肠上皮双氧化酶（DUOX）2在炎症性肠病内质网应激中的作用

The case number of inflammatory bowel disease (IBD) has been rapidly increasing in China over past decades although there is no epidemiological data for the entire country yet. The etiology of IBD is still unknown. The therapy of IBD is currently not satisfied because of the limit of medication and side effects of drugs. As a member of NOX/DUOX oxidase family, DUOX2 of intestinal epithelial cells generates hydrogen peroxide (H2O2), which is a major source of H2O2 for the intestinal epithelium. DUOX2 supports antimicrobial defense, affects adjustment of intestinal microbial flora and amplifies diverse immune signaling so DUOX2 plays an important role in the intestinal innate immune response; however, excessive H2O2 produced by DUOX2 may lead to oxidative stress and cause inflammatory reaction. Both oxidative stress and endoplasmic reticulum (ER) stress can activate NK-κB signaling pathways, resulting in inflammation and damage of epithelium. The influence of DUOX2 and its product H2O2 on ER stress of intestinal epithelial cells has not been clarified yet, and it is also worthy to explore the consequence of ER stress affecting the expression and function of DUOX2. In this study, immunological and molecular biology methods will be used in different animal models and intestinal epithelial cell line. Firstly adenovirus mediated overexpression of DUOX2 will be made in glutathione peroxidase 1 and 2 duoble knokout mouse colitis model and Dextran Sulfate Sodium (DSS) induced mouse colitis model to observe how DUOX2 exacerbate colitis in these mice, and oxidative stress and ER stress molecules will be measured to explore the influence of DUOX2 on ER stress. Secondly, using siRNA technique to interfere DUOX2 or CHOP gene in Caco-2 cell line respectively, the mechanism of DUOX2 and ER stress chaperone molecules affecting one another will be tested. Finally different interventions will be used in animal and cell line to observe the effect of DUOX2 activity on ER stress.The mechanism of DUOX2 affecting on ER stress and its role in pathogenesis of IBD will be discussed with the results of the study and it may be possible to find a new molecular target for the treatment of IBD from this study.

我国炎症性肠病（IBD）的发病呈现升高趋势，目前治疗措施往往达不到满意的疗效，严重影响了患者的生活质量。DUOX2是 NOX/DOUX氧化酶家族蛋白成员之一，由其产生的H2O2是肠黏膜H2O2的主要来源，DUOX2具有调控肠道菌群和抗菌作用，但DUOX2过度活动产生过多的H2O2则可能导致肠道的氧化应激。氧化应激及内质网应激均可激活NK-κB信号通路，促进炎症发生，但DUOX2及其产物H2O2对肠上皮细胞内质网应激的影响以及它们之间的具体作用机制尚未阐明。本课题将应用不同小鼠肠炎模型，诱导DUOX2过表达,观察各种干预前后肠黏膜炎症过程和内质网应激途径中相关分子表达改变；另外，分别敲减肠上皮细胞株DUOX2、CHOP基因，观察不同干预措施对DUOX2活动和内质网应激的作用，探讨DUOX2对内质网应激的影响及其在IBD疾病发生发展中的作用机制，以期为IBD的治疗提供新的理论依据和方向。

炎症性肠病（inflammatory bowel disease, IBD）是慢性非特异性肠道炎性疾病。在IBD发生和发展过程中，氧化应激被认为是导致肠道损伤的关键因素。内质网应激在IBD的发生和发展中的作用越来越受到重视，且氧化应激是内质网应激的主要诱因之一。双氧化酶（DUOX）2等NADPH氧化酶和内质网应激参与IBD的发病机理。研究内容：1）应用急慢性小鼠肠炎模型，研究肠道氧化应激及内质网应激对细胞的炎性损伤作用及机理；2）研究5-羟色胺（5-HT）加重葡聚糖硫酸钠（DSS）诱导的小鼠肠炎以及对人HT-29和Caco-2结肠细胞、U937白细胞株影响；3）小鼠结肠癌模型的研究；4）NADPH氧化酶在人IBD的表达研究；5）NADPH氧化酶和内质网应激蛋白PERK、ATF6 、IREα/β和黏蛋白（Mucin）2在人结肠癌的表达研究；6）DUOX2 在Barrett 食管、胃癌、结肠癌和肝癌中作用的研究。.主要结果及意义：1）DSS诱导小鼠肠炎时NADPH氧化酶Nox1和Duox2在轻度炎症时表达升高5.3和6.7倍。2）IRE1β和MUC2表达均明显降低，慢性炎症组基因表达仅为对照组的 31% 和25%，表明IRE1β和MUC2可能与结肠上皮的功能维持有关，IRE1β/MUC2通路功能受到抑制促进炎症的发生。3）5-HT能够诱导小鼠结肠组织中基质金属蛋白酶（MMP）-3和-9的表达，也可以分别刺激Caco-2细胞和人白细胞株U937中MMPs的表达增加。4）人结直肠肿瘤内质网应激IRE1α-XBP1通路被激活，提示该通路参与了结直肠肿瘤的发生发展。内质网应激蛋白PERK、ATF6、IRE1在结肠癌组织中的表达下降，而IRE1α、XBP1u、 XBP1s表达水平没有差异，表明IRE1β可通过诱导MUC2的表达从而保护结肠黏膜，内质网应激抑制可能是结肠肿瘤发生发展的因素。5）NOX2和NOX3 mRNA 在胃癌组织中的表达明显增加，表面它们在肿瘤的发生中起作用。6）DUOX2 蛋白仅表达在Barrett 食管，而正常上皮细胞无表达，因此DUOX2 可能参与Barrett 食管的癌变。DUOX2蛋白还可作为结直肠癌疾病进展的一个重要指标。这些结果表明NADPH氧化酶和内质网应激在炎症肿瘤的发生过程中发挥作用，并为肠道炎症及发生机制提供了思路，可能为治疗找到新的途径。