ARE Binding Activity And Changes Of HO-1 Expression In Colonic Mucosa Of Ulcerative Colitis Patients

Ulcerative colitis(UC) is a chronic non-specific inflammatory disease, which cause is not yet clear and the main features are continuity, diffuse inflammatory changes. The main clinical symptoms are persistent or recurrent abdominal pain. diarrhea. stool with pus, blood and mucous, tenesmus ect. This disease principal affect the distal colon and the rectum, and expand to the proximal colon even involve the entire colon. Over the past decade in the wake of people lifestyles and diet change, amount of clinical studies indicate that the incidence of the disease is raising. UC is regarded as one of the contemporary refractory disease by WHO, because its etiology and pathogenesis is not yet clear and the pathophysiology complex, the clinical cure rate is low. In recent years, the closely relationship has been found between oxidative stress with pathogenesis and disease development of UC, the over expression of oxygen free radicals and lipid peroxidation induced by oxidative stress play an important role in the development of the disease. Other studies found that immune factor is also the important factor in mechanisms of UC.The Keap1/Nrf2/ARE pathway is one of the most important anti-oxidative stress pathway, and it plays an important role in the pathogenesis of multiple systemic disease. This pathway is involved in human anti-tumor, anti-apoptotic, anti-inflammatory, regulating cardiovascular reactions, anti-atherosclerosis and so on. Nrf2(NF-E2 related factor 2) belongs to one of the CNC(cap’ n’ collar) transcription factor family and is widely present in multiple tissues and organs of the body, Kelch-like ECH associating protein1(Kelch-like epichlorohydrin associating protein 1, Keap l) is an important receptor of Nrf2, under the physiological conditions, Nrf2 is located in the cytoplasm and conjunct with Keap1 in the suppressed states. When suffering from the oxidative stress stimulation, Nrf2 quickly uncouple with allosteric Keap1 and translocates into the nucleus by steady state, combine with small Maf proteins to form allodimer then combined with the antioxidant response element(ARE), binding to the GCTGAGTCA sits of ARE, and participate in the transcription and regulation of a series of phase Ⅱ detoxifying enzymes and antioxidant enzymes genes. ARE is a specific DNA promoter binding sequence, which is located in the 5’ end start-up sequence of the regulated antioxidant genes and phaseⅡ detoxifying enzymes. The necessary gene sequences for ARE transcription’ gene sequences are TGACnnn GC, which was demonstrated by Rushmore in 1991. Subsequent researches found a series of antioxidant stress genes which promoter regions have the same sequences all can combine with the transcription factor Nrf2. More and more studies show that ARE play an important role in induction and transcription of the antioxidant genes including phase Ⅱ detoxifying enzymes. The target gene drived by ARE include γ-glutamyl cysteine synthetase(γ-GCS), Glutathione-s-transferase(GST), benzene Kun reductase1(NQO1), UDP-glucuronosyl transferases(UGT), heme oxygenase-1(HO-1) etc. The expression of these proteins can effectively prevent the production of free radicals, and play a role in antioxidant stress.HO-1 is an extremely important phase Ⅱdetoxifying enzymes in antioxidant stress, heme oxygenase is the initial enzyme and rate-limiting enzyme of the heme catabolism, which can break down the hemeglobin to equimolar amount of biliverdin(BV), free iron(Fe2+) and carbon monoxide(CO), in mammals biliverdin is converted to bilirubin(BR) by biliverdin reductase. Fe2+ can induce the generation of ferritin in vivo. HO have three isoenzymes: HO-1, HO-2, HO-3. Expression of HO-1 can affect against oxidant stress, inflammation, ischemia/reperfusion injure, apoptosis, transplant rejection reactions and so on, which play a protective role of antioxidant stress in numerous disease.Lots of studies have found that the relationship of Nrf2 and inflammatory disease is very closely which involved in the development of inflammation. In inflammatory bowel disease especially ulcerative colitis, the researches have found that Nrf2 is highly expressed in the colon mucosa of UC patient, Nrf2 and the severity of UC are positively correlated. At present there is no research about ARE and HO-1 with UC, We will do research on the ARE binding activity and changes of HO-1 expression of UC, then analyze the respectively revelance of ARE, HO-1 with severity of UC.Purpose: To detect the ARE binding activity and changes of HO-1 expression in colonic mucosa of UC patient, analyze the respectively revelance of ARE, HO-1 with severity of UC.Methods: This study collects 42 patients with the clinical diagnosis of active ulcerative colitis as case group and 25 patients with colonic polyps as control group, biopsys colonic mucosal tissue(rectum- sigmoid junction) was biopsied as 4 pieces under colonoscope. The patients are grouped after scored according to Sutherland DAI standard(Group A, 25 cases in control group. Group B, 22 cases in mild-to-moderate group. Group C, 20 cases in severe group). To detect ARE binding activity by electrophoretic mobility shift Assay(EMSA) and the expression of HO-1 detected by immunohistochemistry staining methods in colonic mucosa tissues.Results:1 According to Sutherland DAI standard, UC disease activity Truelove and Witts classification, The UC patients are divided into three groups of mild, moderate and severe. The average scores of three groups are 4.8±0.2 points, 8.6±0.7 points and 11.5±0.4 points. The clinical data of the three groups patients see Table 1.2 The results of histopathological staining(HE): The nucleus is purple blue, cytoplasm, basement membrane and collagen fibers are pink. The Colonic mucosal epithelium is intact in the normal control group, a few of inflammatory cells infiltration, and glandular cells arranged neat rows, a lot of goblet cells; A large number of inflammatory cells infiltration are shown in UC groups musoca, such as eosinophils, neutrophils, lymphocytes, macroplages cells, crypt structure are disordered, crypts, crypt epithelium are visible inflammatory cells infiltration, epithelial goblet cells are decreased, Paneth cells metaplasia are occasionally seen, the severe patients’ epithelial appear missing, shedding.3 The test results of ARE binging activity in colonic mucosa tissues.The results show that ARE binging activity in UC colonic mucosa is higher than the normal control group, ARE binging activity in each group are as follows: severe active group > mild- moderate active group > control group. Compared with any two groups, the difference is of statistically significance(P <0.01).4 The HO-1expression in colonic mucosal tissueThe Immunohistochemistry results of HO-1 in colonic mucosa shows that the HO-1 expression in normal mucosa is less than others, mainly located in cytoplasm, but in the UC groups HO-1 expression is significantly increased, which is expressed both in nucleus and cytoplasm; levels of HO-1 expression in each group are as follows: severe active group> mild-moderate active group >control group, and the comparation between any two groups is with statistically significance(P <0.01).5 Correlation AnalysisThe change of ARE binging activity and HO-1 expression in UC patients is of positive correlation, the correlation coefficient is 0.946, P<0.01, with notable statistically significance.Conclusion: With the further enhancement of the UC disease severity, the ARE binging activity in UC patients’ colonic mucosa growing. The HO-1 expression detected by Immunohistochemistry is also incresase. The relationship between ARE with HO-1 is of positive correlation. The ARE binging activity and HO-1 expression reflect the severity of UC in a certain extent. The pathogenesis of UC is relationship with the Keap1/Nrf2/ARE /HO-1 signal pathway.