芸薹属紫菜蔬对小鼠炎症性肠病 NLRP3炎性体的调控研究

Recently, modulation of the NLRP3 inflammasome overacrivation is a potential therapeutic target in preventing inflammatory bowel disease(IBD) and it associated colonic carcinogenesis. Some studies suggested the overgenerated ROS plays a very important role during the NLRP3 inflammasome activation. Decreasing the colonic ROS levels was able to reduce the experimental mice IBD and IBD-associated colon carcinogenesis. Purple Brassica rapa L vegetables, including Brassica campestris L. var. purpurea Bailey, purple Brassica rapa var. rapa, Brassica oleracea var. capitata f. rubra and Brassica oleracea L. var. acephala DC were enriched in anthocyanisns which with a highly antioxidant and anti-inflammatory activity. However, the IBD preventive effect of these purple vegetables has not been study. In our previous studies, we found the Brassica oleracea var. capitata f. rubra (red cabbage) was significantly reduced the secretion levels of IL-1β and IL-18, which are both the major physiological effector during the activation of NLRP3 inflammasome in human monocytic leukemia THP-1 cells. In addition, red cabbage also significantly decreased the serum levels of IL-1β, and reduced its colonic mRNA expressions on the DSS-induced colitis mice. Based on these results, this study was first to investigate the antioxidant and anti-inflammatory activities of these purple vegetables using in vitro test and in cell models. Secondly, we focused on investigating the effects of purple purple Brassica rapa L vegetables on regulating the NLRP3 inflammasome activation on 2,4,6-trinitro picrylsulfonic acid(TNBS) and dextran sulfate sodium(DSS) induced inflammatory bowel disease mice model, respectively. The aim of this study was to reveal the molecular mechanisms of these purple vegetables on reregulating the NLRP3 inflammasome activation on experimental IBD mice model. In addition, these results from the present study will provide some scientific evidences for human IBD prevention and clinical therapy.

调控NLRP3炎小体的异常活化已成为干预炎症性肠病(IBD)及其相关肠癌变的一个潜在治疗靶点。过量的ROS是诱导NLRP3炎小体活化的要因之一，而降低肠组织内ROS水平可明显缓解小鼠实验性IBD及其相关的结肠癌变。芸薹属紫色菜蔬(紫菜薹、紫芜菁、紫甘蓝和羽衣甘蓝)是一类富含强抗氧化和抗炎能力物质花青素的十字花科特色蔬菜，其对IBD的干预效果还未被系统研究过。前期研究中，我们发现芸薹属紫甘蓝能明显抑制人白血病THP-1细胞中NLRP3炎小体活化后主要生理效应分子IL-1β和IL-18的分泌，且明显抑制结肠炎小鼠血清及结肠组织中IL-1β的表达。因此，本研究设想以体外化学法研究样品的抗氧化和抗炎效果，结合细胞模型，并利用三硝基苯磺酸和葡聚糖硫酸钠分别诱导的小鼠实验性IBD模型，揭示这四类芸薹属紫菜蔬对IBD小鼠中NLRP3炎小体的分子调控机制，为其在人类IBD的防治层面提供一定的科学依据。

本研究发现，紫菜薹、紫芜菁和羽衣甘蓝乙醇提取物均具较强的体外抗氧能力。特别是紫芜菁和紫菜薹乙醇提取物相对于标准抗氧化剂TBHQ具有较强的DPPH和•OH自由基的清除能力及较强的总还原力。在H2O2所致肠上皮Caco-2细胞损伤模型中，紫菜薹，紫芜菁和羽衣甘蓝乙醇提取物显著提高损伤细胞的生存率，抑制损伤标志物LDH溢出。紫菜薹，紫芜菁和羽衣甘蓝乙醇提取物还能提升受损细胞中SOD，GSH-Px和catalase活性，抑制细胞内ROS水平升高，降低MDA生成，并抑制IL-1β和IL-8的升高。而紫菜薹与紫甘蓝总花色苷还能抑制LPS诱导RAW264.7巨噬细胞中NO和PGE2生成并抑制iNOS和COX-2的mRNA表达。此外，紫菜薹与紫甘蓝总花色苷均能有效的抑制TNF-α，IL-1β，IL-6和IL-8的分泌并抑制其mRNA表达。紫菜薹，紫芜箐，紫甘蓝及羽衣甘蓝乙醇提取物显著抑制DSS致结肠炎小鼠体重降低，改善结肠炎小鼠结肠长度的缩短，降低结肠重量/结肠长度比。4种紫菜蔬提取物处理显著抑制DSS致结肠炎小鼠结肠组织的破坏，抑制模型小鼠结肠组织中MPO酶水平升高，降低小鼠血清中TNF-α，IL-1β，IL-6和IL-18水平。4种紫菜蔬提取物能够抑制结肠炎小鼠结肠组织中TNF-α，IL-1β，IL-6，IL-18以及NLRP3、ASC和caspase-1的mRNA表达，显著提升结肠炎小鼠结肠SOD，GSH-Px，catalase及GSH水平，降低组织中iNOS和COX-2的mRNA表达。在ATP+LPS诱导的THP-1细胞模型中，萝卜硫素抑制细胞内TLR4、MyD88的蛋白表达，抑制NF-κBp65磷酸化，降低IL-1β和IL-18水平，有效抑制细胞模型中NLRP3, caspase-1, ASC的表达。萝卜硫素还能增强细胞中SOD活力和GSH水平，降低MDA和NO生成。同时，萝卜硫素显著抑制DSS致结肠炎小鼠体重降低，抑制结肠炎小鼠结肠长度的缩短，降低结肠重量/结肠长度比，改善DAI指数。萝卜硫素改善DSS所致小鼠结肠组织破坏，降低结肠组织中MPO酶血清LPS水平的升高。萝卜硫素能降低结肠炎小鼠血清TNF-α，IL-1β，IL-6和IL-18水平，抑制结肠组织中NF-κBp65，IκB和IKKα的磷酸化；抑制NLRP3、ASC和caspase-1的蛋白表达而发挥抗炎功效。