HPA-axis responses during experimental colitis in the rat

We investigated the responses of the hypothalamic-pituitary-adrenal (HPA) axis during experimental colitis induced by intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid in the rat. On days 3 and 7 after induction of colitis, the corticotropin-releasing hormone (CRH) mRNA level in the parvocellular paraventricular nucleus (pPVN) of the hypothalamus was reduced, the plasma ACTH level remained at the basal level, and the plasma corticosterone (Cort) level was high. Induction of colitis on day 3 after adrenalectomy with Cort pellet replacement (ADX + Cort) resulted in a marked increase in CRH mRNA on day 7 after induction of colitis compared with noncolitic ADX + Cort animals. Pair feeding to match the food intake of the colitic animals resulted in no significant change in CRH mRNA in the pPVN, plasma ACTH, and Cort compared with healthy control animals. These findings indicated that CRH mRNA expression in the pPVN was inhibited by glucocorticoid feedback during this experimental colitis, and the decrease in food intake during colitis was not simply responsible for the expression of CRH mRNA. It is inferred that the HPA axis including the CRH level in the pPVN is altered in patients with inflammatory bowel disease.     

Increased disease activity in eNOS-deficient mice in experimental colitis

Oral dextran sodium sulfate (DSS, 3%) produces experimental colitis with many features of human inflammatory bowel disease (IBD), (leukocyte extravasation, cachexia, and histopathology). Previous studies suggest that the inducible nitric oxide synthase (iNOS) in blood cells or in the endothelium contribute to this injury. However, until now no study has been performed to directly evaluate the role of endothelial nitric oxide synthase (eNOS) in IBD. We compared disease activity in wild-type (eNOS^+/+) and eNOS-deficient (eNOS^−/−) mice in the DSS model of colitis. Administration of DSS induced weight loss, stool blood, and overt histopathology in both mouse strains. Disease activity was dramatically increased in eNOS^−/− mice compared to wild types. Histologically, eNOS-deficient mice had greater leukocyte infiltration, gut injury, and expressed higher levels of the mucosal addressin, MAdCAM-1. These results demonstrate that eNOS plays an important role in limiting injury to the intestine during experimental colitis and altered eNOS content and/or activity may contribute to human IBD.     

Increased activity and expression of matrix metalloproteinase-9 in a rat model of distal colitis

Matrix metalloproteinases may play a role in tissue remodelling and destruction associated with inflammation. We investigated activity and expression of matrix metalloproteinases in a rat model of colitis and tested the therapeutic potential of a synthetic inhibitor (CGS-27023-A). Colitis was induced by dextran sulphate sodium (at 5% in drinking water for 5 days) in a group of eight rats, whereas a matched control group received plain water. Activity and expression of matrix metalloproteinases were measured in colonic tissue homogenates using zymography and Western blot on days 3 and 5 after induction of colitis. In another set of experiments, two groups of colitic rats (20 per group) were treated with CGS-27023-A (20 mg/kg) or vehicle, respectively. On days 5 and 14, colonic mucosal lesions were blindly scored by microscopic examination. Induction of colitis led to a significant upregulation of matrix metalloproteinase-9 protein and its activity, but no change in matrix metalloproteinase-2 activity was observed. Treatment with CGS-27023-A significantly decreased the extent and severity of epithelial injury but did not influence mucosal repair. We conclude that increased activity of matrix metalloproteinases may contribute to epithelial damage in this model of colitis.     

Selective expression of detoxifying glutathione transferases in mouse colon: effect of experimental colitis and the presence of bacteria

Glutathione transferases (GSTs) play a central role in the cellular defense against harmful endogenous compounds and xenobiotics in mouse and man. The gastrointestinal channel is constantly exposed to bacteria, bacterial products, and xenobiotics. In the present study the distribution of alpha, mu, and pi class GSTs was examined immunohistologically in the colon of conventional and germ-free (GF) mice subjected to experimental colitis. The tissues samples were from conventional mice with and without colitis induced by dextran sulfate sodium (DSS); GF mice treated with DSS or carrageenan; and GF mice inoculated with normal mouse bacterial flora as well as with Lactobacillus GG. In conventional as well as in GF mice the mu and pi class GSTs showed reduced intestinal expression when colitis was induced. In contrast, the level of GSTs reacting with antibodies directed against the alpha class, in particular mGST A4-4, was elevated after induction of inflammation. Of special interest is mGST A4-4 because of its high catalytic activity with toxic products of lipid peroxidation. In the colon of conventionalized GF mice that were given mouse intestinal flora, the mGST A4-4 expression was increased with time for several weeks, but then showed a decrease to a normal level. Additionally, the inoculation of GF mice with Lactobacillus GG induced all the intestinal GSTs studied.     

