自噬功能损伤在链球菌肽聚糖多糖诱导TLR2缺陷小鼠中耳炎发病机制中作用的研究

The effect of otitis media is great. The long-term recurrent attacks can cause the intracranial complications and hearing loss. The precise molecular mechanism of otitis media is still not clear. Toll like receptor 2 (TLR2) signal plays a key role in the infection of otitis media. In previous studies, Tlr2-/- mice were inoculated with streptococcal peptidoglycan-polysaccharide (PGPS) into their middle ears of by tympanic membrane puncture. We successfully established a mice model similar to human otitis media. It was suggested that due to the deficiency of TLR2, PGPS cannot active the downstream factors through the TLR2 pathway, causing oxidative stress and apoptosis in the middle ear infection and epithelial cells. Recently, it was reported that TLR2 is involved in a variety of inflammatory responses, and its mechanism may be associated with autophagy. The previous results showed that in Tlr2-/- mice, the autophagy initiating signal protein LC3 increased, but the expression of autophagy substrate protein p62 increased. This indicates that autophagy degradation function may be damaged. The hearing function of mice was obviously improved after the treatment of rapamycin，an autophagy inducer. In this study, we will verify this hypothesis that the impaired function of autophagy in middle ear epithelial cell may be involved in the otitis media. Repair the autophagy degradation function may be one of the effective targets for the therapy of otitis media. These studies will provide new targets to explore protective therapies for otitis media.

中耳炎的危害较大，长期反复发作会引起颅内的并发症和听力损失，中耳炎精确的分子机制尚不明确。Toll样受体2（TLR2）信号在中耳炎感染起着关键调控作用。前期研究中，我们选用Tlr2-/-小鼠通过鼓膜穿刺的方法向中耳注射链球菌肽聚糖多糖（PGPS），构建了类似人类急性化脓性中耳炎的模型，有可能是由于TLR2的缺失，致下游因子失活，造成中耳感染和上皮细胞出现氧化应激、凋亡。近年来有研究报道，TLR2参与多种炎症反应，其机制可能与细胞自噬相关。前期结果表明，Tlr2-/-中耳炎小鼠的自噬起始信号蛋白LC3升高，但自噬底物p62蛋白表达也升高，这表明自噬降解功能有可能损伤。自噬诱导剂雷帕霉素治疗后小鼠听力功能明显改善。本研究我们将验证假设：中耳上皮细胞的自噬功能损伤有可能参与中耳炎产生的机制，修复自噬降解功能或许是治疗中耳炎的有效靶点之一，为今后寻找和研发更多新的中耳炎药物提供依据。

中耳炎（OM）是一种常见的疾病，有可能引起听力损失和严重的并发症。在前期研究中，我们利用链球菌肽聚糖多糖（PGPS）对Tlr2tm1Kir（TLR2−/−）小鼠进行鼓室注射，已成功建立模拟人类中耳炎的小鼠模型。在本项目中，我们的研究结果发现雷帕霉素（Rapamycin, RPM），一种广泛使用的雷帕霉素机能靶点（mTORC1）的抑制剂和自噬诱导剂，对OM小鼠的听力损失和OM感染均有明显减轻作用。首先，我们通过测试p-S6、Raptor和mTOR蛋白表达评估mTORC1的活性。结果表明TLR2−/−小鼠注射PGPS后，中耳组织中的p-S6、Raptor和mTOR蛋白表达水平降低。自噬起始相关蛋白LC3-II、Beclin-1、ATG7和自噬底物蛋白p62在OM组小鼠的累积水平更高。另外OM组小鼠中溶酶体相关蛋白LAMP1、组织蛋白酶B和组织蛋白酶D的表达增加。但是自噬体与溶酶体融合所必需两种蛋白质Rab7和Syntaxin 17，在OM组小鼠中表达量减少。此外，实验结果表明RPM治疗后，p-S6，mTOR和Raptor的蛋白表达水平低于PGPS组。RPM治疗组中LC3-II、Beclin-1和ATG7的表达降低。Rab7和Syntaxin17的表达在RPM治疗后显著增加。我们的研究结果表明，自噬损伤机制可能与PGPS诱导产生的中耳炎有关，RPM可通过减轻自噬损伤缓解OM。通过RPM调节自噬活性可能是中耳炎的一种有效治疗策略。