芥子碱调控TLR4/NF-кB/MLCK-pMLC通路干预溃疡性结肠炎小鼠肠黏膜通透性的分子机制

Sinapine is an alkaloid mainly present in cruciferae vegetables, which exhibited a greater antioxidant activity. The activation of myosin light chain kinase (MLCK) and myosin light chain (MLC) phosphorylation were both important factors during the tight junction (TJ) dysfunction and abnormal permeability in intestinal mucosa. Maintaining both the normal function of the intestinal epithelial cell TJ, and repeating the intestinal barrier has been identified as a novel strategy for ulcerative colitis (UC) in the clinic. The sinapine enriched Brassica seed extract was capable of reduce the lipopolysaccharide induced the abnormal permeability of Caco-2 intestinal epithelial cells. Brassica extract also increased the colonic mRNA expressions of some TJ factors (such as ZO-1, claudin-1 and occludin) to reduce intestinal permeability and attenuated the dextran sulfate sodium(DSS) induced mice colitis. We suggested that the effect of the Brassica extract to attenuate the UC was associated with its contained sinapine which exhibited an activity to regulate the protein expression of TJ and modulate intestinal permeability in colitis mice. Currently, the effect of sinapine to reduce the mice colitis has not been studied yet. The TLR4/NF-кB pathway played an important role in the pathophysiological process of UC. Hence, the present study was to investigate the molecular mechanisms of sinapine on the intestinal permeability by regulating the TLR4/NF-кB/MLCK-pMLC pathway in the UC mice model and intestinal epithelial cells. The results of this study will present the scientific evidences in human UC prevention and therapy.

芥子碱是一种主要存在于十字花科蔬菜中的生物碱，具有较好的抗氧化活性。目前，抑制肌球蛋白轻链激酶(MLCK)和肌球蛋白轻链(MLC)的异常磷酸化，维持肠上皮细胞紧密连接(TJ)完整，修复肠黏膜屏障已成为治疗溃疡性结肠炎(UC)的新策略。前期发现，富含芥子碱的芸薹子提取物能改善脂多糖所致肠上皮细胞屏障功能障碍；通过上调TJ蛋白ZO-1、claudin-1和occludin的mRNA转录，抑制肠黏膜通透性升高，缓解葡聚糖硫酸钠(DSS)诱发的小鼠结肠炎。我们推测芸薹子所含的芥子碱可能在修复UC受损肠黏膜屏障方面起一定作用。芥子碱对UC的干预效果还未被系统研究过。而TLR4/NF-кB又是UC病生过程中的重要信号通路。因此，本研究拟由TLR4/NF-кB/MLCK-pMLC通路入手，从细胞和整体动物两个层面，探讨芥子碱干预结肠炎肠黏膜高通透性的分子机制，为其在人UC的防治层面提供一定的科学依据。

寻找有效抗炎天然产物是炎症性肠病防治的热点。本研究提示芥子碱能调控Inos和Cox2的mRNA表达来降低LPS致RAW264.7细胞中NO和PGE2分泌，并抑制TNF-α，IL-1β，IL-6和IL-8的过度分泌及其mRNA表达。利用DSS诱导小鼠结肠炎模型观察发现，芥子碱显著抑制结肠炎小鼠体重降低及其结肠长度缩短，降低其结肠重量/结肠长度比。芥子碱显著抑制结肠炎小鼠结肠组织的破坏与MPO水平的升高，降低肠组织中TNF-α，IL-1β，IL-6和IL-18水平。芥子碱可抑制肠炎小鼠肠组织中炎性因子（Tnfα，Il6，Il17α，Il18，Inos和Cox2）和NLRP3小体因子（Nlrp3、Il1β、Asc和Caspase1）mRNA表达。而芥子碱处理还提升肠炎小鼠结肠SOD，GSH-Px，catalase、GSH及粪便中总短链脂肪酸，乙酸，丙酸和丁酸水平。.利用LPS诱发Caco-2细胞肠上皮屏障损伤模型研究发现，芥子碱重要原型物芥子酸能有效抑制模型细胞内NF-κBp65核转位，抑制炎症因子Tlr4、Nfκbp65、Il1β和Il8的mRNA水平，抑制TLR4和MyD88的表达及NF-κBp65磷酸化。通过增加模型细胞中ZO-1、Claudin-1和Occludin的mRNA和蛋白表达，芥子酸促进细胞间Claudin-1和ZO-1分布来抑制LPS致Caco-2细胞的膜通透性改变。而在DSS诱发结肠炎小鼠模型中，芥子酸处理显著抑制结肠炎小鼠体重的降低，改善其DAI水平，抑制结肠炎小鼠结肠长度缩短，降低结肠重量与结肠长度比。芥子酸显著抑制结肠炎小鼠结肠组织破坏，降低结肠组织炎症评分，抑制肠组织损伤指标（MPO和MDA）与血清LPS和FD-40水平的升高。同时，芥子酸能够分别调控结肠炎小鼠血清及组织中炎性细胞因子（TNF-α，IL-1β，IL-6，IL-17α，IL-18和IFN-γ）和抗炎细胞因子（IL-4和IL-10）的水平，并恢复其肠组织SOD、GSH-Px、catalase和GSH水平。此外，芥子酸能抑制结肠炎小鼠肠内NLRP3、ASC、IL-β和Caspase-1的蛋白表达而发挥抗炎作用，并经增强ZO-1、Claudin-1和Occludin的mRNA和蛋白表达来维持肠上皮屏障的完整和稳态。以上研究为芥子碱和芥子酸用于炎性肠病的临床防治提供了一定的基础支撑。