Expression Of STAT3 In Colonic Mucosa Of Patients With Ulcerative Colitis

Ulcerative colitis (UC) was characterized by chronic nonspecific inflammation of the intestine, and infectious, hereditary and immune factors were considered to be involved in its pathogenesis. Newly identified signal transducers and activators of transcription (STAT) family, which was involved in the signal transduction of multiple cytokines and growth factors, have been demonstrated to take part in regulating immune reactions, inflammatory reactions and the growth and differentiation of cells. Among STAT family STAT3 was considered to have intimate relationship with inflammation and might play an important role in the pathogenesis of UC. Increased expression and activation of STAT3 had been demonstrated in mucosa of patients with UC, but its effects had not yet been understood. Vitro research revealed that STAT3 was activated mainly by cytokines of IL-6 family through binding to gp130 receptors and activating Janus tyrosine kinases (JAK) which then induced phosphorylation of STAT3. Phosphorylated STAT3 transfered into the nucleus and induced transcription of target genes. However, the activating pattern of STAT3 in UC still kept unknown.Objective: The aim of our study was to investigate the expression and activation of STAT3 in colonic mucosa of patients with UC and analyze their correlations with clinical and endoscopic activity. The activating mechanism of STAT3 was also explored by investigating expression of IL-6 and analyzing the correlation between IL-6 and STAT3 as well as the correlation between IL-6 and p-STAT3.Methods: Mucosal samples were taken from freshly obtained intestinal resection specimens of 47 patients with ulcerative colitis (UC) and 13 normal controls. The patients were divided into classⅠgroup, classⅡgroup and classⅢgroup according to endoscopic activity and into mild group, moderate group and severe group according to clinical activity. Expression of IL-6, STAT3 and phosphorylated STAT3 was assayed by Western blotting and RT-PCR assays on protein level and mRNA level in extracts of endoscopic colonic biopsies. In addition, their cellular localization was determined by immunohistochemistry (IHC).Results1 Clinical and pathological performance of patients with UC 26 of 47 UC patients mainly suffered from bloody purulent stool, 14 from hematochezia and 7 only from abdominal pain. Fever occurred in 8 patients.Coloscopy showed that in normal controls, the nucosa was smooth with clear vascular lake there were hyperemia, edema, erosion or ulcer in the mucosa with cover of bloody purulent secretion in moderate and severe patients, and in mild patients, grave edema and hyperemia were the main appearances which made the mucosa grainy, fragile and more bleeding.[0] 4 of 47 patients had polypi formation.Optical microscope observation revealed that in normal mucosa the colon glands were well-arranged with epithelial cells on surface and crypt complete, but in UC mucosa, the tissue present hyperemia, edema, erosion, ulcer and the loss of goblet cells with considerable inflammatory cells infiltration in lamina propria. We also detected analosis of glands, thickening of colon paries and formation of crypt abscess. Atypical hyperplasia and formation of fibrous connective tissue also could be found in the mucosa of patients with polypi.2 Expression of STAT3 and p-STAT3 in colonic mucosa of normal controls and patients with UC2.1 IHC revealed that in normal mucosa, STAT3 was detected mainly in the cytoplasm of diffused distribution mononuclear[0] cells in lamina propria and also sometimes in neutrophils and epithelial cells, In the patients'mucosa, STAT3 was located in the same types of cells as the controls but positive cells increased obviously with stronger staining density. In contrast, p-STAT3 was detected both in the nucleus and cytoplasm of lamina propria mononuclear cells while mainly in the nucleus. p-STAT3 positive cells could hardly be found, or only be detected in a hand of diffused mononuclear cells in normal mucosa. But in UC patients, p-STAT3 was expressed in a great