Nrf2在溃疡性结肠炎患者中的表达及其与氧化应激的关系

  本文关键词：Nrf2在溃疡性结肠炎患者中的表达及其与氧化应激的关系，由笔耕文化传播整理发布。

        溃疡性结肠炎（ulcerative colitis，UC）是一种病因尚不十分清楚的直肠和结肠慢性非特异性炎症性疾病。其主要症状为腹痛、腹泻、粘液脓血便等，主要侵犯远端结肠及直肠的粘膜层及粘膜下层，并向近端结肠扩展，以致侵及整个结肠。近年来大量临床资料显示该病的发病率显著增高。UC病程长，病变范围广泛，呈慢性持续性，且易癌变，越来越受到国内外人士的广泛重视，被WHO视为最难治的疾病之一。由于其确切的发病机制尚不明确，临床上至今没有特异性的根治措施。一些研究发现UC的抗氧化剂防御网络的作用与活性氧（Reactive Oxygen Species，ROS）及活性氮（Reactive Nitrogen Species，RNS）的产生过程中的作用是不平衡的。氧化应激引起氧自由基的大量表达，往往会导致脂肪、蛋白质和DNA的损伤，在UC的发病中起着重要的作用（诱导或加重UC），被认为是参与肠道炎症发生发展的一个重要因素。Nrf2(NF-E2related factor2)属于CNC(cap＇n＇collar)转录因子家族成员之一，广泛存在于机体多个组织器官中，有亮氨酸拉链结构，能活化ARE而启动多种抗氧化反应和解毒基因，从而调节体内多种细胞预防蛋白，对外源性有毒物质尤其敏感，在参与细胞抗氧化应激诱导的外源性有毒物质的防御机制中发挥重要的作用。Kelch样ECH结合蛋白1(Kelch-likeECH associating protein1，Keap1)是Nrf2的重要受体，影响Nrf2的表达。生理状态下，Nrf2位于细胞质中，与Keap l结合，处于被抑制状态，且被泛素蛋白酶体途径迅速降解。当受到氧化应激信号刺激后，Nrf2迅速与变构的Keap1解耦连，以稳定状态转位进入细胞核，与小Maf蛋白结合形成异二聚物，再与基因中的抗氧化反应元件(ARE)结合，增加ARE介导的靶基因表达，从而调控抗氧化基因的转录与翻译，如谷氨酰半胱氨酸合成酶(γ-glutamyl cysteine synthetase，γ-GCS)，谷胱甘肽-s-转移酶(GST)，苯鲲还原酶1(NQO1)，UDP-葡萄糖苷酸转移酶(UDP-glucuronosyltransferases，UGT)，环氧化物水解酶(epoxide hydrolase)等。Nrf2的激活受多个水平的调控，主要涉及Nrf2与Keap1的相互作用以及依赖于介导Nrf2稳定性的机制。Nrf2从Keap1上解离有两种机制：亲核物质或ROS的直接攻击作用；Keap1被磷酸化的间接作用。目前一些研究认为蛋白激酶C(PKC)、促分裂原活化蛋白激酶(MAPKs)和磷脂酰肌醇-3-激酶(PI3K)等途径也参与了Nrf2/ARE信号通路的激活并调控其依赖的基因的表达。此外，TauCI（牛磺醋氯胺，被激活的中性粒细胞中的一个产物）能增加Nrf2向核内转移，增加Nrf2与ARE的结合；在内质网中，增加的非折叠蛋白可经由胰腺内质网激酶(PERK)途径磷酸化Nrf2而激活Nrf2。最近多项研究发现，Nrf2与炎症性疾病关系密切，参与炎症的发生及发展。Nrf2在大多数炎症性疾病中高表达，抑制炎症的进一步发展，但Nrf2与UC的严重程度的关系需进一步研究。我们将就Nrf2在UC患者中的表达及对UC的发生发展进行研究。目的：检测UC患者结肠组织中Nrf2的表达，分析Nrf2与UC病情严重程度的相关性，探讨Nrf2在UC发生发展中的作用。方法：本研究共收集临床确诊为溃疡性结肠炎患者35例（病例组）、结肠息肉患者21例（对照组=A组），抽取其清晨空腹外周静脉血4ml、肠镜下活检结肠粘膜组织（直-乙交界处）4块。按照Sutherland DAI评分标准对患者进行评分后予以分组（轻中度组20例=B组、重度组15例=C组）。