Expression Of Nrf2in Patients With Ulcerative Colitis And The Relationship With Oxidative Stress

The main symptoms of Ulcerative Colitis (UC) are abdominal pain,diarrhea, and stool with pus, blood and mucous etc., and mainly affects thedistal colon and submucosa of the rectum, then extends to the proximal colonuntil pervades the entire colon. In recent years, large numbers of literaturereports show that the incidence of the disease has become significantly higher.It is regarded as one of the contemporary refractory disease by WHO due to itslong duration, wide range of lesions, chronic persistence and easy canceration.Therefore, more and more extensive attention has been paid to the diseaseboth at home and abroad. However, since the exact pathogenesis is unclear,there have been no specific clinical curative measures available. Someresearch found that the generation process of antioxidant defense network andReactive Oxygen Species (ROS) as well as Reactive Nitrogen Species(RNS)is unbalanced in UC. Abundant expression of Oxygen Free Radicals (OFR）caused by oxidative stress usually leads to the damage of fat, protein and DNAwhich plays an important role in the pathogenesis of UC (induce or aggravateUC), and considered to be an important factor in the development of intestinalinflammation.Nrf2(NF-E2related factor2) belongs to the CNC (cap’ n’ collar)transcription factor family and widely exists in multiple tissues and organswith leucine zipper structure, which can activate ARE to initiate a variety ofantioxidant response and detoxification genes and accordingly regulatesprevention protein of a variety of cells in the body. It is the receptors ofexogenous toxic substances and oxidative stress, and plays an important rolein the main defense mechanisms of cellular anti-oxidative stress and inductionof exogenous toxic substances.Kelch-like ECH associating protein1(Keap l) is an important receptor of Nrf2affecting the expression of Nrf2. In the physiological conditions, Nrf2and Keap l combined in the cytoplasm with state of deactivated and degradedrapidly by the ubiquitin-proteasome pathway. When stimulated by signals ofoxidative stress, Nrf2rapidly uncoupling with allosteric Keap1andtranslocates into the nucleus with steady state, combine with small Mafproteins to form allodimer which then combine with antioxidant responseelement (ARE) to increase the target gene expression which was mediated byARE, thereby to regulate expression of various of antioxidant genes includingγ-glutamyl cysteine synthetase (γ-GCS), Glutathione-s-transferase (GST),NQO1, UDP-glucuronosyl transferases (UGT) and epoxide hydrolase etc..The activation of Nrf2is regulated at multiple levels, mainly includeinteractions between Nrf2and Keap1and mechanism depending on thestability of Nrf2. There are two distinct mechanisms for Nrf2dissociationfrom Keap1: direct attacks of nucleophilic substances or ROS; indirect effectof phosphorylation. Currently, several protein kinase pathways such as proteinkinase C (PKC), mitogen-activated protein kinases (MAPKs) andphosphatidylinositol3-kinase (PI3K) are also regarded to be involved in theNrf2/ARE activation and regulation of its dependent gene expression. Besides,TauCI (product of activated neutrophils) can increase the transferation of Nrf2into the nucleus and the combination of Nrf2and ARE; the accumulation ofunfolded proteins in the endoplasmic reticulum can directly phosphorylateNrf2for activation by pancreatic endoplasmic reticulum kinase (PERK).Multiple recent researches show that Nrf2and inflammatory diseases areclosely related and involved in the occurrence and development ofinflammation. High expression of Nrf2in most inflammatory diseases inhibitsfurther development of inflammation, but the further research of relationshipbetween Nrf2and the severity of UC is needed. We will do research onexpression of Nrf2in UC patients and the occurrence and development of UC.Purpose: To detect the expression of Nrf2in UC patient, analyze thecorrelation of Nrf2and disease severity of UC, to explore the effect of Nrf2inUC occurrence and development. Methods: This research collects35patients (case group) with clinicaldiagnosed ulcerative colitis and21patients (control group=Group A) withcolonic polyps, and draw4ml of fasting peripheral venous blood inthe morning and biopsy colonic mucosal tissue (rectum-sigmoid junction)4pieces under endoscope. The patients are grouped after scored according toSutherland DAI standard (20cases in mild-to-moderate group=Group B,15cases in severe group=Group C). To detect T-SOD activity and MDA contentin serum by