Inhibition of arginase ameliorates experimental ulcerative colitis in mice

Abstract

Nitric oxide (NO) is produced from the conversion of L-arginine by NO synthase (NOS) and regulates a variety of processes in the gastrointestinal tract. Considering the increased activity of arginase in colitis tissue, it is speculated that arginase could inhibit NO synthesis by competing for the same L-arginine substrate, resulting in the exacerbation of colitis. We examined the role of arginase and its relationship to NO metabolism in dextran sulfate sodium (DSS)-induced colitis. Experimental colitis was induced in mice by administration of 2.5% DSS in drinking water for 8 days. Treatment for arginase inhibition was done by once daily intraperitoneal injection of N^ω-hydroxy-nor- arginine (nor-NOHA). On day 8, we evaluated clinical parameters (body weight, disease activity index, and colon length), histological features, the activity and expression of arginase, L-arginine content, the expression of NO synthase (NOS), and the concentration of NO end-product (NOx: nitrite + nitrate). Administration of nor-NOHA improved the worsened clinical parameters and histological features in DSS-induced colitis. Treatment with nor-NOHA attenuated the increased activity of arginase, upregulation of arginase Ι at both mRNA and protein levels, and decreased the content of L-arginine in colonic tissue in the DSS-treated mice. Conversely, despite the decreased expression of NOS2 mRNA, the decreased concentration of NOx in colonic tissues was restored to almost normal levels. The consumption of L-arginine by arginase could lead to decreased production of NO from NOS, contributing to the pathogenesis of the colonic inflammation; thus, arginase inhibition might be effective for improving colitis.     

Immunopathogenesis of experimental ulcerative colitis is mediated by eosinophil peroxidase

The precise role that individual inflammatory cells and mediators play in the development of gastrointestinal (GI) dysfunction and extraintestinal clinical manifestations of ulcerative colitis (UC) is unknown. In this study, we have used a mouse model of UC to establish a central role for eotaxin and, in turn, eosinophils in the development of the immunopathogenesis of this disease. In this model the administration of dextran sodium sulfate (DSS) induces a prominent colonic eosinophilic inflammation and GI dysfunction (diarrhea with blood and shortening of the colon) that resembles UC in patients. GI dysfunction was associated with evidence of eosinophilic cytolytic degranulation and the release of eosinophil peroxidase (EPO) into the colon lumen. By using IL-5 or eotaxin-deficient mice, we show an important role for eotaxin in eosinophil recruitment into the colon during experimental UC. Furthermore, using EPO-deficient mice and an EPO inhibitor resorcinol we demonstrate that eosinophil-derived peroxidase is critical in the development of GI dysfunction in experimental UC. These findings provide direct evidence of a central role for eosinophils and EPO in GI dysfunction and potentially the immunopathogenesis of UC.     

Role of anti-angiogenic factor endostatin in the pathogenesis of experimental ulcerative colitis

AimsVascular endothelial growth factor (VEGF) and pathologic angiogenesis have been demonstrated to play a pathogenic role in the development and progression of inflammatory bowel disease. Thus, we hypothesized that the potent anti-angiogenic factor endostatin might play a beneficial role in experimental ulcerative colitis (UC).Main methodsWe used three animal models of UC: (1) induced by 6% iodoacetamide (IA) in rats, or (2) by 3% dextran sulfate sodium (DSS) in matrix metalloproteinase-9 (MMP-9) knockout (KO) and wild-type mice, and (3) interleukin-10 (IL-10) KO mice. Groups of MMP-9 KO mice with DSS-induced UC were treated with endostatin or water for 5 days.Key findingsWe found concomitant upregulation of VEGF, PDGF, MMP-9 and endostatin in both rat and mouse models of UC. A positive correlation between the levels of endostatin or VEGF and the sizes of colonic lesions was seen in IA-induced UC. The levels and activities of MMP-9 were also significantly increased during UC induced by IA and IL-10 KO. Deletion of MMP-9 decreased the levels of endostatin in both water- and DSS-treated MMP-9 KO mice. Treatment with endostatin significantly improved DSS-induced UC in MMP-9 KO mice.Significance1) Concomitantly increased endostatin is a defensive response to the increased VEGF in UC, 2) MMP-9 is a key enzyme to generate endostatin which may modulate the balance between VEGF and endostatin during experimental UC, and 3) endostatin treatment plays a beneficial role in UC. Thus, anti-angiogenesis seems to be a new therapeutic option for UC.     

