Butyrate Attenuates Lipopolysaccharide-Induced Inflammation in Intestinal Cells and Crohn's Mucosa through Modulation of Antioxidant Defense Machinery

Oxidative stress plays an important role in the pathogenesis of inflammatory bowel disease (IBD), including Crohn's disease (CrD). High levels of Reactive Oxygen Species (ROS) induce the activation of the redox-sensitive nuclear transcription factor kappa-B (NF-κB), which in turn triggers the inflammatory mediators. Butyrate decreases pro-inflammatory cytokine expression by the lamina propria mononuclear cells in CrD patients via inhibition of NF-κB activation, but how it reduces inflammation is still unclear. We suggest that butyrate controls ROS mediated NF-κB activation and thus mucosal inflammation in intestinal epithelial cells and in CrD colonic mucosa by triggering intracellular antioxidant defense systems. Intestinal epithelial Caco-2 cells and colonic mucosa from 14 patients with CrD and 12 controls were challenged with or without lipopolysaccaride from Escherichia Coli (EC-LPS) in presence or absence of butyrate for 4 and 24 h. The effects of butyrate on oxidative stress, p42/44 MAP kinase phosphorylation, p65-NF-κB activation and mucosal inflammation were investigated by real time PCR, western blot and confocal microscopy. Our results suggest that EC-LPS challenge induces a decrease in Gluthation-S-Transferase-alpha (GSTA1/A2) mRNA levels, protein expression and catalytic activity; enhanced levels of ROS induced by EC-LPS challenge mediates p65-NF-κB activation and inflammatory response in Caco-2 cells and in CrD colonic mucosa. Furthermore butyrate treatment was seen to restore GSTA1/A2 mRNA levels, protein expression and catalytic activity and to control NF-κB activation, COX-2, ICAM-1 and the release of pro-inflammatory cytokine. In conclusion, butyrate rescues the redox machinery and controls the intracellular ROS balance thus switching off EC-LPS induced inflammatory response in intestinal epithelial cells and in CrD colonic mucosa.

氧化应激（Oxidative stress）在包括克罗恩病（CrD）在内的炎症性肠病（IBD）的发病机制中发挥重要作用。高水平活性氧（ROS）可激活氧化还原敏感型核转录因子κB（NF-κB），进而触发炎症介质的活化与释放。丁酸盐可通过抑制NF-κB活化，降低克罗恩病患者固有层单核细胞的促炎细胞因子表达，但其缓解黏膜炎症的具体机制仍不明确。本研究提出，丁酸盐可通过激活细胞内抗氧化防御系统，在肠上皮细胞及克罗恩病患者结肠黏膜中调控ROS介导的NF-κB活化，从而抑制黏膜炎症。本研究以肠上皮Caco-2细胞、14例克罗恩病患者及12例健康对照者的结肠黏膜为研究对象，在添加或不添加丁酸盐的条件下，用大肠杆菌脂多糖（EC-LPS）刺激或不予刺激，分别处理4小时与24小时。采用实时荧光定量PCR（real time PCR）、蛋白质免疫印迹（western blot）及共聚焦显微镜技术（confocal microscopy），探究丁酸盐对氧化应激水平、p42/44丝裂原活化蛋白激酶（MAPK）磷酸化、p65-NF-κB活化及黏膜炎症的影响。研究结果显示：EC-LPS刺激可显著降低Caco-2细胞及克罗恩病患者结肠黏膜中谷胱甘肽S-转移酶α亚型（GSTA1/A2）的mRNA水平、蛋白表达量与催化活性；EC-LPS诱导的ROS水平升高，可介导Caco-2细胞及克罗恩病患者结肠黏膜中的p65-NF-κB活化与炎症应答。此外，丁酸盐处理可恢复GSTA1/A2的mRNA水平、蛋白表达及催化活性，同时抑制NF-κB活化、环氧合酶2（COX-2）与细胞间黏附分子1（ICAM-1）的表达，并减少促炎细胞因子的释放。综上，丁酸盐可修复氧化还原调控系统，维持细胞内ROS平衡，从而阻断EC-LPS诱导的肠上皮细胞及克罗恩病患者结肠黏膜的炎症应答。