/drug effects , Intestine, Small/transplantation , Animals , Blotting, Western , Body Weight , Disaccharides/metabolism , Glucose/metabolism , Intestine, Small/drug effects , Membrane Glycoproteins/metabolism , Monosaccharide Transport Proteins/metabolism , Rats , Rats, Inbred Lew , Sodium-Glucose Transporter 1 , Statistics, NonparametricABSTRACT
Epidermal growth factor (EGF) is one of the trophic factors for intestinal adaptation after small bowel transplantation (SBT). A recent report indicates that nitric oxide (NO) has cytoprotective effects on bacterial translocation (BT) after SBT. We hypothesized that EGF stimulates the expression of the inducible NO synthase (iNOS) gene in the graft after SBT, followed by increased production of NO, resulting in the decrease of BT. Intestinal epithelial cells (IEC)-6 were treated with EGF and/or IL-1beta in the presence and absence of phosphatidylinositol 3-kinase (PI3-kinase) and EGF receptor kinase inhibitors (LY-294002 and tyrphostin A25). The induction of NO production and iNOS and its signal molecules, including the inhibitory protein of NF-kappaB (IkappaB), NF-kappaB, and Akt, were analyzed. IL-1beta stimulated the degradation of IkappaB and the activation of NF-kappaB but had no effect on iNOS induction. EGF, which had no effect on the NF-kappaB activation and iNOS induction, stimulated the upregulation of type 1 IL-1 receptor (IL-1R1) through PI3-kinase/Akt. Simultaneous addition of EGF and IL-1beta stimulated synergistically the induction of iNOS, leading to the increased production of NO. Our results indicate that EGF and IL-1beta stimulate two essential signals for iNOS induction in IEC-6 cells: the upregulation of IL-1R1 through PI3-kinase/Akt and the activation of NF-kappaB through IkappaB kinase, respectively. Simultaneous addition of EGF and IL-1beta can enhance the production of NO, which may contribute to the cytoprotective effect of EGF against intestinal injury.
Subject(s)
Epidermal Growth Factor/pharmacology , Epithelial Cells/metabolism , Interleukin-1/pharmacology , Intestinal Mucosa/metabolism , Nitric Oxide/biosynthesis , Animals , Blotting, Northern , Blotting, Western , Cells, Cultured , Chromones/pharmacology , Drug Synergism , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , ErbB Receptors/antagonists & inhibitors , I-kappa B Proteins/metabolism , Intestines/cytology , Intestines/drug effects , Morpholines/pharmacology , NF-kappa B/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type II , Phosphoinositide-3 Kinase Inhibitors , Protein Serine-Threonine Kinases/biosynthesis , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins c-akt , RNA, Messenger/biosynthesis , Rats , Staphylococcal Protein A/pharmacologyABSTRACT
BACKGROUND/AIMS: Nuclear translocation and DNA binding of NF-kappaB is essential, as interleukin-1beta (IL-1beta) stimulates the induction of inducible nitric oxide synthase (iNOS) gene expression in hepatocytes. However, recent evidence indicates that the activation of NF-kappaB is not sufficient to induce the NF-kappaB-dependent transcription, and the existence of a second signaling is postulated. METHODS: Primary cultured hepatocytes were treated with IL-1beta, and the expression of iNOS and type 1 IL-1 receptor (IL-1R1) was analyzed in the presence of antisense of IL-1R1, phosphatidylinositol 3-kinase (PI3K) inhibitor, proteasome inhibitor and hypoxia. Moreover, the activities of Akt and NF-kappaB were recorded and the cotransfection was carried out. RESULTS: Antisense experiment revealed that IL-1R1 was required for iNOS transcription. IL-1beta markedly stimulated the induction of IL-1R1, which preceded the induction of iNOS. The IL-1R1 induction was found to be PI3K/Akt-dependent but NF-kappaB-independent. The up-regulation of IL-1R1 was associated with the second activation of Akt, which accelerated the phosphorylation of NF-kappaB p65 subunit. Cotransfection experiments revealed that Akt increased the transcriptional activity of iNOS gene promoter. CONCLUSIONS: These results indicate that the up-regulation of IL-1R1 in concert with the activation of NF-kappaB is required for the transcriptional activation of iNOS gene.
Subject(s)
Hepatocytes/metabolism , Nitric Oxide Synthase/genetics , Receptors, Interleukin-1/genetics , Animals , Base Sequence , Cells, Cultured , DNA/genetics , Hepatocytes/drug effects , Interleukin-1/pharmacology , Models, Biological , NF-kappa B/metabolism , Nitric Oxide Synthase Type II , Oligodeoxyribonucleotides, Antisense/genetics , Oligodeoxyribonucleotides, Antisense/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Receptors, Interleukin-1 Type I , Recombinant Proteins/pharmacology , Signal Transduction , Up-RegulationABSTRACT
We reported that epidermal growth factor (EGF) stimulated graft adaptation in a rat model of syngeneic small bowel transplantation. However, graft rejection is a severe problem with clinical small bowel transplantation, because small intestinal wall contains large amounts of lymphoid tissue. Studies were performed to investigate the effect of EGF on allogeneic graft adaptation after small bowel transplantation in rats treated with an immunosuppressant FK506. The transplanted animals received intraperitoneally EGF or saline (untreated) after surgery and were examined for analysis one week later. EGF-treated group markedly enhanced the water absorption and induction of sodium glucose cotransporter (SGLTI) as compared with EGF-untreated group. EGF-treated group also increased the mucosal crypt depth and its cell proliferating rate, although there was no significant difference in the mucosal villus height between the two groups. These results indicate that EGF accelerates intestinal allograft adaptation in part by the recovery of mucosal structure and function after small bowel transplantation in rats. EGF may have relevance to promote graft function in clinical small intestinal transplantation.
