AhR活化对结肠炎性损害的缓解作用及机制研究

发布时间：2018-06-10 12:55

本文选题：芳香烃受体 + 锌指蛋白　；参考：《第三军医大学》2017年博士论文

【摘要】：背景及目的炎症性肠病(Inflammatory bowel disease, IBD),包括溃疡性结肠炎(Ulcerative colitis, UC)和克罗恩病(Crohn's disease, CD),是一种慢性肠道特发性疾病。大量研究表明遗传、环境及免疫因素均与IBD相关。其中,免疫调节紊乱目前被认为是IBD发病的主要病因,但其发病机制仍未研究透彻。肠粘膜屏障(Intestinal mucosal barrier,IMB)包括机械屏障、免疫屏障、生物屏障及化学屏障,是肠上皮抵抗外源物质的重要屏障。肠粘膜机械屏障由肠上皮细胞(Intestinal epithelial cell, IEC)及细胞之间的紧密连接(Tightjunction, TJ)构成。其中,TJ起着重要的作用。研究表明,肠道炎症与TJ之间有着密切的关系。IBD患者的肠道紧密连接严重破坏,肠道通透性明显增加。另外,肠道紧密连接屏障的破坏会导致肠腔内有毒分子的渗透,诱导粘膜免疫系统和炎症反应,引发肠道疾病甚至全身系统性疾病如全身炎症反应综合征(Systemic inflammatory response syndrome,SIRS)及多器官功能衰竭(Mutiply organ failure,MOF)。因此,目前认为有效的抑制炎症及维持肠粘膜屏障功能是目前治疗IBD的主要策略。芳香烃受体(Aryl hydrocarbon receptor,AhR),是一种广泛表达于机体各种细胞的转录因子。AhR通过与配体结合活化的方式入核,与AhR核转录子(AhR nuclear translocator,ARNT)形成异源二聚体,特异性结合于AhR反应原件,诱导包括细胞色素酶4501A1 (Cytochrome P450 1A1,CYP1A1)等一系列下游基因的表达。6-甲酰基吲哚并[3,2-B]咔(6-formylindolo(3,2-b) carbazole,FICZ)是 AhR 的一种内源性配体,研究表明,FICZ可以通过活化AhR参与细胞生理功能的维持,包括调控细胞增殖、凋亡,抑制免疫反应等。目前多项研究表明AhR活化可以抑制肠道炎症,但是其具体机制仍不清楚。另一方面,我们实验室的前期工作表明,AhR可以在肠梗阻模型中通过调节紧密连接蛋白的表达和定位缓解肠粘膜屏障功能异常。因此,本课题通过模拟IBD动物模型及体外细胞模型,采用蛋白质免疫印迹(Western blotting,WB)、实时荧光定量聚合酶链式反应(Real-time quantitation PCR,Real-time Q-PCR)、免疫组化(Immunohistochemical,IHC)、免疫荧光(Immunofluorescence,IF)、跨膜电阻测量等技术,研究AhR活化减轻IBD肠道炎症及缓解肠粘膜屏障功能异常的具体机制。方法1.建立小鼠DSS诱导的结肠炎模型,通过给予小鼠FICZ腹腔注射处理,用小鼠体重改变、结肠长度、死亡率、结肠形态学改变及炎症因子表达来评估小鼠肠道炎症的炎性损害情况。采用 Western blotting、Real-time Q-PCR、IHC 等技术检测 CYP1A1、(tristetraprolin) TTP、ZO-1、claudin-1、occludin 等的表达变化。使用 Ussing Chamber检测小鼠结肠跨上皮电阻。观察AhR活化对结肠炎炎症及肠粘膜屏障功能的影响。2.建立肠上皮细胞系LPS干预模型,通过给予FICZ处理、siRNA干扰AhR处理后,采用 Western blotting、Real-time Q-PCR、IF 等技术检测 CYP1A1、TTP、MAPKAPK2、p-MAPKAPK2的表达,探讨AhR活化缓解结肠炎炎症的机制。3.建立肠上皮细胞系TNF-α/IFN-γ干预模型,通过给予FICZ处理,用Western blotting、Real-time Q-PCR、IF 等技术检测 CYP1A1、ZO-1、claudin-1、occludin 等的表达变化,并检测细胞的跨上皮电阻(TER),探讨AhR活化维持肠粘膜屏障功能的具体机制。主要结果1.小鼠DSS模型中,相比于Sham组,DSS处理组小鼠体重明显降低,死亡率增加,结肠长度明显缩短,肠粘膜正常形态学改变(表现为炎症细胞浸润、上皮细胞融合脱落等)。小鼠结肠跨上皮电阻下降,肠粘膜通透性增加,肠粘膜屏障功能受损。而给予小鼠AhR激动剂FICZ后,同DSS组相比较,小鼠体重有所回升,死亡率降低,结肠长度明显增加,而肠粘膜损伤也有所缓解。小鼠结肠跨上皮电阻增加,肠粘膜屏障功能有所恢复。2.小鼠DSS模型中,给予FICZ可以显著上调CYP1A1的表达,活化AhR。并且可以上调RNA结合蛋白TTP的表达。3.体外LPS干预模型中,FICZ可以显著上调TTP表达。siRNA干扰AhR后,TTP表达有所下降,并且给予FICZ不能上调TTP的表达。另一方面,FICZ可以抑制MK2的磷酸化。siRNA干扰AhR后,FICZ不再能抑制MK2的磷酸化。4.小鼠DSS模型中,DSS诱导的结肠炎结肠上皮中紧密连接蛋白如ZO-1、claudin-1、occludin的表达有下降。而给予FICZ处理后可以显著上调ZO-1、claudin-1、occludin的表达。5.体外TNF-α/IFN-γ干预模型中,相比于TNF-α/IFN-γ处理组,给予FICZ可以上调细胞的跨上皮电阻,并且可以缓解ZO-1的结构破坏。另一方面,FICZ可以增加MLC的磷酸化。结论1. FICZ在体内体外模型中均可以活化AhR。2. AhR活化可以缓解DSS诱导的结肠炎炎症及肠粘膜屏障功能异常。3. MK2/p-MK2 /TTP通路参与了 AhR调控肠道炎症的过程。4. AhR活化可以减少炎症因子导致的肠上皮中紧密连接蛋白表达的下降及定位的改变。5. MLCK/p-MLC通路可能参与了 AhR调控紧密连接蛋白,缓解肠粘膜屏障功能异常的过程。