NF1 gene expression in mouse fracture healing and in experimental rat pseudarthrosis.

Neurofibromatosis type 1 (NF1) is an inherited disease with an incidence of about 1:3000 worldwide. Approximately half of all patients with NF1 present osseous manifestations, which can vary from mild to severely debilitating changes such as congenital pseudarthrosis. In the present study, fracture healing of mouse tibia was followed and specimens were collected 5, 9, 14, and 22 days postoperatively. Experimental pseudarthrosis of rat was followed up to 15 weeks postoperatively. In situ hybridization and immunohistochemistry were used to demonstrate expression of NF1 tumor suppressor and phosphorylated p44/42 mitogen-activated protein kinase (MAPK), an indicator of the Ras-MAPK pathway. The results showed that ossified callus was formed in mouse fracture 22 days after the operation. The final outcome of rat pseudarthrosis was detected 9 weeks after the operation, presenting abundant cartilaginous callus at the pseudarthrosis. NF1 gene expression was noted in the maturing and in the hypertrophic cartilages during normal mouse fracture healing, and in rat pseudarthrosis. Phosphorylated p44/42 MAPK was detected in a subpopulation of the hypertrophic chondrocytes in both models. Furthermore, positive labeling for NF1 mRNA and protein was detected in endothelium in both the pseudarthrosis and in the fracture. In conclusion, NF1 gene expression and function are needed for normal fracture healing, possibly restraining excessive Ras-MAPK pathway activation.     

Increased somatostatin mRNA expression in periventricular nucleus of rat hypothalamus during hypoxia

We reported that hypoxia inhibited the growth hormone (GH) and induced somatostatin (SS) release from the hypothalamic median eminence (ME) of rats. This study is designed to examine the SS mRNA alterations in the periventricular nucleus (PeN) of the hypothalamus in rats and the possible involvement of glucocorticoid (GC) during hypoxia. Rats were exposed to hypoxia in a simulated hypobaric chamber. SS mRNA levels in the PeN were tested by in situ hybridization. Hypoxia of 5-km altitude (10.8% O(2)) for 2, 5 and 24 h increased the SS mRNA expression by 34.72%, 50.31% and 95.05% (p<0.05), respectively. Severe hypoxia of 7-km altitude (8.2% O(2)) enhanced the SS expression by 79.08% (p<0.01), 74.90% (p<0.01) and 71.40% (p<0.05), respectively. Prolonged hypoxia (5 km for 5 days) exposure augmented a 2.5-fold SS mRNA (p<0.001). One week post adrenalectomy (ADX), SS mRNA level was significantly increased. During hypoxia, 5 km for 5 h, SS mRNA in ADX rats was not further increased. An increased SS mRNA was showed by pretreatment with low dose of dexamethasone (DEX) (125 microg/kg, i.p.) to ADX animals but this increase was depressed by a high dose of DEX (500 microg/kg, i.p.). The data suggested that (1) hypoxia stimulated the expression of SS mRNA in the PeN of rat hypothalamus. (2) Increased circulating GC levels might play a role in upregulating the SS mRNA in the rat PeN during hypoxia.     

Expression of angiotensin II AT2 receptors in the rat skin during experimental wound healing.

We localized and characterized angiotensin II AT1 and AT2 receptors in the skin of 2-week-old rats during experimental wound healing. Both AT1 and AT2 were present in the skin. Three days after wounding, the expression of angiotensin II receptors was significantly enhanced in the dermis as well as in a localized band within the superficial dermis of the skin surrounding the wound. The major proportion of this increase was due to angiotensin II AT2 receptors. Our results suggest a physiological role for AT2 receptors in the process of tissue repair.     

