TNF-α 에 의해 유발된 청각세포 손상에서 α-Lipoic acid 의 세포 보호 효과

Abstract

As our society becomes more industrialized and aging, the number of people experiencing hearing loss is increasing globally. This can be attributed to factors such as increased noise exposure, age-related deterioration, and the growing usage of ototoxic drugs like anti-cancer drugs. It is a matter of concern because a higher population of individuals with hearing impairments brings significant economic and social costs. However, there has been limited availability of possible treatment and prevention for hearing loss, such as avoidance of ototoxic drugs or noise, hearing rehabilitation through hearing aids, and cochlear implantation in profound hearing loss.

Therefore, extensive research has been conducted to understand the mechanisms behind hearing loss to prevent and treat it effectively.

Reactive oxygen species (ROS) and TNF-α related apoptosis of inner ear cells is a common mechanism of sensorineural hearing loss of various causes.

α-Lipoic acid (ALA) inhibits cell membrane injury caused by reactive oxygen species (ROS) as a powerful antioxidant. Thus, previous studies reported that it has a protective effect on various types of hearing loss, including ototoxic, age-related, noise-induced, and sudden sensorineural hearing losses. Also, TNF-α, one of the major inflammatory mediators, promotes the production of ROS and causes cellular apoptosis of inner ear cells.

ALA has antioxidant action and a cytoprotective mechanism that prevents the accumulation of inflammatory cells. So, studies on the mechanism that inhibits the over-expression of ROS and its relation to TNF-α can play a positive role in future sensorineural hearing loss prevention and treatment strategies.

In this study, the generation of ROS, genes, and proteins involved in the Nrf2/KEAP1 signaling pathway, and glutathione (GSH) and glutathione disulfide (GSSG) were quantified in the TNF-α treated and ALA pre-treated House-Ear Institute-Organ of Corti 1 (HEI-OC1) cells. Using the results, we tried to measure the antioxidative action through the Nrf2/KEAP1 signaling pathway and intracellular redox signal regulation mechanism of ALA and to figure out the possibility of application to the prevention and treatment of hearing loss. The experimental concentration of TNF-α and ALA was determined by quantification of the cell viability and ROS production in the HEI-OC1 cells treated with different concentrations of TNF-α and ALA. Next, cytoprotective effect and ROS reduction were measured and compared within the control, TNF-α alone, and the ALA preconditioned group. In addition, Western blot was performed in three fractionated cell groups in the nucleus and cytoplasm, the Nrf2 in the nucleus and KEAP1 in the cytoplasm, SOD1, GCLC, HMOX1, and NQO1 were quantified using RT-qPCR. Then, the expressed proteins SOD1, γ-GCSc, NQO1, and HO-1 were measured. Furthermore, the redox potential of ALA was also confirmed by measuring glutathione (GSH), glutathione disulfide (GSSG), and the GSH/GSSG ratio in the three groups.

As a result, cell viability was about 17% higher, and the production of ROS was about 64% lower in the ALA preconditioning group than in the TNF-α alone group. And the Nrf2/KEAP1 signaling-related genes SOD1, GCLC, NQO1, HMOX1, Proteins SOD1, γ-GCSc, NQO1, HO- 1, and nuclear Nrf2 expressed all significantly higher in ALA preconditioning group. KEAP1, an Nrf2 inhibitory protein, was lower than 1/3 of the TNF-α-only treated group in the ALA preconditioning group. In addition, the intracellular glutathione (GSH) was about 4 times higher, the glutathione disulfide (GSSG) was about 30% lower, and the GSH/GSSG ratio was about 6 times higher in the ALA preconditioning group than in the TNF-α alone group.

Based on these findings, it has been suggested that ALA has a cytoprotective effect. It inhibits the generation and accumulation of ROS, which are responsible for TNF-α-induced oxidative stress. Additionally, it activates the Nrf2/KEAP 1 signaling system. The increase in intracellular redox potential could be a mechanism that contributes to hair cell preservation of ALA pre- treatment.

However, because the above results are in vitro data on the HEI-OC1, they could be primary data for in vivo experiments. Therefore, it may be helpful to prepare further research for the possibility that ALA administration can be applied as a preventive and therapeutic strategy for hearing loss.