Caffeine Ameliorates Age-Related Hearing Loss by Downregulating the Inflammatory Pathway in Mice

Abstract

Objective: Age-related hearing loss (ARHL), also known as presbycusis, is a debilitating sensory impairment that affects the elderly population. There is currently no ideal treatment for ARHL. Long-term caffeine intake was reported to have anti-aging effects in many diseases. This study is to identify whether caffeine could ameliorate ARHL in mice and analyze its mechanism.

Methods: Caffeine was administered in drinking water to C57BL/6J mice from the age of 3 months to 12 months. The body weight, food intake and water intake of the mice were monitored during the experiment. The metabolic indicators of serum were detected by ELISA. The function of the hearing system was evaluated by ABR and hematoxylin and eosin staining of the cochlea. Genes' expression were detected by Q-PCR, immunofluorescencee and Western blot.

Results: The results showed that the ARHL mice exhibited impaired hearing and cochlear tissue compared with the young mice. However, the caffeine-treated ARHL mice showed improved hearing and cochlear tissue morphology. The expression of inflammation-related genes, such as TLR4, Myd88, NF-κB, and IL-1β, was significantly increased in the cochleae of ARHL mice compared with young mice but was down-regulated in the caffeine-treated cochleae.

Conclusions: Inflammation is involved in ARHL of mice, and long-term caffeine supplementation could ameliorate ARHL through the down-regulation of the TLR4/NF-κB inflammation pathway. Our findings provide a new idea for preventing ARHL and suggest new drug targets for ARHL treatment.

Fig. 1.. Effects of caffeine on ABR thresholds of mice.

(A-D): ABR thresholds were measured by click and tone bursts (8, 16, 32 kHz) in mice at the ages of 3 months, 9 months and 12 months. Numerical values are means ± SDs or p value. 3 months and 9 months: n=15. 12 months: 12m group, n=15; 12m-caffeine group, n=13. *: p < 0.05.

Fig. 2.. Effects of caffeine on the cochlear histomorphology of mice.

The morphology of the Corti organ. Black arrow: OHCs; red arrow: IHCs. (D-F) The morphology of the spiral ganglion. *: SGNs. (G-I) The morphology of SVs. Blue arrow: SVs. (J, K) Quantification analysis of the SGN density and SV thickness. Data are shown as the mean ± SD. n=6. *: p < 0.05, **: p <0.01. Scale bar = 50 μm.

Fig. 3.. mRNA expression levels of Nfkbia, IL-1β, Nfkb1and Icam4 analyzed by Q-PCR.

Data shown as mean ± SD. n = 3; *: p < 0.05.

Fig. 4.. Protein expression levels of TLR4, Myd88, NF-κB and IL-1β analyzed by western blot.

(A) Immunoblotting of TLR4, Myd88, NF-κB and IL-1β protein levels. (B) Quantification analysis of TLR4, NF-κB, Myd88 and IL-1β protein levels. Data are shown as the mean ± SD. n = 3; *: p < 0.05, **: p < 0.01.

Fig. 5.. Protein expression of NF-κB in the cochlea analyzed by immunofluorescence.

(A(a-l)) Immunostaining for NF-κB in the HCs of the cochleae of the three groups. (A(m-x)) Immunostaining for NF-κB in the SGNs of the cochleae of the three groups. (B, C, D) Line scans of NF-κB immunofluorescence intensity within the IHCs and SGNs in 2 m, 12 m, and 12 m caffeine mice. (E) Quantitative analysis of the fluorescence intensity of NF-κB in IHCs and SGNs. Data are shown as the mean ± SD. n= 4, *: p <0.05, **: p <0.01. Scale bar=20 μm.

Fig. 5.. Protein expression of NF-κB in the cochlea analyzed by immunofluorescence.

(A(a-l)) Immunostaining for NF-κB in the HCs of the cochleae of the three groups. (A(m-x)) Immunostaining for NF-κB in the SGNs of the cochleae of the three groups. (B, C, D) Line scans of NF-κB immunofluorescence intensity within the IHCs and SGNs in 2 m, 12 m, and 12 m caffeine mice. (E) Quantitative analysis of the fluorescence intensity of NF-κB in IHCs and SGNs. Data are shown as the mean ± SD. n= 4, *: p <0.05, **: p <0.01. Scale bar=20 μm.