HMGB1通过Nrf2/HO-1通路调控自身免疫性甲状腺炎铁死亡的作用及其分子机制

Autoimmune thyroiditis (AIT) is an organ-specific disorder due to the interplay between environmental and genetic factors. However, the mechanism of AIT is still unclear.It has been previously demonstrated that DAMP（Damage-associated molecular pattern) derived from iodine-induce oxidative damage of thyrocytes and the disfunction of T lymphocytes play an important role in the development of AIT. Ferroptosis is a recently recognized form of regulated cell death and is morphologically, biochemically, and genetically distinct from apoptosis,necrosis, and autophagy. The process of Ferroptosis is characterized by the accumulation of lethal reactive oxygen species (ROS). Ferroptosis is gradually becoming a new target for the treatment of cancer and acute sterility inflammtion, such as tissue acute damage. There are accumulating evidences that Ferroptosis may play a vital role in the pathogenesis of AIT.HMGB1 released from tissue damage might reduce the releasing of ROS and prevent the production of oxidative stress and inhibit the occurrence of Ferroptosis by active Nrf2/HO-1 signaling pathway, thus exert an apparent protective effect on AIT. The present study uses AIT patients and mouse model both in vivo and in vitro and aim to: (1) Explore the potential mechanisms of iron metabolism, Ferroptosis in the pathogenesis of AIT.(2)Investigate the roles of HMGB1 released from damaged thyrocytes of AIT in the link of Nrf2/HO-1 signaling pathway activation, iron metabolism, Ferroptosis and T lymphocytes differentiation.Our research will give theoretical evidences for finding novel therapeutic targets of AIT by blocking HMGB1-mediated Nrf2 signaling or inhibiting the Ferroptosis of thyrocytes.

自身免疫性甲状腺炎是一种遗传和环境因素共同作用引起的器官特异性自身免疫疾病，目前发病机制尚未明确。碘所致氧化应激导致的甲状腺细胞损伤，引起损伤相关分子模式DAMP释放，T细胞调节失衡是AIT重要的免疫学发病机制。铁死亡是一种氧化性细胞死亡方式，其发展进程以ROS异常增多引起的氧化还原失衡为特点。组织损伤释放的HMGB1可能是通过激活Nrf2/HO-1抗氧化途径，增强细胞清除氧自由基的能力，抑制铁死亡的发生，从而发挥对AIT的保护作用。本研究将利用AIT患者及其动物模型以及体外细胞作为研究对象，探讨在AIT发生发展中，是否存在铁稳态的失衡以及铁死亡现象，探讨氧化损伤引起甲状腺细胞释放的HMGB1在调控Nrf2/HO-1通路、铁死亡以及T细胞活化等环节的作用，为寻找AIT治疗方法提供新的理论依据。

有研究表明，Nrf2/HO-1通路在调控机体自身免疫反应中发挥重要作用。该通路在铁死亡通路中发挥了作用。HMGB1可以通过调控Nrf2信号通路介导铁死亡的发生。但其具体机制未明确。.本研究通过HT患者、AIT小鼠体内实验以及体外培养甲状腺细胞，研究对铁死亡、HMGB1或Nrf2信号通路任一靶点进行干预，来探讨其参与AIT的机制研究。明确AIT中，存在铁死亡的功能变化以及铁稳态的失衡。进一步验证AIT以及细胞中Nrf2信号通路以及下游通路及靶基因的表达和定位。然后进一步在小鼠体内实验干预，利用AIT动物模型，Tg免疫诱导AIT小鼠以及Nrf2敲除小鼠，观察HMGB1与Nrf2信号通路在AIT中相互影响以及其对铁死亡的调控。测定甲状腺局部炎症，血清TgAb水平，线粒体功能的改变，炎性因子，Nrf2/HO-1信号通路蛋白及铁死亡的表达。然后以原代培养的小鼠甲状腺细胞为研究对象，进行体外实验。分别给予高碘或细胞因子诱导或铁死亡抑制剂干预，论证AIT引起细胞ROS产生增加，引起DAMP（HMGB1）释放，诱导细胞铁死亡，同时HMGB1可以活化氧化应激通路Nrf2/HO-1，进一步调控铁死亡的发生。.结果显示：铁死亡抑制剂能明显抑制SAT小鼠的炎症反应，HMGB1的抑制剂同样可以抑制其炎症反应。而HMGB1抑制剂可以抑制SAT小鼠铁死亡，Nrf2敲除小鼠给予高碘诱导后其炎症反应显著低于野生组。研究表明Nrf2通路在AIT中的作用，主要是通过HMGB1蛋白的直接调节。微环境的改变可以引起甲状腺细胞膜电位升高，甲状腺细胞中出现铁死亡并受Nrf2/HO-1通路调节。同时RNA测序的结果分析，Nrf2可能对铁死亡通路的表达有直接作用。Nrf2敲除能通过调节铁死亡，上调趋化因子的作用。NOD.H-2h4小鼠甲状腺细胞中的体外研究，HMGB1以及Gpx4在中存在相互作用。HMGB1对Gpx4的激活可能主要是通过Nrf2通路实现的。.以上结果提示，本研究将有助于研究AIT的发病机制，为治疗以Nrf2信号通路为靶点治疗自身免疫病调控铁死亡提供理论依据。