Increased expression of lipocalin-type-prostaglandin D synthase in ulcerative colitis and exacerbating role in murine colitis

The pathogenesis of ulcerative colitis (UC) is unclear, but enhancement of disease activity by usage of nonsteroidal anti-inflammatory drugs suggests involvement of prostanoid in its pathophysiology. However, biological effect of prostaglandin (PG) D(2) on intestinal inflammation remains unknown. We investigated the expression of enzymes for PGD(2) synthesis, prostaglandin D synthase (PGDS), and its relation to the activity of colitis in UC patients. The role of lipocalin-type PGDS (L-PGDS) using a murine colitis model was also assessed. Tissue samples were obtained by colonic biopsies from patients with UC. Expression levels of mRNAs for L-PGDS and hematopoietic-type PGDS were investigated by quantitative RT-PCR. COX-2 and L-PGDS expression was investigated by immunohistochemistry. Localization of L-PGDS expression was also determined by in situ hybridization. In experimental study, mice were treated with dextran sodium sulfate in the drinking water to induce colitis. The degree of colonic inflammation was compared with L-PGDS(-/-) mice and control mice. The level of L-PGDS mRNA expression was increased in UC patients in parallel with disease activity. Colocalization of L-PGDS and cyclooxygenase (COX) 2 was observed in lamina proprial infiltrating cells and muscularis mucosa in UC patients. The level of hematopoietic PGDS mRNA expression did not differ from control mucosa. Dextran sodium sulfate treatment to L-PGDS(-/-) mice showed lower disease activity than control mice. We reported for the first time the presence of L-PGDS in the COX-2-expressing cells in the mucosa of active UC patients and that only L-PGDS increased with disease activity. An animal model study suggests that PGD(2) derived from L-PGDS-expressing cells plays proinflammatory roles in colitis.     

Increased transglutaminase activity during skin wound healing in rats

Outer, middle and inner layers from wounded or unwounded rat dorsal skin were separated and extracted first with buffer and then with Triton X-100 and dithiothreitol. The extracts and residues were assayed for transglutaminase activity and tissue transglutaminase antigen. Transglutaminase activities in all skin layers are increased in the period 1-5 days after wounding. Most of the increased activity is in the buffer-soluble fraction in the inner skin layer though there is no corresponding increase in antigen in this fraction. This suggests that there is production of activated soluble tissue transglutaminase in the wounded inner layer. In the 3-5 day wounded outer layer the largest fraction of both activity and antigen is associated with the insoluble residue remaining after extraction with Triton X-100. On DEAE-cellulose chromatography Triton X-100 extracts of the inner layer of wounded skin showed a single major peak of activity, corresponding approximately with rabbit liver transglutaminase; the outer layer showed the same peak plus a different one, eluting at lower salt concentration, which is thought to be epidermal transglutaminase.     

中性粒细胞弹性蛋白酶在小鼠实验性结肠炎中的表达及意义

目的：观察中性粒细胞弹性蛋白酶（NE）在葡聚糖硫酸钠（DSS）诱导的小鼠实验性结肠炎中的表达情况并探讨其表达的意义。方法：健康雄性BALB/c小鼠随机分为正常对照组、模型组。模型组小鼠予5%DSS溶液自由饮用制备小鼠急性实验性结肠炎模型,正常对照组小鼠予蒸馏水自由饮用。每日观察小鼠的一般状况及疾病活动指数（DAI）评分,于实验第5、9天分批处死小鼠,取小鼠结肠行组织学损伤评分;ELISA法检测小鼠血浆中性粒细胞弹性蛋白酶浓度;Western blot法检测远端结肠组织中NE蛋白的表达。结果：与正常对照组相比,模型组小鼠在实验第5天及第9天DAI评分显著升高（P〈0.001）、结肠组织学损伤评分明显升高（P〈0.01）、血浆NE浓度明显升高（P〈0.01）、远端结肠NE蛋白表达增加显著（P〈0.001）。在实验第5天及第9天,模型组小鼠DAI评分与组织学损伤评分呈显著正相关（P〈0.05）,血浆中NE浓度及结肠中NE蛋白的表达与DAI评分、组织学评分均呈显著正相关（P〈0.05）。结论：NE在实验性结肠炎小鼠血浆及结肠组织中表达明显增加,且与疾病活动指数及组织学评分呈正相关,提示NE可能参与实验性结肠炎小鼠的发病。     

Transiently truncated and differentially regulated expression of midkine during mouse embryogenesis

Midkine (MK) is a retinoic acid response cytokine, mostly expressed in embryonic tissues. Aberrant expression of MK was found in numerous cancers. In human, a truncated MK was expressed specifically in tumor/cancer tissues. Here we report the discovery of a novel truncated form of MK transiently expressed during normal mouse embryonic development. In addition, MK is concentrated at the interface between developing epithelium and mesenchyme as well as highly proliferating cells. Its expression, which is closely coordinated with angiogenesis and vasculogenesis, is spatiotemporally regulated with peaks in extensive organogenesis period and undifferentiated cells tailing off in maturing cells, implying its role in nascent blood vessel (endothelial) signaling of tissue differentiation and stem cell renewal/differentiation. Cloning and sequencing analysis revealed that the embryonic truncated MK, in which the conserved domain is in-frame deleted, presumably producing a novel secreted small peptide, is different from the truncated form in human cancer tissues, whose deletion results in a frame-shift mutation. Our data suggest that MK may play a role in epithelium-mesenchyme interactions, blood vessel signaling, and the decision of proliferation vs differentiation. Detection of the transiently expressed truncated MK reveals its novel function in development and sheds light on its role in carcinogenesis.     

