本文關(guān)鍵詞：Nrf2在潰瘍性結(jié)腸炎患者中的表達(dá)及其與氧化應(yīng)激的關(guān)系，由筆耕文化傳播整理發(fā)布。

        潰瘍性結(jié)腸炎（ulcerative colitis，UC）是一種病因尚不十分清楚的直腸和結(jié)腸慢性非特異性炎癥性疾病。其主要癥狀為腹痛、腹瀉、粘液膿血便等，主要侵犯遠(yuǎn)端結(jié)腸及直腸的粘膜層及粘膜下層，并向近端結(jié)腸擴(kuò)展，以致侵及整個(gè)結(jié)腸。近年來(lái)大量臨床資料顯示該病的發(fā)病率顯著增高。UC病程長(zhǎng)，病變范圍廣泛，呈慢性持續(xù)性，且易癌變，越來(lái)越受到國(guó)內(nèi)外人士的廣泛重視，被WHO視為最難治的疾病之一。由于其確切的發(fā)病機(jī)制尚不明確，臨床上至今沒(méi)有特異性的根治措施。一些研究發(fā)現(xiàn)UC的抗氧化劑防御網(wǎng)絡(luò)的作用與活性氧（Reactive Oxygen Species，ROS）及活性氮（Reactive Nitrogen Species，RNS）的產(chǎn)生過(guò)程中的作用是不平衡的。氧化應(yīng)激引起氧自由基的大量表達(dá)，往往會(huì)導(dǎo)致脂肪、蛋白質(zhì)和DNA的損傷，在UC的發(fā)病中起著重要的作用（誘導(dǎo)或加重UC），被認(rèn)為是參與腸道炎癥發(fā)生發(fā)展的一個(gè)重要因素。Nrf2(NF-E2related factor2)屬于CNC(cap＇n＇collar)轉(zhuǎn)錄因子家族成員之一，廣泛存在于機(jī)體多個(gè)組織器官中，有亮氨酸拉鏈結(jié)構(gòu)，能活化ARE而啟動(dòng)多種抗氧化反應(yīng)和解毒基因，從而調(diào)節(jié)體內(nèi)多種細(xì)胞預(yù)防蛋白，對(duì)外源性有毒物質(zhì)尤其敏感，在參與細(xì)胞抗氧化應(yīng)激誘導(dǎo)的外源性有毒物質(zhì)的防御機(jī)制中發(fā)揮重要的作用。Kelch樣ECH結(jié)合蛋白1(Kelch-likeECH associating protein1，Keap1)是Nrf2的重要受體，影響Nrf2的表達(dá)。生理狀態(tài)下，Nrf2位于細(xì)胞質(zhì)中，與Keap l結(jié)合，處于被抑制狀態(tài)，且被泛素蛋白酶體途徑迅速降解。當(dāng)受到氧化應(yīng)激信號(hào)刺激后，Nrf2迅速與變構(gòu)的Keap1解耦連，以穩(wěn)定狀態(tài)轉(zhuǎn)位進(jìn)入細(xì)胞核，與小Maf蛋白結(jié)合形成異二聚物，再與基因中的抗氧化反應(yīng)元件(ARE)結(jié)合，增加ARE介導(dǎo)的靶基因表達(dá)，從而調(diào)控抗氧化基因的轉(zhuǎn)錄與翻譯，如谷氨酰半胱氨酸合成酶(γ-glutamyl cysteine synthetase，γ-GCS)，谷胱甘肽-s-轉(zhuǎn)移酶(GST)，苯鯤還原酶1(NQO1)，UDP-葡萄糖苷酸轉(zhuǎn)移酶(UDP-glucuronosyltransferases，UGT)，環(huán)氧化物水解酶(epoxide hydrolase)等。Nrf2的激活受多個(gè)水平的調(diào)控，主要涉及Nrf2與Keap1的相互作用以及依賴于介導(dǎo)Nrf2穩(wěn)定性的機(jī)制。Nrf2從Keap1上解離有兩種機(jī)制：親核物質(zhì)或ROS的直接攻擊作用；Keap1被磷酸化的間接作用。