NF-κB介导的IL-6/JAK/STAT3通路在色胺酮抗溃疡性结肠炎中的作用研究

The clinical symotoms of patients with ulcerative colitis was improved significantly by oral administration with Qing-Dai. Tryptanthrin is one of indoloquinazoline alkaloids from Qing-Dai which has excellent anti-inflammatory activity and good clinical safety. Our previous study has demonstrated that with tryptanthrin treatmentment，the intestinal mucosal injury and the health conditions of mice with DSS induced colitis were significantly improved, the levels of TNF-α and IL-6 in serum were reduced, and the degradation of IκBα and the phosphorylation of STAT3 were inhibited. These finding indicating that the anti-ulcerative colitis of tryptanthrin might be mediated via inhibiting the expression of NF-κB and then down-regulating the activation of IL-6/JAK/STAT3 pathway to reduce the intestinal mucosal injury. Therefore, in this project，the anti-ulcerative colitis effects of tryptanthrin will be validated in vitro and vivo through regulating the expression and transcriptional activity of NF-κB, as well as the activities of NF-κB mediated IL-6/JAK/STAT3 signaling pathway by using molecular biological techniques including the infection of slow virus, RNA interference, immunofluorescence, immunoprecipitation and iTRAQ. The achievement of the project will provide scienctific basis for tryptanthrin against ulcerative colitis as well as the clinical application of Qing-Dai.

临床发现溃疡性结肠炎患者口服青黛可显著改善其临床症状，色胺酮为青黛中一种吲哚喹啉类生物碱，具有明确的抗炎活性且安全性较好。课题组前期研究发现，色胺酮能够显著改善DSS诱导溃疡性结肠炎小鼠的肠黏膜损伤，提高生存质量，降低小鼠血清中TNF-α和IL-6的水平，同时抑制NF-қB抑制因子IκBα和STAT3的磷酸化。我们推测：色胺酮可能通过抑制NF-κB入核转录从而下调IL-6的表达及其下游通路JAK/STAT3的活化进而减轻肠黏膜炎症反应。基于此，本项目拟通过慢病毒感染、RNA干扰、免疫荧光、免疫共沉淀及iTRAQ等技术，在体内和体外炎症模型上研究色胺酮对NF-κB表达及转录活性的影响，对IL-6及其下游JAK/STAT3通路的调控作用，以及NF-κB对IL-6/JAK/STAT3通路的影响，以期初步阐明色胺酮抗溃疡性结肠炎的分子作用机制，为青黛的临床应用提供科学依据。

临床发现溃疡性结肠炎（UC）患者口服青黛可显著改善其临床症状，色胺酮为青黛中一种吲哚喹啉类生物碱，具有明确的抗炎活性且安全性较好。①本项目采用 LPS 诱导人单核巨噬细胞 RAW264.7 炎症模型，采用酶联免疫吸附法(ELISA)，蛋白免疫印迹法(Western blot)等技术在体外阐明NF-κB与IL-6/JAK/STAT3通路在色胺酮抗炎作用中的调控作用。结果表明，色胺酮可以通过抑制 IκBα的降解，下调 NF-κB p65 的转录活性，进而抑制 IL-6/JAK/STAT3 信号通路的活性发挥抗炎作用，同时可以抑制MAPK 通路的激活。② 采用TMT相对定量蛋白组学筛选色胺酮处理后与抗炎作用相关的显著性差异蛋白总计12个，其中转铁蛋白受体蛋白1（Tfrc）和高亲和力免疫球蛋白ε受体亚基γ(Fcer1g)与NF-κB信号通路相关。另外，细胞色素b-245轻链（Cyba）和细胞色素b-245重链（Cybb）蛋白在色胺酮处理后显著下调表明色胺酮的抗炎作用与氧化应激相关。③ 采用葡聚糖硫酸钠（DSS）诱导的UC小鼠C57BL/6J模型在体内验证色胺酮抗UC的分子机制。结果表明，色胺酮能够显著改善UC小鼠的肠黏膜损伤，提高生存质量。可显著抑制IκBα泛素化进而抑制NF-κB p65入核，下调炎症因子IL-6和TNF-α水平，进而抑制JAK/STAT3信号通路关键分子STAT3的磷酸化水平，并发挥抗UC作用。④ 利用Illumina MiSeq测序平台采用细菌的16S rRNA进行 V4-V5区高通量测序研究色胺酮对UC小鼠肠道菌群的影响。结果表明色胺酮处理能够一定程度恢复肠炎中厚壁菌门/拟杆菌门比例，回调结肠炎小鼠菌群的多样性，色胺酮可能通过促进肠道菌群结构恢复减轻结肠炎症。肠道菌群组间差异分析结果表明4个菌属Erysipelotrichaceae_UCG_003、Sellimonas、Rikenellaceae_RC9_gut_group和Tyzzerella丰度可能与色胺酮对UC治疗作用相关。采用反溶剂沉淀法制备的色胺酮纳米结晶粒径分布在100~300 nm之间，PDI小于0.3，分布较均匀。本项目的研究结果为天然产物色胺酮的活性挖掘与开发利用提供了理论依据，为中医临床使用青黛治疗UC提供了科学依据。