Green tea polyphenol extract attenuates colon injury induced by experimental colitis

Inflammatory bowel disease (IBD) is characterised by oxidative and nitrosative stress, leukocyte infiltration, and up-regulation of intercellular adhesion molecule 1 (ICAM-1) expression in the colon. The aim of the present study was to examine the effects of green tea extract in rats subjected to experimental colitis induced by intracolonic instillation of dinitrobenzene sulphonic acid (DNBS). At 4 days after DNBS administration the rats were sacrificed. Treatment with green tea extract significantly attenuated diarrhoea and loss of body weight. This was associated with a remarkable amelioration of the disruption of the colonic architecture, significant reduction of colonic myeloperoxidase (MPO) and tumor necrosis factor-alpha (TNF-alpha) production. Green tea extract also reduced the appearance of nitrotyrosine immunoreactivity in the colon and reduced the up-regulation of ICAM-1.     

Increased cell surface EGF receptor expression during the butyrate-induced differentiation of human HCT-116 colon tumor cell clones

Several clonal sublines of HCT-116 human colon adenocarcinoma cells were isolated and characterized on the basis of their growth characteristics, intrinsic enterocyte-like differentiation (as assessed by alkaline phosphatase and lactase activities), and responses to butyrate, an inducer of colon tumor cell maturation. The HCT-116 sublines were found to be heterogeneous and several phenotypically distinct clones were identified. Further characterization of these clones indicated that the effects of butyrate on cell growth, alkaline phosphatase activity, and lactase activity were distinct and separable. The growth of all of the clones were inhibited by butyrate (IC50 values varied from 0.44 to 1.5 mM), but the effects of this agent on alkaline phosphatase and lactase activities varied widely. In several sublines butyrate had no effect on either enzyme while in others one or both activities were induced. Additionally, the binding of 125I-epidermal growth factor (EGF) to cell surface receptors was found to be proportional to the expression of lactase activity in the cell. The D3 clone and other sublines with intrinsic lactase activities greater than 100 nmol/mg/min expressed a class of high-affinity EGF receptors (e.g., D3 cells had 3.48 X 10(4) EGF receptors/cell with a kd of 0.61 nM). Other clones with less lactase activity had undetectable levels of 125I-EGF binding. In clones which exhibited greater than twofold increases in lactase activity in response to butyrate, the expression of a large number of low-affinity EGF receptors was also induced. In one such clone, the P1 subline, lactase activity was increased from 70 nmol/mg/min to 230 nmol/mg/min after 96 h in 2 mM butyrate, and the expression of EGF receptors was increased from undetectable levels to 1.18 X 10(5) EGF receptors/cell (kd of 3.2 nM). Northern blot analysis indicated that the increased 125I-EGF binding after butyrate treatment may have been due, in part, to a greater than twofold accumulation of EGF receptor mRNA. In addition, the expression of the messages for transforming growth factor alpha (TGF-alpha) and transforming growth factor beta (TGF-beta) was examined in butyrate-treated cells. While TGF-alpha mRNA levels were found to correlate with EGF receptor message levels in the HCT-116 clones, TGF-beta mRNA expression was not found to correlate with the butyrate-induced growth inhibition or with increases in EGF receptor expression, alkaline phosphatase activity, or lactase activity in these cells.     

Soybean curd refuse alleviates experimental tumorigenesis in rat colon

Adult Fischer-344 rats which underwent administration of azoxymethane were fed diets containing soybean curd refuse (SCR) or a high-molecular-weight fraction of soy protein digest (HMF), or Hammarsten casein (CAS) as a protein source over a period of 34 weeks. All the living rats of each group at 22, 28 or 34 weeks were endoscopically inspected for tumor incidence in the colon. SCR turned out to be comparable to HMF in anti-tumorigenicity, or rather better than HMF.