生物钟调控BAFF介导IBD肠道免疫失调的机制研究

It is generally known that the development of intestinal inflammation in IBD attributes to the excessive immune response induced by mucosal immune disorder. Studies have shown that the external environment significantly participate in the pathogenesis of IBD and circadian clock is an environmently important factor of immune system, but there is no verified mechanism how circadian clock affect intestinal inflammation in IBD. The abnormal expression of clock genes in intestinal tract of IBD patients and the increased susceptibility of DSS in Bmal1-/- mice indicated that circadian disorder involve in the pathopoiesis of IBD. The further research illustrated that the expression of BAFF in monocyte/macrophage may be regulated by clock gene Bmal1. BAFF that play an important role in the pathopoiesis of autoimmune diseases was recently found as a new potential biomarkers. On the basis of this studies, we hypothesize that the expression of BAFF regulated by clock gene may be the environmental pathopoiesis of intestinal inflammation in IBD and chronotherapy based on circadian clock is potentially novel administration method of biological agents. This study will illustrate how circadian disruption induce intestinal inflammation in IBD through BAFF in systemic level, cellular level and molecular level, using body specimens and mouse model through chromatin immunoprecipitation, luciferase reporter gene transfection, magnetic activated cell sorting and other experiment methods. Furthermore, we will evaluate the efficacy of chronotherapy in BAFF targeted therapy in order to discover new therapeutic targets and biological treatment.

IBD发生的关键是肠粘膜免疫失调引发过度的免疫应答。环境是影响IBD发病的重要因素，生物钟是环境影响人体免疫的重要环节，但生物钟是否调控IBD肠道炎症目前不明。我们发现生物钟基因在IBD患者肠道表达降低，且核心生物钟基因Bmal1敲除小鼠对DSS易感性增加，提示生物钟紊乱参与IBD发生。进一步研究发现，人和小鼠单核/巨噬细胞BAFF表达可能受Bmal1调控。BAFF参与多种自身免疫性疾病发生，是IBD新的生物标记。我们推测生物钟调控BAFF引起肠道免疫紊乱可能是环境影响IBD肠道炎症的重要机制，同时，基于生物钟理论的时辰疗法可能是生物制剂给药方式的革新。本课题在人体标本和生物钟紊乱动物模型基础上，采用CHIP、luciferase报告基因转染等手段，从分子、细胞和整体水平阐明生物钟调控BAFF在IBD中的作用机制，同时运用时辰疗法进行BAFF靶向治疗，探索新的IBD治疗靶点和生物治疗方法。

一、粪便B细胞活化因子对腹部不适患者的诊断价值。我们发现了一种出色的新型粪便生物标志物BAFF，它在诊断肠道炎症方面与粪便钙卫蛋白一样有效。本研究前瞻性纳入230例腹部不适患者，在内镜检查前24小时内采集粪便样本。我们发现，UC、CD、胃癌和结直肠癌的粪便BAFF高于健康组，而胃息肉、结直肠息肉、食管炎/胃炎/十二指肠炎和消化性溃疡患者的值在健康人范围内。219·5 pg/g粪便BAFF的最佳截断值对IBD或癌产生的敏感性、特异性、阳性预测值和阴性预测值分别为 85%、91%、84% 和 92%。因此，我们的结果表明粪便BAFF作为IBD 和胃肠癌的敏感筛查参数的潜在作用。.二、BAFF阻断减弱小鼠内毒素血症模型中的炎症反应和肠屏障功能障碍。本研究旨在研究BAFF在实验性内毒素血症模型中的表达和功能，并确定潜在机制。我们通过施用LPS建立了内毒素血症小鼠模型。LPS攻击后，BAFF产量系统性和局部性提高。BAFF 阻断提高了存活率、全身炎症和多器官损伤。此外，BAFF阻断可减轻肠道炎症和肠道通透性受损。BAFF阻断上调ZO-1和occludin蛋白水平通过NF-κB/MLCK/MLC信号通路。这些结果表明，BAFF 阻断剂至少部分通过减轻炎症、多器官损伤和改善肠道屏障功能来预防致命的内毒素血症，并为脓毒症的进一步研究和临床治疗的实验证据提供了新的焦点。.三、BAFF阻断通过抑制NLRP3炎症小体和NF-κB激活来减弱DSS诱导的慢性结肠炎。本研究旨在研究BAFF在实验性结肠炎中的表达和功能以及潜在的机制。在DSS小鼠中施用小鼠来源的BAFF抗体。结果表明，BAFF阻断通过缓解炎症，抑制肠道NLRP3炎症小体和巨噬细胞的NF-κB信号通路来部分预防结肠炎。BAFF在IBD炎症调节中起着重要作用，从而为结肠炎的进一步研究和IBD新潜在治疗靶点的实验证据提供了新的思路。