quantity of infiltrative mononuclear cells with stronger staining density than controls.Expression of STAT3 in classⅠgroup, classⅡgroup and classⅢgroup was significantly increased as compared to controls (24.88 vs 14.50, 33.75 vs 14.50, 41.18 vs 14.50), p<0.05, p<0.01, p<0.01, respectively, but there were no statistic differences among classⅠgroup, classⅡgroup and classⅢgroup, P>0.05.At the same time, compared with the normal controls, the expression of p-STAT3 in classⅡgroup and classⅢgroup was also significantly upregulated (34.11 vs 15.00, 42.09 vs 15.00), p<0.01. The p-STAT3 expression of classⅢgroup and classⅡgroup was higher than classⅠgroup, P<0.05, P<0.01, respevtively. No statistic differences could be found between classⅡgroup and classⅢgroup as well as between classⅠgroup and normal controls (21.13 vs 15.00), P>0.05.Compared with controls, Expression of STAT3 in mild group, moderate group and severe group had an obvious increasing tendency (28.70 vs 13.50, 35.70 vs 15.00, 44.78 vs 13.50), p<0.01. The STAT3 level of severe group was higher than that of mild group, p<0.05, while no statistic differences could be detected between mild group and moderate group as well as between moderate group and severe group, P>0.05.The expression of p-STAT3 in mild group, moderate group and severe group increased largely as compared to normal controls (29.00 vs 14.00, 35.96 vs 14.00, 42.89 vs 14.00), p<0.01, while the p-STAT3 level of severe group was higher than that of mild group and moderate group, p<0.05. There were no statistic differences between mild group and moderate group, P>0.05.2.2 Expression of STAT3 and p-STAT3 proteins in colonic mucosa of normal controls and patients with UCUsing western blotting, both STAT3 and p-STAT3 proteins were assessed in biopsy specimens, STAT3 or p-STAT3 band of 90 kD andβ-actin band of 43 kD were showed. The data showed that the expression of STAT3 protein in classⅠgroup, classⅡgroup and classⅢgroup was significantly increased as compared to controls (25.19 vs 8.04, 29.89 vs 8.04, 50.97 vs 8.04), P<0.01. Along with the aggravation of the disease, the STAT3 level of classⅢgroup grew higher than classⅠgroup and classⅡgroup,P<0.05, P<0.01, respectively. No statistic differences could be detected between classⅠgroup and classⅡgroup, P>0.05.In contrast to the scanty expression of p-STAT3 in normal controls, its expression in classⅠgroup, classⅡgroup and classⅢgroup promoted largely (17.75 vs 7.00, 33.61 vs 7.00, 50.44 vs 7.00),P<0.01, and there were significant statistic differences between every two groups, P<0.01.Compared with normal controls, expression of STAT3 protein in mild group, moderate group and severe group was significantly increased (26.30 vs 8.04, 38.72 vs 8.04, 48.94 vs 8.04). The STAT3 protein level of moderate group and severe group promoted significantly than mild group along with the severity, p<0.01, and no statistic differences could be found between moderate group and severe group, P>0.05.The expression of p-STAT3 protein in mild group, moderate group and severe group had the same tendency as STAT3 protein (21.70 vs 7.00, 42.33 vs 7.00, 48.89 vs 7.00).2.3 Expression of STAT3 mRNA in colonic mucosa of normal controls and patients with UCThe STAT3 andβ-actin mRNA levels were performed by RT-PCR assay. It showed that there were the STAT3 band of 275 bp and theβ-actin band of 500 bp in patients with UC and normal controls. Expression of STAT3 mRNA in classⅡgroup and classⅢgroup was significantly increased as compared to controls (31.16 vs 12.85, 46.03 vs 12.85), P<0.01. The STAT3 expression of classⅡgroup and classⅢgroup was higher than classⅠgroup while classⅢgroup was also higher than classⅡgroup, p<0.01. There were no statistic differences between classⅠgroup and normal controls (18.88 vs 12.85), P>0.05.Compared with normal controls, expression of STAT3 mRNA in mild group, moderate group and severe group increased significantly (28.50 vs 12.85, 35.22 vs 12.85, 47.28 vs 12.85), P<0.01. The STAT3 level of severe group was higher than that of mild group and moderate group, p<0.05, and no statistic differences could