GSK-3β信号通路在炎症性肠病Th17/Treg免疫失衡中的作用及中药靛玉红的干预效应研究

The etiology of inflammatory bowel disease (IBD) is still unclear, but immune imbalance of IL-17 producing T heper cells (Th17) and regulatory T helper cells (Treg) plays an important role in the pathogenesis of IBD, as many researches shown. Recently, it has been reported that the inhibitor of glycogen synthase kinase-3 beta (GSK-3β) can inhibit Th17 differentiation and enhance the suppressive effect of Treg. However, it is still unknown that whether GSK-3β signal involves in the immune imbalance of Th17/Treg of IBD. In the preliminary study, we found GSK-3β inhibitor LiCl and natural compound indirubin significantly ameliorated the severity of DSS-induced colitis and decreased the production of IL-17A/F in the colon in vivo; as well, indirubin also significantly suppressed Th17 differentiation and greatly enhanced the expression of FoxP3 in Tregs in vitro. In the present study, we will plan to investigate the role of GSK-3β signaling in regulation of Th17/Treg immune balance of IBD using GSK-3β inhibitor as a molecular probe and investigate the regulating effects and underlying mechanisms of indirubin on Th17 and Treg in Th17/Treg imbalance of IBD in vivo and in vitro. The aim of this study is to explore a new strategy for the treatment of IBD and provide a basis for the development of innovative drugs.

越来越多的证据表明调节性T细胞（Treg）与分泌IL-17的效应性T细胞(Th17)免疫失衡在IBD发病中扮演着重要作用。新近表明，糖原合成酶-3β(GSK-3β)抑制剂能抑制Th17的分化，增强Treg的免疫抑制活性。然而，GSK-3β信号是否参与调节IBD肠粘膜Th17/Treg免疫失衡尚不清楚。前期，我们发现GSK-3β抑制剂LiCl及靛玉红可显著减轻急性结肠炎小鼠的疾病程度、降低结肠中IL-17A/F的水平；体外发现靛玉红可抑制Th17分化，延缓Treg细胞FoxP3表达的丧失。本研究拟运用GSK-3β抑制剂为分子探针研究GSK-3β信号通路在结肠炎Th17/Treg免疫失衡中的作用；基于GSK-3β信号通路从体内和体外两方面探讨靛玉红对结肠炎中Th17/Treg免疫失衡中Th17和Treg细胞的调节作用及机制，为IBD的治疗提供新的策略，并为创新药物的研制提供研究基础。

目前临床已用的抗IBD药物主要有氨基水杨酸、糖皮质激素、免疫抑制剂和生物制剂类等; 因IBD患者需长期用药，以上药物虽然有效，但长期应用存在各种副作用，无法满足临床需求，深入挖掘和研究IBD治疗新靶点或策略仍是当前面临的重大社会命题。本项目体外发现GSK-3β抑制剂可抑制CD4T细胞激活；抑制Th17分化，增加Treg分化；提高Treg的免疫抑制能力；体内采用GSK-3β抑制剂为分子探针，发现抑制GSK-3β信号可减轻DSS诱导的急性和慢性结肠炎小鼠的体重降低、腹泻及便血等临床症状，抑制结肠的缩短及肠道损伤程度；抑制肠道中Th17相关细胞因子水平及基因表达，降低肠系膜淋巴结及脾脏中Th17数目，提升结肠炎小鼠Treg细胞的免疫抑制功能；其作用机制与抑制Th17中p-STAT3表达，提升Treg中p-STAT5表达有关；提示靶向GSK-3β信号通路可作为调节Th17/Treg免疫失衡，改善结肠炎的重要治疗策略。基于该策略，我们发现中药青黛主要活性成分靛玉红可显著改善急性和慢性结肠炎小鼠的疾病严重程度及结肠和肠系膜淋巴结中Th17/Treg免疫失衡；体外，靛玉红可抑制CD4T细胞激活；抑制Th17分化，提升Treg分化及其免疫抑制能力；此外，靛玉红可抑制Th17及Treg中p-GSK-3β的表达，同时抑制Th17中p-STAT3表达，提高Treg中p-STAT5表达；提示靛玉红可通过GSK-3β-STAT3/STAT5调节Th17/Treg免疫失衡发挥其治疗结肠炎的作用。同时，发现中药青黛还可通过激活AMPK/Nrf-2信号通路抑制肠道氧化应激及抑制STAT1/STAT3通路减轻肠道Th1/Th17免疫反应而改善结肠炎。通过以上研究，明确了靶向GSK-3β信号通路可作为调节Th17/Treg免疫失衡改善结肠炎的一种重要治疗策略；同时，明确了中药青黛及其活性成分靛玉红抗炎症性肠病的作用及机制，为其临床应用奠定了理论基础。通过该项目实施，已发表SCI论文4篇，国际会议论文1篇；晋升副高职称2人，中级职称1人，培养硕士研究生1名，本科生2名，较好地完成了项目的研究任务和目标。

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