l-N-acetylcysteine protects outer hair cells against TNFα initiated ototoxicity in vitro

Abstract

Objective: The present study is aimed at determining the efficacy and exploring the mechanisms by which l-N-acetylcysteine (l-NAC) provides protection against tumor necrosis factor-alpha (TNFα)-induced oxidative stress damage and hair cell loss in 3-day-old rat organ of Corti (OC) explants. Previous work has demonstrated a high level of oxidative stress in TNFα-challenged OC explants. TNFα can potentially play a significant role in hair cell loss following an insult to the inner ear. l-NAC has shown to provide effective protection against noise-induced hearing loss in laboratory animals but mechanisms of this otoprotective effect are not well-defined.

Design: Rat OC explants were exposed to either: (1) saline control (N = 12); (2) TNFα (2 μg/ml, N = 12); (3) TNFα+l-NAC (5 mM, N = 12); (4) TNFα+l-NAC (10 mM, N = 12); or (5) l-NAC (10 mM, N = 12). Outer hair cell (OHC) density, levels of reactive oxygen species (ROS), lipid peroxidation of cell membranes, gluthathione activity, and mitochondrial viability were assayed.

Results: l-NAC (5 and 10 mM) provided protection for OHCs from ototoxic level of TNFα in OC explants. Groups treated with TNFα+l-NAC (5 mM) showed a highly significant reduction of both ROS (p < 0.01) and 4-hydroxy-2-nonenal immunostaining (p < 0.001) compared to TNFα-challenged explants. Total glutathione levels were low in TNFα-challenged explants compared to control and TNFα+l-NAC (5 mM) treated explants (p < 0.001).

Conclusions: l-NAC is a promising treatment for protecting auditory HCs from TNFα-induced oxidative stress and subsequent loss via programmed cell death.

Chinese abstract

目的：本研究的目的是探讨L-乙酰半胱氨酸（L-NAC）在3天大的老鼠皮质器官（OC）外植体内对肿瘤坏死因子-α（TNFα）诱导的氧化应激损伤和毛细胞损失的防护机制, 并确定其有效性。以前的工作证明在TNFα激发的OC外植体中存在高度氧化应激。在内耳受到损害后, TNFα可能在毛细胞损失上起重要作用。 L-NAC已显示它对实验室动物可以提供有效的防止噪音诱导的听力损失的作用, 但是这种耳保护作用的机制尚不明确。

设计：将大鼠OC外植体置于下列一种物质中：（1）作对照的盐水（N = 12）; （2）TNFα（2μg/ml, N = 12）; （3）TNFα+ L-NAC（5mM, N = 12）; （4）TNFα+ L-NAC（10mM, N = 12）;或（5）L-NAC（10mM, N = 12）。检测外毛细胞（OHC）密度、活性氧（ROS）水平、细胞膜的脂质过氧化作用谷胱甘肽活性和线粒体活力。

结果：在OC外植体中, L-NAC（5和10mM）为OHC提供了防止TNFα耳毒性水平的保护作用。与TNFα激发的外植体相比, 用TNFα+ L-NAC（5mM）处理过的组显示ROS（p < 0.01）和4-羟基-2-壬烯醛免疫染色（p < 0.001）的显著降低。与对照组和TNFα+ L-NAC（5mM）处理的外植体相比, TNFα激发的外植体中的总谷胱甘肽水平低（p < 0.001）。

结论：对于保护听觉HC免受TNFα诱导的氧化应激以及随后由于程序化细胞死亡导致的听力损失, L-NAC是一种有前景的治疗方法。

Keywords:

• l-N-Acetylcysteine
• antioxidant
• TNFα ototoxicity

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The research work in Dr Adrien A Eshraghi’s lab is supported by Medel Corporation and HERA Foundation.