| BackgroundInflammatory bowel disease (IBD) is a complex inflammatory disease of the gastrointestinal tract with unknown cause that lacks molecular markers for diagnosis. Crohn's disease(CD) and ulcerative colitis (UC) are the two major forms of IBD. The activity and severity of IBD is of great importance in diagnosis and treatment monitoring. CRP is one of the acute-phase proteins mostly studied in IBD, being used as a good inflammation marker for diagnosis, the evaluation of disease activity and risk of complications, the prediction of relapse, and treatment monitoring. Some studies have evaluated the association between CRP and clinical scores in IBD, showing a strong association of CRP with disease activity in Crohn's disease and modest association in ulcerative colitis. Despite having some advantages compared with other indices, CRP is far from an ideal parameter for these conditions. To evaluate the activity and severity of IBD, Many kinds of DAI (disease activity index) were presented.The Mayo Index is the most widely used one in UC,and BEST CDAI in CD.Regenerating gene IV (REG IV), within the calcium depending lectin gene super superfamily, is mainly involved in the liver, pancreatic, gastrointestinal cell proliferation, differentiation or inflammation. Several studies have shown that Reg IV may be involved in inflammatory bowel disease, but its role is not clear.Material and methods1.MaterialSixty-one IBD patients (38 ulcerative colitis,23 Crohn's disease] and twenty-five healthy controls were included in the study Biopsy specimens from patients with IBD and healthy controls were used for evaluating the expression of REGⅣ. The clinical activity of disease was determined by the Crohn's disease activity index (CDAI) and the colitis activity index (Mayo Index). The protocol of this study was approved by the Ethics Committee of Nanfang hospital.2.ImmunohistochemistryImmunohistochemical staining for REGⅣwas performed with an SABC Kit Briefly, the paraffin-embedded sections were deparaffinized in xylene and rehydrated in graded ethanol solutions (100%,95%,80%,70%) and phosphate-buffered saline (PBS). Subsequently they were subjected to heating antigen retrieval using a retrieval solution. Incubation with anti-Reg IV monoclonal antibody (SantaCruz Biotechnology, Santa Cruz, California, USA) was carried out at 1/50-1/100 dilution for 12 hours at 4℃. Detection was achieved using a standard staining system and antigen localization was visualized with DAB. The sections were counterstained with hematoxyline-eosin and mounted. we scored both staining qualities as either negative or weakly, moderately, or strongly positive.3. The clinical activity of diseaseDetermination of CRP concentrations was performed at the Department of Clinical Chemistry at Nanfang hospital. Serum CRP concentrations were measured using a latex based immunoassay (N Latex CRP mono, Dade Behring, Behring Laser Nephelometer, Marburg, Germany). At the day of the CRP measurement, the CDAI or Mayo Index was determined in these patients.4.Reverse transcription-polymerase chain reaction (RT-PCR)Total RNA was extracted from cells using Trizol (Invitrogen). RNA samples (3μg) were subjected to reverse transcription using a RevertAidTM First Strand cDNA Synthesis Kit (Fermentas). The PCR was initiated by 5 min incubation at 94℃, ended after a 10 min extension at 72℃,36 cycles for denaturation at 94℃for 30s, annealing at 55℃, and extension 72℃for 1 min using PCR kit (SBS Genetech Co. Ltd., Beijing, China).5. Ethical considerationsAll patients in the study consented to participate, and the study was approved by the Regional Ethical Committee (REC) at the Nanfang hospital.6. Statistical analysisThe Mann-Whitney U-test were used for single comparisons, and One way ANOVA was used for multiple comparisons. The association of CDAI or Mayo Index with CRP was assessed by means of Pearson's correlation coefficient. Data are presented as mean and s.d. if not otherwise stated. P-values less than 0.05 were considered significant.Results1. The expression of RegⅣImmunohistochemical staining of RegⅣwas performed in normal controls and patients with UC or CD. RegⅣexpression was categorized as 1+(weak); 2+ (moderate); 3+(strong). The immunostaining of RegⅣin normal tissues showed no signal stain in 0 cases, weak stain in 19 cases (76%), moderate stain in 4 cases (16%) and strong stain in 8%(2 cases). In contrast, RegⅣimmunostaining in IBD tissues showed no signal stain in 0 cases (0%), weak stain in 17 cases (27.9%), moderate stain in 17 cases (27.9%) and strong stain in 27 cases (44.2%) RegⅣexpression was significantly upregulated in IBD, compared with the control group (P<0.05). There were no significantly differences of RegⅣexpression between the UC and the CD (P=0.503). Specimens of active UC had significantly different RegⅣexpression compared with that of inactive UC (P<0.05). RegⅣexpression in inactive UC mucosa had no significantly differences compared with normal controls (P=0.406). Specimens of active CD had no significant difference with that of inactive CD (P=0.515)..The result of the RT-PCR also showed that the level of the RegⅣmRNA was corresponding with the Immunohistochemical staining of RegⅣ2. The relationship between RegⅣexpression and activity parameters in IBDRegⅣexpression and serum CRP concentrations were examined in patients with IBD. Among patients with UC, the serum CRP concentration was 88.33±47.99mg/l for strong RegⅣstaining,13.73±10.86 mg/1 for moderate staining and for weak staining 5.59±4.59mg/l (P<0.05). Among patients with CD,the serum CRP concentration was 41.90±41.48mg/l for strong RegⅣstaining,43.66±34.24 mg/l for moderate staining and for weak staining 18.84±20.60mg/l (P=0.455). For UC patients, CRP concentration was significantly higher in patients with strong RegⅣstaining than in patients with weak staining (P<0.05). With regard to CD patients, there was no significantly difference in patients with strong RegⅣstaining than in patients with moderate or weak staining (P=0.455).Next, we examined the relationship between clinical disease activities and RegⅣexpression. There was a significant difference in Mayo Index between weak, moderate and strong Reg IV groups(4.58±1.88,6.20±1.32 and 8.06±2.38 respectively, P<0.05). There was no significant difference in CDAI between weak, moderate and strong Reg IV groups(148.40±38.12,179.14±64.87 and 241.16±136.95 respectively, P=0.226). The concentration of CRP positivly correlated with Mayo Index (r= 0.627, P<0.05),and CDAI (r=0.624, P<0.05).ConclusionThe results of this study showed that RegⅣexpression in mucosa of IBD patients was significantly higher than that of healthy controls.We also found that there was a low expression of RegⅣin normal colonic mucosa, but inflammation caused a significant increase in its expression. The expression of RegⅣincreased with an increasing degree of inflammation in UC.For UC patients, CRP concentration was significantly higher in patients with strong RegⅣstaining than in patients with weak staining.With regard to CD, There was no significantly difference in patients with strong RegⅣstaining than in patients with moderate or weak staining.We also examined the relationship between the activity of IBD and RegⅣexpression. There was a significant difference in Mayo Index between weak, moderate and strong RegⅣgroups, There was no significant difference in CDAI between weak, moderate and strong RegⅣgroups.In our study, we found positive correlation between CRP concentration and the activity of IBD, as represented by Mayo Index and CDAI.In summary, this study suggested that the expression of RegⅣwas increased in IBD patients compared with that of healthy controls.And RegⅣcould be an independent predictor of disease activity in UC patients. |
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