spectrophotometry and adopt Western blot andImmunohistochemistry staining methods to detect the expression of Nrf2in colonic mucosa. According to the test results, the relationship of Nrf2content and severity of UC will be statistically analyzed.Results：1. According to Sutherland DAI standard (see Table1) and UC disease activityTruelove and Witts classification (Table.2), UC patients are divided into threegroups of mild, moderate and severe. The average scores of each group are4.5points,8.9points,11.2points orderly. The clinical data of patients with threegroups see Table3.2. Test results of T-SOD, MDA in serum2.1T-SOD activity in serum of the case group is significantly lower than thatof the control group (P <0.01), and T-SOD activity in serum in case groupwith different disease severity is also different which severe UC group islower than mild-moderate UC group. The comparative difference between twogroups is with statistical significance (P <0.01).2.2The MDA content in serum of the case group is higher than that in thecontrol group (P <0.01), and the MDA content in serum in case group withdifferent disease severity is also different, which severe UC group is higherthan mild-moderate UC group. Comparisons between any two groups:comparative difference between mild-moderate UC group and the controlgroup is not significant (P>0.05), which is of no statistically significance; Thecomparative difference between severe UC group and mild-moderate UCgroup as well as the control group is of statistical significance (P <0.01). 3. Histopathological staining(HE) changes in colonic mucosa:The nucleus is purple blue; cytoplasm, basement membrane and collagenfibers were pink. Colonic mucosal epithelial is integrity, a small amount ofinflammatory cells infiltration in the lamina propria, and glands arranged neatrows in normal; A large number of inflammatory cells immersed in the laminapropria in UC groups, such as eosinophils, neutrophils, lymphocytes, plasmacells, infiltration of monocytes and other cells, crypt structural disorder,destruction, crypts, crypt epithelium massive neutrophil infiltration, Panethcell metaplasia, goblet cells decreased, severe mucosal epithelial shedding.4. Detection of Nrf2in colonic mucosa4.1Immunohistochemistry of Nrf2in colonic mucosa shows that there is lessexpression of Nrf2protein in cytoplasm in normal mucosa. However, theexpression in UC group is significantly increased which is expressed both innucleus and cytoplasm; and levels of Nrf2protein expression in each groupare as follows: active severe case group> active mild-moderate group>control group, and the comparative difference between any two groups is withstatistically significance (P <0.01).4.2Western blot shows that the levels of Nrf2protein in UC colonic mucosa ishigher than that in the normal control group (P <0.01), and levels of Nrf2protein expression in each group are as follows: active severe group> activemild-moderate group> control group. Comparisons between any two groups:comparative difference between mild-moderate group and the control group isof no statistical significance with P>0.05; comparative difference betweensevere group and mild-moderate group as well as the control group is ofstatistically significance (P <0.01).5. Correlation Analysis5.1The Nrf2protein changes in UC colonic mucosa detected byimmunohistochemical staining is of negative correlation with T-SOD activitychanges in serum, and the correlation coefficient is-0.775(P <0.01); the Nrf2protein changes in UC colon tissue detected by Western blot is also ofnegative correlation with T-SOD activity changes, and the correlation coefficient is-0.649(P <0.01).5.2The Nrf2protein changes in UC colonic mucosa detected byimmunohistochemical staining is of positive correlation with MDA contentchanges in serum, and the correlation coefficient is0.768(P<0.01); the Nrf2protein changes in UC colonic mucosa detected by Western blot is also ofpositive correlation with MDA content changes, and the correlation coefficientis0.718(P<0.01).Conclusion：1. With the further enhancement of the disease severity (level of oxidativestress), the expression of Nrf2protein in UC patients’ colonic mucosaincreases. Therefore, Nrf2can reflect the severity of UC to some extent, andthe UC incidence may be related with the signal pathway of Nrf2/ARE.2. Both methods detecting the expression of Nrf2in colonic mucosa at thesame time display that the changes of Nrf2protein in colonic mucosa is ofnegative correlation with T-SOD activity changes in serum, and is of positivecorrelation with MDA content changes in serum(P <0.01).