通过分光光度法检测血清中的T-SOD活力、MDA含量，采用Western blot、免疫组织化学染色方法检测结肠粘膜中Nrf2的表达。根据检测结果，对Nrf2含量与UC严重程度的关系进行统计学分析。结果：1.依据Sutherland DAI评分（见Table1）及UC病情活动度Truelove和Witts分级(Table2)，将UC患者分为轻度、中度、重度3组，3个组的评分均数依次为4.5分、8.9分、11.2分。入选患者的临床资料见Table3。2.血清中T-SOD、MDA的检测结果2.1病例组血清中T-SOD活力明显低于对照组（P＜0.01），且病例组中病情严重程度不同的组间血清中T-SOD活力也有差异，重度UC组＜轻中度UC组，两组间比较差异有统计学意义（P＜0.01）。2.2病例组血清中MDA含量高于对照组（P＜0.01），且病例组中病情严重程度不同的组间血清中MDA含量不同，重度UC组＞轻中度UC组，任意两组间比较：轻中度UC组与对照组比较差异不明显，P＞0.05，差异无统计学意义；重度UC组与轻中度UC组、对照组比较，差异均有统计学意义（P＜0.01）。3. HE染色结果：细胞核呈紫蓝色，细胞浆、基底膜及胶原纤维呈粉红色。正常人结肠粘膜上皮完整，固有层内少量炎性细胞浸润，腺体排列整齐；UC组粘膜弥漫性炎症，固有层内大量炎性细胞如嗜酸性细胞、中性粒细胞、淋巴细胞、浆细胞、单核细胞等细胞浸润，隐窝结构紊乱、破坏，隐窝内、隐窝上皮可见大量中性粒细胞浸润，潘氏细胞化生，杯状细胞减少，，重度者出现粘膜上皮脱落。4.结肠组织中Nrf2检测4.1结肠粘膜Nrf2免疫组化显示Nrf2在正常粘膜较少表达，主要位于细胞浆中，而UC组Nrf2表达明显增多，细胞核、胞浆中均有表达；病例组中重度活动组＞轻中度活动组＞对照组，任意两组间相比差异具有统计学意义(P<0.01)。4.2Western blot显示：UC各组结肠组织中Nrf2的水平高于正常对照组，P<0.01，各组Nrf2的表达水平依次为：重度活动组＞轻中度活动组＞对照组。任意两组间相比：轻中度组与对照组比较P＞0.05，差异无统计学意义；重度组与轻中度组、对照组比较，差异具有统计学意义(P<0.01)。5.相关性分析5.1免疫组织化学染色检测的UC组结肠组织中Nrf2的变化与其血清中T-SOD活力的变化成负相关，相关系数为-0.775(P<0.01)；应用Western blot方法检测的UC组结肠组织中Nrf2的变化与其血清中T-SOD活力的变化成负相关，相关系数为-0.649(P<0.01)。5.2免疫组织化学染色检测的UC组结肠组织中Nrf2的变化与其血清中MDA含量的变化成正相关，相关系数为0.768(P<0.01)；应用Western blot方法检测的UC组结肠组织中Nrf2的变化与其血清中MDA含量的变化成正相关，相关系数为0.718(P<0.01)。结论：1.随着病情严重程度（氧化应激水平）的进一步增强，UC患者结肠粘膜组织中Nrf2的表达不断增多，Nrf2在一定程度上可以反映UC的严重程度，UC的发病可能与Nrf2/ARE信号通路有关。2.两种检测结肠粘膜Nrf2蛋白表达的方法均显示结肠组织中Nrf2的变化与血清中T-SOD活力的变化成负相关，与血清中MDA含量的变化成正相关（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在溃疡性结肠炎患者中的表达及其与氧化应激的关系

摘要4-8ABSTRACT8-12前言13-14材料与方法14-25结果25-28附图28-32附表32-34讨论34-37结论37参考文献37-41综述 Nrf2 在消化系统疾病中的研究进展41-57    参考文献49-57致谢57-58个人简历58

  本文关键词：Nrf2在溃疡性结肠炎患者中的表达及其与氧化应激的关系，由笔耕文化传播整理发布。