[Abstract]:Background and objective Inflammatory bowel disease (IBD), including ulcerative colitis (Ulcerative colitis, UC) and Crohn's disease (Crohn's disease, CD). It is a chronic intestinal idiopathic disease. A large number of studies have shown that heredity, environment and immuno factor are associated with IBD. The main cause of disease, but its pathogenesis is still not thoroughly studied. The intestinal mucosal barrier (Intestinal mucosal barrier, IMB), including mechanical barriers, immune barriers, biological barriers and chemical barriers, is an important barrier to the resistance to exogenous substances in the intestinal epithelium. The intestinal mucosal mechanical barrier consists of the intestinal epithelial cells (Intestinal epithelial cell, IEC) and the cells. Tightjunction (TJ) composition. Among them, TJ plays an important role. Studies have shown that intestinal inflammation and TJ have a close relationship with the severe disruption of the intestinal close connections in.IBD patients and the obvious increase in intestinal permeability. In addition, the destruction of the intestinal tight junction barrier can lead to the infiltration of toxic molecules in the intestinal cavity and induce the mucosal immune system. Systemic and inflammatory reactions lead to intestinal diseases and even systemic diseases such as systemic inflammatory response syndrome (Systemic inflammatory response syndrome, SIRS) and multiple organ failure (Mutiply organ failure, MOF). Therefore, it is considered that effective inhibition of inflammation and intestinal mucosal barrier function is the main strategy for the treatment of IBD. Aryl hydrocarbon receptor (AhR) is a transcriptional factor that is widely expressed in various cells of the body and.AhR is activated by ligand binding to the ligand and forms a heterogenous two polymer with the AhR nuclear transcriptome (AhR nuclear translocator, ARNT). P450 1A1, CYP1A1), such as the expression of a series of downstream genes,.6- formyl indole and [3,2-B] carbazole (6-formylindolo (3,2-b) carbazole, FICZ) is an endogenous ligand for AhR. The study shows that FICZ can be involved in the maintenance of cell physiological function by activating AhR, including regulating cell proliferation, apoptosis, inhibiting immune response and so on. AhR activation can inhibit intestinal inflammation, but its specific mechanisms are still unclear. On the other hand, our laboratory earlier work showed that AhR could alleviate intestinal mucosal barrier dysfunction by regulating the expression and localization of tight connexin in the intestinal obstruction model. Therefore, this lesson is done by simulating the IBD animal model and in vitro cell model, Western blotting (WB), real-time quantitative polymerase chain reaction (Real-time quantitation PCR, Real-time Q-PCR), immunohistochemistry (Immunohistochemical, IHC), immunofluorescence (Immunofluorescence, IF), and transmembrane resistance measurements were used to study the reduction of intestinal inflammation and intestinal mucosal barrier work Method 1. the model of DSS induced colitis in mice was established. By intraperitoneal injection of FICZ in mice, the mice weight change, colon length, death rate, colonic morphological change and inflammatory factor expression were used to evaluate the inflammatory damage of intestinal inflammation in mice. Western blotting, Real-time Q-PCR, IHC