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. 2020 Nov-Dec;86(6):703-710.
doi: 10.1016/j.bjorl.2019.04.012. Epub 2019 Jun 8.

Heat shock response in noise-induced hearing loss: effects of alanyl-glutamine dipeptide supplementation on heat shock proteins status

Marcos Soares  1 , Analu B Dos Santos  2 , Tainara M Weich  2 , Gabriela Gomes Mânica  2 , Paulo Ivo Homem de Bittencourt Junior  3 , Mirna Stela Ludwig  2 , Thiago Gomes Heck  4
Affiliations

Heat shock response in noise-induced hearing loss: effects of alanyl-glutamine dipeptide supplementation on heat shock proteins status

Marcos Soares et al. Braz J Otorhinolaryngol. 2020 Nov-Dec.

Abstract
in English, Portuguese

Introduction: The 72kDa heat shock protein, HSP72, located intracellularly provides cochlear cytoprotective and anti-inflammatory roles in the inner ear during stressful noise challenges. The expression of intracellular HSP72 (iHSP72) can be potentiated by alanyl-glutamine dipeptide supplementation. Conversely, these proteins act as pro-inflammatory signals in the extracellular milieu (eHSP72).

Objective: We explore whether noise-induced hearing loss promotes both intracellular and extracellular HSP72 heat shock response alterations, and if alanyl-glutamine dipeptide supplementation could modify heat shock response and prevent hearing loss.

Methods: Female 90 day-old Wistar rats (n=32) were randomly divided into four groups: control, noise-induced hearing loss, treated with alanyl-glutamine dipeptide and noise-induced hearing loss plus alanyl-glutamine dipeptide. Auditory brainstem responses were evaluated before noise exposure (124dB SPL for 2h) and 14days after. Cochlea, nuclear cochlear complex and plasma samples were collected for the measurement of intracellular HSP72 and extracellular HSP72 by a high-sensitivity ELISA kit.

Results: We found an increase in both iHSP72 and eHSP72 levels in the noise-induced hearing loss group, which was alleviated by alanyl-glutamine dipeptide treatment. Furthermore, H-index of HSP72 (plasma/cochlea eHSP72/iHSP72 ratio) was increased in the noise-induced hearing loss group, but prevented by alanyl-glutamine dipeptide treatment, although alanyl-glutamine dipeptide had no effect on auditory threshold.

Conclusions: Our data indicates that cochlear damage induced by noise exposure is accompanied by local and systemic heat shock response markers. Also, alanyl-glutamine reduced stress markers even though it had no effect on noise-induced hearing loss. Finally, plasma levels of 72kDa heat shock proteins can be used as a biomarker of auditory stress after noise exposure.

Introdução: A proteína de choque térmico de 72 kDa, HSP72 localizada intracelularmente (iHSP72) possui papéis citoprotetores e anti-inflamatórios cocleares na orelha interna durante situações de ruído estressantes. A expressão dessa proteina pode ser potencializada pela suplementação com dipeptídeo de alanil-glutamina. Por outro lado, essas proteínas atuam como sinais pró-inflamatórios no meio extracelular (eHSP72).

Objetivo: Investigar se a perda auditiva induzida por ruído promove alterações tanto das proteínas HSP72 intracelulares quanto extracelulares na resposta de choque térmico e se a suplementação com alanil-glutamina pode modificar a resposta de choque térmico e evitar a perda auditiva.

Método: Ratos Wistar fêmeos, com 90 dias de idade (n = 32) foram divididas aleatoriamente em quatro grupos: controle, perda auditiva induzida por ruído, tratados com alanil-glutamina e perda auditiva induzida por ruído mais alanil-glutamina. Os potenciais evocados auditivos do tronco encefálico foram avaliados antes da exposição ao ruído (124 dB NPS por 2 h) e 14 dias após. A cóclea, o complexo nuclear coclear e amostras de plasma foram coletadas para mensuração de HSP72 intra e extracelular com um kit Elisa de alta sensibilidade.

Resultados: Houve um aumento nos níveis de HSP72 intra e extracelular no grupo perda auditiva induzida por ruído, que foi minimizado pelo tratamento com alanil-glutamina. Além disso, o índice H das HSP72 (razão eHSP72 no plasmacom alanil-glutamina, embora/cóclea) aumentou no grupo perda auditiva induzida por ruído, mas foi limitado pelo tratamento com alanil-glutamina, embora o alanil-glutamina não tenha efeito no limiar auditivo.

Conclusões: Nossos dados indicam que o dano coclear induzido pela exposição ao ruído é acompanhado por marcadores da resposta de choque térmico locais e sistêmicos. Além disso, alanil-glutamina reduziu os marcadores de estresse, mesmo não tendo efeito sobre a perda auditiva induzida por ruído. Finalmente, os níveis plasmáticos de proteínas de choque térmico de 72 kDa podem ser usados como biomarcador do estresse auditivo após a exposição ao ruído.

Keywords: Alanyl-glutamine dipeptide; Dipeptídeo de alanil-glutamina; HSP72; Heat shock response; Noise-induced hearing loss; Perda auditiva induzida por ruído; Resposta de choque térmico.

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Figures

Figure 1
Figure 1
Experimental design. Seven days before the induction of the animal model of noise-induced hearing loss, treatment with alanyl-glutamine (DIP and DIP-NIHL groups) or water (CON and NIHL groups) was started. One day before, auditory evaluation with ABR was performed to determine basal hearing thresholds. At day zero, the NIHL and DIP-NIHL groups were exposed to 124 dB SPL for 2 h. After 14 days, ABR was performed in all animals, followed by extraction of the cochlea, cochlear nuclei, and plasma for measurement of HSP72 concentration.
Figure 2
Figure 2
Effects of alanyl-glutamine dipeptide (DIP) treatment on noise-induced hearing loss (NIHL). (A) Hearing threshold and (B) Hearing threshold shift. NIHL and DIP+NIHL showed an increase in hearing threshold (ap < 0.05 vs. same group before noise exposure) and hearing threshold shift (bp < 0.05 vs. groups without noise exposure).
Figure 3
Figure 3
Effects of alanyl-glutamine Dipeptide (DIP) treatment on Noise-Induced Hearing Loss (NIHL) Heat Shock Response (HSR). (A) NIHL promoted an increase in cochlear iHSP72 expression (ap < 0.05 vs. control). (B) No changes in iHSP72 expression were observed in cochlear nuclear complex. (C) Plasma eHSP72 concentrations were higher in the NIHL group than all groups combined (bp < 0.05).
Figure 4
Figure 4
Effects of alanyl-glutamine Dipeptide (DIP) treatment on Noise-Induced Hearing Loss (NIHL) eHSP72/iHSP72 ratio (H-index). (A) plasma/cochlear HSP70 ratio and (B) plasma/nuclear cochlear complex HSP70 ratio. NIHL promoted an increase in both plasma/cochlear and plasma/cochlear nuclear complex eHSP72/iHSP72 ratio (ap < 0.05 vs. all groups).
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