Mast cells in ulcerative colitis

The changes in the number and ultrastruture of mast cells were studied in 37 colonoscopical biopsies from patients with ulcerative colitis. Changes in the active stage of the disease and during remission were compared. Cell counts were performed on semithin sections stained with Giemsa after osmium tetroxide fixation. This method overcome the uncertain staining found after formalin fixation. Accumulation of mast cells accompanied by intense degranulation was found to be significant in the active stage of the disease. Two forms of degranulation were observed: discharge of the individual granules and protrusion and detachment of the cytoplasmic processes containing granules. The latter was a sign of rapid degranulation, as described earlier in animal experiments. Mast cells were closely associated with capillary blood vessels, Schwann cells, neural fibres, myofibroblasts and collagenous fibres, and were also present between epithelial cells. It is assumed that close topographic contact may also imply a functional correlation.     

Sulfasalazine-induced colitis complicating idiopathic ulcerative colitis.

A diagnosis of idiopathic ulcerative colitis was made in a previously healthy 9-year-old boy. Symptoms persisted despite therapy with sulfasalazine, 50 mg/kg daily, but they eventually responded to treatment with parenteral nutrition and prednisone, 40 mg daily. Metronidazole was also given to eradicate persistent Dientamoeba fragilis from the stools. The symptoms resolved over 3 weeks, and the daily dose of prednisone was tapered. On two subsequent occasions a challenge with sulfasalazine caused an immediate recurrence of loose, blood-streaked stools and of nonspecific histologic features of ulcerative colitis, which resolved when the sulfasalazine was discontinued.     

A role of a lymphocyte tryptase, granzyme A, in experimental ulcerative colitis

In the large-intestinal mucosae of rats orally administered dextran sulfate sodium, which induces an enteritis resembling ulcerative colitis (UC), the activity for granzyme A, a lymphocyte tryptase, increased at an earlier stage than that at which UC markers (growth-regulated gene product/cytokine-induced neutrophil chemoattractant-1 and caspase-3) increased. This suggests involvement of the enzyme in the exacerbation and perpetuation of enteritis.     

Oxygen radicals in ulcerative colitis.

This article reviews the pathophysiologic concept that superoxide and hydrogen peroxide, generated by activated leukocytes, together with low-molecular-weight chelate iron derived from fecal sources and from denatured hemoglobin, amplify the inflammatory response and subsequent mucosal damage in patients with active episodes of ulcerative colitis. The putative pathogenic mechanisms reviewed are as follows: (1) Dietary iron is concentrated in fecal material owing to normally limited iron absorption. (2) Mucosal bleeding, characteristic of ulcerative colitis, as well as supplemental oral iron therapy for chronic anemia, further conspire to maintain or elevate mucosal iron concentration in colitis. (3) Fenton chemistry, driven especially by leukocyte-generated superoxide and hydrogen peroxide, leads to formation of hydroxyl radicals. (4) The resultant oxidative stress leads to the extension and propagation of crypt abscesses, either through direct membrane disruption by lipid peroxidation or through generation of secondary toxic oxidants such as chloramines. (5) Chemotactic products of lipid peroxidation, including 4-hydroxynonenal, provide positive feedback to accelerate this inflammatory/oxidative process, leading to acute exacerbations of the disease. (6) Other oxidized products, such as oxidized tryptophan metabolites, created by free radical mechanisms in or near the mucosa, may act as carcinogens or tumor promotors that contribute to the exceedingly high incidence of colon carcinoma in patients suffering from chronic ulcerative colitis. In this way, self-sustaining cycles of oxidant formation may amplify flare-ups of inflammation and mucosal injury in ulcerative colitis. This concept, if proved correct by subsequent research, would provide a rationale for several novel clinical approaches to the management of ulcerative colitis, including use of SOD mimetics, iron chelators, and chain-breaking antioxidants.     