and so on were used. CYP1A1, (tristetraprolin) TTP, ZO-1, claudin-1, occludin and other expression changes. Use Ussing Chamber to detect the cross epithelial resistance of the colon in mice. Observe the effect of AhR activation on inflammation of colitis and intestinal mucosal barrier function,.2. to establish the LPS intervention model of the intestinal epithelial cell line. Western blotting, Real-time Q-PCR, IF and other techniques were used to detect the expression of CYP1A1, TTP, MAPKAPK2, p-MAPKAPK2, and to explore the mechanism of AhR activation to alleviate inflammation of the colitis. The expression changes of DIN and other cell resistance (TER) were detected and the specific mechanism of AhR activation to maintain intestinal mucosal barrier function was explored. The main results in 1. mice DSS model, compared with the Sham group, the weight of the mice in the DSS treatment group decreased significantly, the mortality rate increased, the colon length shortened obviously, and the intestinal mucosa normal morphological changes (showing inflammation fine). The resistance of the colon of mice decreased, the permeability of intestinal mucosa increased and the intestinal mucosal barrier function was damaged. After the mice were given AhR agonist FICZ, the weight of mice increased, the death rate decreased, the colon length increased obviously, and the intestinal mucosa damage was also relieved. The colon of mice was on the upper part of the colon. The skin resistance increased and the intestinal mucosal barrier function recovered in the DSS model of.2. mice. FICZ could significantly up-regulate the expression of CYP1A1, activate AhR. and increase the expression of RNA binding protein TTP in the LPS intervention model in vitro. FICZ can significantly increase TTP expression.SiRNA interference. On the other hand, FICZ can inhibit MK2 phosphorylation.SiRNA interference AhR, FICZ no longer inhibits the DSS model of MK2 phosphorylated.4. mice, and DSS induced colonic colonic epithelium in colon epithelium, such as ZO-1, claudin-1, decreased. In the TNF- alpha /IFN- gamma intervention model of.5. in vitro, compared to the TNF- alpha /IFN- gamma treatment group, FICZ can increase the cross epithelial resistance of the cells and alleviate the structural damage of ZO-1. On the other hand, FICZ can increase the phosphorylation of MLC. Conclusion 1. FICZ can be activated by activation of AhR.2. AhR in vivo and in vitro models. Inflammation of enteritis and intestinal mucosal barrier function.3. MK2/p-MK2 /TTP pathway participates in the process of AhR regulation of intestinal inflammation,.4. AhR activation can reduce the decrease and localization of the expression of tight connexin in the intestinal epithelium caused by inflammatory factors, the.5. MLCK/p-MLC pathway may participate in the AhR regulated tight connexin and alleviate the intestinal mucosal screen. The process of abnormal dysfunction.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R574

【相似文献】