Necrosulfonamide通过抑制细胞焦亡防治老年性聋的实验研究

Age-related hearing loss (ARHL) is one of the most prevalent deficits. ARHL has been shown to be independently associated with many physiological psychological conditions. Pyroptosis plays an important role in neurodegenerative diseases, but its role in ARHL is not clear. Previous studies of the applicant have shown that NLRP3 expression is enhanced in the cochlea of elderly mice and benzyl alcohol can increase the permeability of round window membrane. Along with the growth of the age, after intratympanic injection with pyroptosis inhibitor necrofonamide (NSA) , or after intratympanic injection with NSA and benzyl alcohol, this project intends to detect the changes of molecules related to pyroptosis, such as NLRP3, ASC, Caspase-1, GSDMD, IL-1βand IL-18, at the RNA and protein levels, and also detect the changes of IL-1βand IL-18 in the perilymph. With ABR results together, we explore the role of pyroptosis in ARHL and investigate that RNA can ameliorate ARHL by inhibiting pyroptosis. The pharmacokinetics of the inner ear was studied Objectives: 1. To clarify the role and mechanism of pyroptosis in ARHL. 2. To clarify the effect of pyroptosis inhibitor on the amelioration of ARHL. 3. To clarify that benzyl alcohol can enhance the amelioration effect of NSA, and optimize the prescription on the basis of pharmacokinetic research. This project will lay a theoretical and experimental foundation for the amelioration of ARHL.

老年性聋发病率高，危害广泛。焦亡在神经退行性疾病中有重要作用，但在老年性聋中的作用尚不清楚。申请人前期研究表明，焦亡相关炎性小体NLRP3在老年小鼠耳蜗中表达明显增强，结合前期研究发现苯甲醇可增加圆窗膜通透性，本项目拟研究随着年龄增长以及鼓室内分别注射焦亡抑制剂necrosulfonamide（NSA）、NSA联合苯甲醇后，通过在RNA和蛋白层面检测基底膜、蜗轴、血管纹中NLRP3、ASC、Caspase-1、GSDMD、IL-1β、IL-18等焦亡相关分子的改变，以及外淋巴中IL-1β和IL-18的改变，结合ABR阈值，探讨焦亡在老年性聋中的作用以及NSA通过抑制焦亡防治老年性聋，并进行内耳药代动力学研究。拟实现目标：1.明确焦亡在老年性聋中的作用及机制；2.明确焦亡抑制剂对老年性聋的防治作用；3.明确苯甲醇可增强NSA的防治作用，优化处方。本项目将为老年性聋的防治奠定理论和实验基础。

老年性聋发病率高，危害广泛。本项目通过蛋白层面检测螺旋神经元中焦亡相关分子NLRP3的年龄相关性变化，免疫荧光染色检测NLRP3在螺旋神经元中的表达改变，结合ABR阈值的改变，研究焦亡在老年性聋发病中作用。利用细胞焦亡抑制剂NSA，经鼓室内注射，研究ABR阈值的改变，研究焦亡抑制剂对C57BL/6J小鼠老年性聋的防治作用。本项目还研究了乙琥胺多囊脂质体温敏凝胶对老年性聋的防治作用，乙琥胺是第一个被报道的对老年性聋有保护作用的药物，但全身给药有毒副作用，限制了其应用。我们制备了包含乙琥胺的多囊脂质体温敏凝胶，病进一步评估了组织学和听觉功能层面的影响。我们制备的多囊脂质体凝胶缓释释放时间超过120小时。治疗8周以后，与口服乙琥胺给药组相比，鼓室内乙琥胺多囊脂质体温敏凝给药组在耳蜗中圈和耳蜗底圈螺旋神经元细胞（对应低中频听力）的损失明显减少，但耳蜗底圈螺旋神经元（对应高频听力）无明显差别。听觉功能方面，与口服乙琥胺给药组相比，鼓室内乙琥胺多囊脂质体温敏凝给药组ABR阈值改变在低中频4, 8, 16 kHz明显降低，但在高频的32 kHz无明显差异。因此，通过制备乙琥胺多囊脂质体温敏凝胶，可以通过鼓室内注射使药物达到耳蜗顶圈，这在口服全身给药中是很难实现的，这个区域对于低频听力的保护具有重要作用。乙琥胺多囊脂质体温敏凝胶，作为一种新的鼓室内给药缓释系统，对老年性聋聋具有明显的保护作用。