Olsalazine in active ulcerative colitis.

Olsalazine (azodisalicylate) is a new drug in which two molecules of 5-aminosalicylic acid are linked by an azo bond. Its role in the treatment of mildly active, distal ulcerative colitis was investigated. Sixty patients were randomly allocated to receive olsalazine 1 g or a placebo as a retention enema nightly for two weeks. Clinical improvement was seen in 19 (66%) and sigmoidoscopic improvement in 17 (59%) of the 29 patients receiving olsalazine compared with 12 (43%) and 11 (39%), respectively, of the 28 in the control group. These differences were not significant. In a second trial 40 patients were randomised to receive oral olsalazine 2 g daily or a placebo capsule for two weeks. Significant clinical and sigmoidoscopic improvement was seen in the patients receiving oral olsalazine compared with the patients receiving placebo capsules. Oral olsalazine may be valuable in the treatment of mildly active ulcerative colitis. Its role in maintaining remission is yet to be determined.     

Colorectal cytology in chronic ulcerative colitis

During a period of ten years, 109 colorectal cytology specimens were obtained from 41 patients with chronic ulcerative colitis. There were 28 male and 13 female patients, whose average age was 45 years. All patients were symptomatic and had ulcerative colitis with repeated attacks of profuse diarrhea on the average for 12 years prior to their first cytologic examination. Eight cytology specimens were positive for malignant cells, 35 had atypia, 58 were negative and 8 were unsatisfactory. The malignant cells showed marked anisocytosis, pleomorphism and nuclear hyperchromasia and appeared in loosely cohesive clusters or in single forms in an inflammatory and necrotic background. Subsequent colectomy revealed invasive carcinoma in five patients and carcinoma in situ in two. Smears that were negative or showed atypia contained abundant chronic inflammatory cells. The atypical colonic epithelial cells contained prominent nuclei and formed cohesive clusters. Surgical biopsy and/or segmental resection revealed the presence of polyps, pseudopolyps, polypoid hyperplasia, mucosal atypia and crypt abscesses in patients with atypical and negative cytologic findings. It is concluded that cytologic examination of the colon can play an important role in the examination of patients with ulcerative colitis and allows for the detection of malignant transformation of the colonic mucosa.     

MiRNA-182-5p aggravates experimental ulcerative colitis via sponging Claudin-2

Tight junction proteins play crucial roles in maintaining the integrity of intestinal mucosal barrier. MiRNA-182-5p is capable of targeting claudin-2 which is one of the vital tight junction proteins and the effect and mechanism of miRNA-182-5p was explored here in the DSS-induced colitis model. The pathological conditions were evaluated via hematoxylin and eosin staining. The gene expression level was assessed via PCR. Quantitative immunohistochemistry analysis was performed for the measurement of claudin-2. microRNA.org online tool was used for target gene prediction. Luciferase reporter assay and RNA pull-down assay were performed to detect the target of miRNA-182-5p. The inflammatory and oxidative stress level were measured using corresponding kits. MiRNA-182-5p was highly expressed in colitis model and miRNA-182-5p inhibitor exerted protective effects on colitis induced by DSS in mice. The protective effects includded improvement of pathological changes, increases in anti-inflammation and anti-oxidative genes, and up-regulation of TGF-β1. Claudin-2 mRNA was predicted as the target of miRNA-182-5p, which was validated via luciferase reporter assay and RNA pull-down assay. Claudin-2 overexpression was found in miRNA-182-5p inhibitor group. Consistent with the role of miRNA-182-5p, claudin-2 overexpression also exerted protective effects on DSS-induced colitis in mice. Inhibition of miRNA-182-5p exerted protective effects on colitis via targeting and upregulating claudin-2. The findings in study provide a new therapeutic strategy for colitis treatment and lay the foundation for future study.