be detected between mild group and moderate group, P>0.05.3 Expression of IL-6 in colonic mucosa of normal controls and patients with UC3.1 IHC revealed that IL-6 was detected mainly in the cytoplasm of a few lamina propria mononuclear cells in normal mucosa and positive cells increased obviously with stronger staining density in UC mucosa.Higher expression of IL-6 was detected in classⅡgroup and classⅢgroup than in normal controls (34.50 vs 16.50, 41.21 vs 16.50), p<0.01. The IL-6 level of classⅡgroup and classⅢgroup increased largely towards classⅠ.group, p<0.05 p<0.01, respectively. There were no statistic differences between classⅠgroup and the normal controls as well as between classⅡgroup and classⅢgroup (19.50 vs 16.50), P>0.05.Expression of IL-6 in mild group, moderate group and severe group was significantly increased as compared to normal controls (27.00 vs 16.50, 34.63 vs 16.50, 46.00 vs 16.50), p<0.05, p<0.01, p<0.01, respectively. The IL-6 level of severe group was higher than that of mild group, p<0.05. There were no statistic differences between mild group and moderate group as well as between moderate group and severe group, P>0.05.3.2 Expression of IL-6 protein in colonic mucosa of normal controls and patients with UCUsing western blotting, IL-6 protein level was assessed in biopsy specimens and the IL-6 band of 26 kD was showed. IL-6 had an increase protein expression in classⅡgroup and classⅢgroup than in normal controls (34.98 vs 10.77, 46.44 vs 10.77), P<0.01, and along with the increase of severity, IL-6 level of classⅢgroup was higher than classⅠgroup and classⅡgroup while classⅡgroup was higher than classⅠgroup, P<0.01. No statistic differences could be detected between classⅠgroup and normal controls (16.38 vs 10.77), P>0.05.Expression of IL-6 protein in mild group, moderate group and severe group was significantly increased as compared to controls (20.20 vs 10.77, 41.02 vs 10.77, 49.28 vs 10.77), P<0.05, P<0.01, P<0.01, respectively. The IL-6 expression in moderate group and severe group had an increase towards mild group, p<0.01. There were no statistic differences between moderate group and severe group, P>0.05.3.3 Expression of IL-6 mRNA in colonic mucosa of normal controls and patients with UCIL-6 andβ-actin mRNAs were performed by RT-PCR assay, and it showed that there were the IL-6 band of 223 bp and theβ-actin band of 500 bp in patient groups and normal controls.Compared with normal controls, the expression of IL-6 mRNA in classⅠgroup, classⅡgroup and classⅢgroup was significantly increased(23.94 vs 11.62, 35.61 vs 11.62, 41.41 vs 11.62), P<0.05, P<0.01, P<0.01, respectively. The IL-6 expression of classⅡgroup and classⅢgroup was higher than classⅠgroup, p<0.01. There were no statistic differences between classⅡgroup and classⅢgroup, P>0.05.Expression of IL-6 mRNA in mild group, moderate group and severe group was significantly increased as compared to controls (27.07 vs 11.62, 34.22 vs 11.62, 54.00 vs 11.62), P<0.01. The IL-6 level of severe group was higher than mild group and moderate group, p<0.01, while no statistic differences could be detected between mild group and moderate group, P>0.05.4 Correlation analysis showed that there was a positive correlation between IL-6 and STAT3 in UC mucosa on protein level and mRNA level respectively, r= 0.671, r=0.485, P<0.01.5 Correlation analysis showed that there was a positive correlation between IL-6 and p-STAT3 on protein level in UC mucosa, r= 0.764, P<0.01.Conclusions1 In colonic mucosa of UC patients, both the expression and activation of STAT3 were gradually increased and well correlated to the clinical and endoscopic activity of the disease, which implicating that STAT3 may reflect the severity of UC to some extent.2 In colonic mucosa of UC patients, the expression of IL-6 was significantly upregulated along with the aggravation of severity of the disease.3 Correlation analysis showed that there was a positive correlation between IL-6 and STAT3 as well as between IL-6 and p-STAT3, suggesting that STAT3 took part in the pathogenesis of UC through IL-6/JAK/STAT3 signaling pathway.