A20调控透明质酸激活的耳蜗天然免疫炎症反应的作用和机制

Sudden sensorineural hearing loss remain as a global problem because of lacking effective treatment. Ischemia, metabolic dysfunction, immune reaction, and inflammatory response are involved in the occurs of the diseases. Our previous studies demonstrated that the substances of pathogen-associated molecular patterns (PAMPs）induced accumulation of hyaluronic acid, one of damage-associated molecular patterns (DAMPs) in the cochlea. The accumulated hyaluronic acid, impaired integrity of inner ear biological barriers, activated pattern recognition receptors (PRRs) of Toll-like receptors (TLRs)-2/4 and the downstream pathway of NF-kB, and activated innate immunity in the cochlea, and caused hearing loss. It was confirmed that A20 is a negative regulator of innate immunity and reduced the severity of inflammation through degradation of the substrates of Ubc13/UbcH5 and prevention of interactions between the substrates and TNF receptor-associated factors (TRAFs） and cellular inhibitor of apoptosis proteins (CIAPs). A20 inhibits The canonical NF-kB pathway and promotes transection from canonical NF-kB pathway to non-canonical NF-kB pathway. The present project will investigate low-molecular weight hyaluronic acid fragments-induced inflammatory response and impairments in model cells and cochlea of animal model using in vitro and in vivo experiments. A baculovirus expression systems for recombinant A20 protein will be established. Liposome nanocarrier will be used to administer A20 protein and A20 siRNA to the model cells and cochlea of animal model. The degradation effect of A20 on the substrates of Ubc13/ UbcH5 will be tested using E2 ubiquitin-loading assay. The impact of A20 protein levels on the low-molecular weight hyaluronic acid fragments-induced inflammation in the model cells and cochlea and the associated molecular mechanism will be elucidated. The successful implementation of the present project will help to understand the immune reaction-mediated impairment mechanism of sensorineural hearing loss and provide theoretic basis for exploring novel therapeutic method and clinically associated drug delivery system in treating sensorineural loss.

感音神经性耳聋与缺血、代谢障碍、创伤和炎症反应有关，治疗缺乏有效手段。我们发现病理相关分子模式诱导耳蜗透明质酸这一损伤相关分子模式聚集，损害内耳生物屏障完整性、激活耳蜗TLR-2/4和下游NF-κB信号通路的天然免疫机制，并导致听力损伤。已证实A20降解底物Ubc13/ UbcH5并防止其与TRAFs和CIAPs相互作用，抑制TLR-2/4下游的NF-κB的规范途径、促进其向非规范途径转换，在控制天然免疫的炎症反应过程中起关键作用。但A 20在耳蜗中是否也通过此通路发挥作用尚不清楚。为此，本项目通过体外和在体实验，研究小分子量透明质酸片段诱导的模型细胞和动物模型耳蜗炎症反应与损伤。用纳米脂质体介导投放A20蛋白及其siRNA，通过E2载荷实验分析A20对底物的降解作用。阐明A20水平干预小分子量透明质酸片段诱导耳蜗炎症反应的分子机制。从而为了解感音神经性耳聋损伤机制并探索新疗法提供理论依据

感音神经性耳聋与缺血、代谢障碍、创伤和炎症反应有关，治疗缺乏有效手段。本课题以噪声性耳聋作为模型研究了大鼠耳蜗的TLR-4/NF-kB信号通路激活机制以及低分子透明质酸对噪声诱导耳蜗炎症的贡献; 进行了梅尼埃病和突发性耳聋的临床转化医学研究，研究了梅尼埃病的炎性损伤机制，优化了内耳增强磁共振检测内淋巴积水和内耳炎症反应的方法。将大鼠暴露于120 dB SPL噪声，每天8小时，共2天诱导永久性阈移，大鼠耳蜗内TLR4、MyD88、IκBα、TNF-α和 IL-1β的表达显著升高，NF-κB p65的DNA结合能力显著增强，胞浆内的NF-κB p65水平保持稳定，在螺旋神经节细胞和螺旋韧带的TLR4、 TNF-α和IL-1β 的表达升高。提示噪声暴露后耳蜗TLR4/NF-κB信号通路被激活，并参与了噪声诱导耳蜗的炎症反应。进一步发现透明质酸广泛分布于大鼠内耳，其总量在噪声暴露后升高不显著。但是，耳蜗内透明质酸合成酶1和3（HAS1、HAS3）在噪声暴露后3-7天显著升高，且与TLR4和 TNF-α的动态变化一致; 透明质酸水解酶2和3（HYAL2、HYAL3）在噪声暴露后立即戏剧性第降低，然后逐渐升高并在第3天超过正常水平，第7天恢复正常。HAS2和HYAL1没有发生变化。向大鼠鼓阶投放低分子透明质酸（LMW-HA）诱导了听力损失，且TNF-α和IL-1β表达显著升高，鼓室投放透明质酸也诱导了类似的变化。但是，鼓阶和鼓室投放高分子透明质酸（HMW-HA）都没有导致特有的听力损失。因此，噪声暴露后大鼠耳蜗内 HAS1、HAS3、HYAL2和HYAL3可以协同诱导LMW-HA升高并参与噪声诱导的耳蜗炎症反应。在临床上，用高通量的Luminex 液相悬浮芯片测试了梅尼埃病患者血清中48种细胞因子/趋化因子，与正常人对比，初步发现梅尼埃病患者MIP-1α、MIP-1β、CTACK (CCL27)、G-CSF、HGF、IL-1 α、TNF-α、IL-8和HGF显著升高，TRAIL和PDGFBB显著降低，目前还在进一步分析该变化与临床表现的关系。通过二代基因测序发现梅尼埃病患者多基因突变，目前还在进一步分析其与临床表现的相关性。建立了新颖的重T2加权3维液体抑制反转恢复幅度重建和零插值重建（heavily T2-weighted 3-dimensional fluid-attenu