DUOX2调控自噬介导的HMGB1释放在炎症性肠病发病中的作用及机制研究

Reactive oxygen species (ROS) are essential components of intestinal mucosal innate immunity. Excessive generation of ROS can cause inflammation, which in turn leads to mucosal damage. Therefore, ROS is suggested to play an important role in the etiology and pathogenesis of inflammatory bowel disease (IBD). Dual oxidase 2 (DUOX2), a new member of NADPH oxidase, which is mainly expressed along the gastrointestinal tract, is highly upregulated in patients with IBD. However, the regulatory mechanism of DUOX2 in IBD is still underdetermined. Our preliminary data showed that DUOX2 derived ROS induced autophagy and aggravated mice colitis. Whereas, deficiency of DUOX2 alleviated the symptoms and intestinal mucosal inflammation of mice colitis. In addition, abnormally activated autophagy has been identified to promote the release of intrinsic inflammatory mediator high mobility group box-1 protein (HMGB1). And we also found that the expression of HMGB1 was significantly elevated in IBD patients. Therefore, we propose a hypothesis that DUOX2 plays an important role in pathogenesis of IBD by regulating autophagy-induced HMGB1 release. In the current study, we will test the regulatory mechanism of DUOX2 in autophagy and HMGB1 by applying multiple techniques including qRT-PCR, immunoblot, immunostaining and RNA interference on IBD patient-derived organoids culture systems and DUOX2 knockout mice systems. The discovery of DUOX2 as novel regulator in the pathogenesis of IBD by promoting autophagy -induced HMGB1 makes it a new candidate mediator of physiopathological processes and potential therapeutic target for IBD patients.

活性氧（ROS）是肠黏膜固有免疫重要组分，但过量ROS是诱发肠黏膜免疫炎症损伤的关键环节，在炎症性肠病（IBD）发病中起重要作用。DUOX2是肠道ROS关键酶还原性辅酶II家族新成员，在IBD患者肠黏膜表达明显增加，但具体机制不明。我们前期发现DUOX2有诱导自噬的作用，抑制DUOX2后可改善结肠炎小鼠的症状及组织学炎症；而异常激活的自噬已被证实可促进内源性炎症介质HMGB1的释放从而介导肠道黏膜免疫炎症，我们已证实HMGB1在IBD患者肠黏膜表达明显增加。基于此，我们推测DUOX2介导的氧化应激可能通过调控自噬通路进而促进HMGB1的释放，在IBD发病中发挥重要作用。本研究拟采用荧光定量PCR、蛋白印迹、免疫组化、RNA干扰等技术，使用患者来源的微小肠及基因敲除动物模型，首次阐明DUOX2通过调控自噬促进HMGB1释放介导肠黏膜炎症发生发展的潜在机制，有望为IBD的治疗提供新靶点。

炎症性肠病（IBD）发病机制不明，目前认为肠黏膜屏障受损、免疫功能失衡和肠道菌群失调是IBD发病的核心环节。氧化应激（Oxidative Stress, OS）反应是肠道固有免疫的重要组分，然而过量氧化应激导致活性氧（ROS）的累积诱发肠道菌群失衡和黏膜免疫损伤，因此氧化应激被认为是IBD发病机制中的关键环节。本项目首先运用多组学孟德尔随机化的研究方法，整合公共数据库和本中心的数据，鉴定出了多个OS家族中与IBD直接相关致病基因、并通过共定位分析进行基因-微生物群相互作用分析发现这些致病OS基因由DNA甲基化和宿主-微生物群相互作用介导。在表达变化显著的基因中，效应值最大的基因即为DUOX2。进而，我们扩大样本量验证了其在IBD中高表达，且与疾病活动呈正相关。进一步我们构建了DUOX2肠上皮特异性敲除小鼠模型，发现敲除DUOX2后小鼠肠炎症状明显减轻。机制研究中发现DUOX2介导肠炎一方面是与宿主互作通过调控肠上皮细胞死亡机制即诱导自噬及凋亡；另一方面则是通过调控肠道菌群，引起菌群失调参与结肠炎致病。本项目对进一步解释了OS家族基因在IBD中的作用，通过构建肠特异性敲除鼠系统研究了DUOX2与肠道稳态之间的密切联系及具体机制。对研究IBD的发病机制、寻找潜在的治疗靶点具有重要的科学意义。