Objective

2

To investigate the mechanism of the cyclic GMP⁃AMP synthase (cGAS)⁃stimulator of interferon genes (STING) signaling pathway in aging cochlear hair cells.

Methods

2

Cochlear hair cells OC⁃1 were induced to senescence using

D

⁃galactose (

D

⁃gal). OC⁃1 cells were treated with different concentrations of

D

⁃gal for 72 h

then the cell viability was assessed by using the cell counting kit⁃8 (CCK⁃8) assay

. After OC⁃1 cells were divided into the Control and

D

⁃gal groups

the β⁃galactosidase staining was adopted to verify the successful establishment of the cellular senescence model. Subsequently

flow cytometry was utilized to assess mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential levels

immunofluorescence was employed to observe mitochondrial DNA (mtDNA) leakage

and Western Blot was conducted to detect the expression levels of cGAS

STING

TANK⁃binding kinase 1 (TBK1)

phosphorylated TBK1 (p⁃TBK1)

nuclear factor (NF)⁃κB p65 subunit

and phosphorylated p65.

Results

2

After treating OC⁃1 cells with various concentrations of

D

⁃gal for 72 hours

the CCK⁃8 assay results showed a gradual decline in cell viability when the concentration of

D

⁃gal exceeded 10 mg⋅mL

-1

(

P

<

0.001). Therefore

15 mg⋅mL

-1

was chosen as the treatment concentration of the

D

⁃gal group in the subsequent experiments. Compared with the cells in the control group

cells in the

D

⁃gal group displayed prominent blue staining

elevated mitochondrial ROS levels (

P

<

0.01)

decreased mitochondrial membrane potential (

P

<

0.01)

and mtDNA leakage. Furthermore

the expression levels of cGAS and STING proteins were upregulated in the

D

⁃gal group (

P

<

0.05)

with increased ratios of p⁃TBK1/TBK1 and p⁃p65/p65 (

P

<

0.05).

Conclusion

2

The cGAS⁃STING pathway may be activated through mtDNA translocation

thereby participating in the development process of age⁃related hearing loss.