目前一些研究認(rèn)為蛋白激酶C(PKC)、促分裂原活化蛋白激酶(MAPKs)和磷脂酰肌醇-3-激酶(PI3K)等途徑也參與了Nrf2/ARE信號(hào)通路的激活并調(diào)控其依賴的基因的表達(dá)。此外，TauCI（牛磺醋氯胺，被激活的中性粒細(xì)胞中的一個(gè)產(chǎn)物）能增加Nrf2向核內(nèi)轉(zhuǎn)移，增加Nrf2與ARE的結(jié)合；在內(nèi)質(zhì)網(wǎng)中，增加的非折疊蛋白可經(jīng)由胰腺內(nèi)質(zhì)網(wǎng)激酶(PERK)途徑磷酸化Nrf2而激活Nrf2。最近多項(xiàng)研究發(fā)現(xiàn)，Nrf2與炎癥性疾病關(guān)系密切，參與炎癥的發(fā)生及發(fā)展。Nrf2在大多數(shù)炎癥性疾病中高表達(dá)，抑制炎癥的進(jìn)一步發(fā)展，但Nrf2與UC的嚴(yán)重程度的關(guān)系需進(jìn)一步研究。我們將就Nrf2在UC患者中的表達(dá)及對(duì)UC的發(fā)生發(fā)展進(jìn)行研究。目的：檢測(cè)UC患者結(jié)腸組織中Nrf2的表達(dá)，分析Nrf2與UC病情嚴(yán)重程度的相關(guān)性，探討Nrf2在UC發(fā)生發(fā)展中的作用。方法：本研究共收集臨床確診為潰瘍性結(jié)腸炎患者35例（病例組）、結(jié)腸息肉患者21例（對(duì)照組=A組），抽取其清晨空腹外周靜脈血4ml、腸鏡下活檢結(jié)腸粘膜組織（直-乙交界處）4塊。按照Sutherland DAI評(píng)分標(biāo)準(zhǔn)對(duì)患者進(jìn)行評(píng)分后予以分組（輕中度組20例=B組、重度組15例=C組）。通過(guò)分光光度法檢測(cè)血清中的T-SOD活力、MDA含量，采用Western blot、免疫組織化學(xué)染色方法檢測(cè)結(jié)腸粘膜中Nrf2的表達(dá)。根據(jù)檢測(cè)結(jié)果，對(duì)Nrf2含量與UC嚴(yán)重程度的關(guān)系進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果：1.依據(jù)Sutherland DAI評(píng)分（見(jiàn)Table1）及UC病情活動(dòng)度Truelove和Witts分級(jí)(Table2)，將UC患者分為輕度、中度、重度3組，3個(gè)組的評(píng)分均數(shù)依次為4.5分、8.9分、11.2分。入選患者的臨床資料見(jiàn)Table3。2.血清中T-SOD、MDA的檢測(cè)結(jié)果2.1病例組血清中T-SOD活力明顯低于對(duì)照組（P＜0.01），且病例組中病情嚴(yán)重程度不同的組間血清中T-SOD活力也有差異，重度UC組＜輕中度UC組，兩組間比較差異有統(tǒng)計(jì)學(xué)意義（P＜0.01）。2.2病例組血清中MDA含量高于對(duì)照組（P＜0.01），且病例組中病情嚴(yán)重程度不同的組間血清中MDA含量不同，重度UC組＞輕中度UC組，任意兩組間比較：輕中度UC組與對(duì)照組比較差異不明顯，P＞0.05，差異無(wú)統(tǒng)計(jì)學(xué)意義；重度UC組與輕中度UC組、對(duì)照組比較，差異均有統(tǒng)計(jì)學(xué)意義（P＜0.01）。3. HE染色結(jié)果：細(xì)胞核呈紫藍(lán)色，細(xì)胞漿、基底膜及膠原纖維呈粉紅色。正常人結(jié)腸粘膜上皮完整，固有層內(nèi)少量炎性細(xì)胞浸潤(rùn)，腺體排列整齊；UC組粘膜彌漫性炎癥，固有層內(nèi)大量炎性細(xì)胞如嗜酸性細(xì)胞、中性粒細(xì)胞、淋巴細(xì)胞、漿細(xì)胞、單核細(xì)胞等細(xì)胞浸潤(rùn)，隱窩結(jié)構(gòu)紊亂、破壞，隱窩內(nèi)、隱窩上皮可見(jiàn)大量中性粒細(xì)胞浸潤(rùn)，潘氏細(xì)胞化生，杯狀細(xì)胞減少，，重度者出現(xiàn)粘膜上皮脫落。4.結(jié)腸組織中Nrf2檢測(cè)4.1結(jié)腸粘膜Nrf2免疫組化顯示Nrf2在正常粘膜較少表達(dá)，主要位于細(xì)胞漿中，而UC組Nrf2表達(dá)明顯增多，細(xì)胞核、胞漿中均有表達(dá)；病例組中重度活動(dòng)組＞輕中度活動(dòng)組＞對(duì)照組，任意兩組間相比差異具有統(tǒng)計(jì)學(xué)意義(P<0.01)。4.2Western blot顯示：UC各組結(jié)腸組織中Nrf2的水平高于正常對(duì)照組，P<0.01，各組Nrf2的表達(dá)水平依次為：重度活動(dòng)組＞輕中度活動(dòng)組＞對(duì)照組。任意兩組間相比：輕中度組與對(duì)照組比較P＞0.05，差異無(wú)統(tǒng)計(jì)學(xué)意義；重度組與輕中度組、對(duì)照組比較，差異具有統(tǒng)計(jì)學(xué)意義(P<0.01)。5.相關(guān)性分析5.1免疫組織化學(xué)染色檢測(cè)的UC組結(jié)腸組織中Nrf2的變化與其血清中T-SOD活力的變化成負(fù)相關(guān)，相關(guān)系數(shù)為-0.775(P<0.01)；應(yīng)用Western blot方法檢測(cè)的UC組結(jié)腸組織中Nrf2的變化與其血清中T-SOD活力的變化成負(fù)相關(guān)，相關(guān)系數(shù)為-0.649(P<0.01)。5.2免疫組織化學(xué)染色檢測(cè)的UC組結(jié)腸組織中Nrf2的變化與其血清中MDA含量的變化成正相關(guān)，相關(guān)系數(shù)為0.768(P<0.01)；應(yīng)用Western blot方法檢測(cè)的UC組結(jié)腸組織中Nrf2的變化與其血清中MDA含量的變化成正相關(guān)，相關(guān)系數(shù)為0.718(P<0.01)。結(jié)論：1.隨著病情嚴(yán)重程度（氧化應(yīng)激水平）的進(jìn)一步增強(qiáng)，UC患者結(jié)腸粘膜組織中Nrf2的表達(dá)不斷增多，Nrf2在一定程度上可以反映UC的嚴(yán)重程度，UC的發(fā)病可能與Nrf2/ARE信號(hào)通路有關(guān)。2.兩種檢測(cè)結(jié)腸粘膜Nrf2蛋白表達(dá)的方法均顯示結(jié)腸組織中Nrf2的變化與血清中T-SOD活力的變化成負(fù)相關(guān)，與血清中MDA含量的變化成正相關(guān)（P<0.01）。

    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).

Nrf2在潰瘍性結(jié)腸炎患者中的表達(dá)及其與氧化應(yīng)激的關(guān)系

摘要4-8ABSTRACT8-12前言13-14材料與方法14-25結(jié)果25-28附圖28-32附表32-34討論34-37結(jié)論37參考文獻(xiàn)37-41綜述 Nrf2 在消化系統(tǒng)疾病中的研究進(jìn)展41-57    參考文獻(xiàn)49-57致謝57-58個(gè)人簡(jiǎn)歷58

  本文關(guān)鍵詞：Nrf2在潰瘍性結(jié)腸炎患者中的表達(dá)及其與氧化應(yīng)激的關(guān)系，由筆耕文化傳播整理發(fā)布。