Trichostatin A protects against cisplatin-induced ototoxicity by regulating expression of genes related to apoptosis and synaptic function

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Abstract

Objective

Although inhibition of histone deacetylases (HDACs) has been shown to protect against cisplatin-induced hearing loss, the underlying mechanism is still poorly understood. In the present study, we aim to investigate the protective effect of trichostatin A (TSA), a specific inhibitor of HDACs, on cisplatin-induced ototoxicity and to determine the differentially expressed genes involved in this process.

Methods

The basilar membrane of the cochlea was isolated from 3-day newborn Wistar rats. Organotypic cultures were treated with 150 μM cisplatin or 200 nM TSA. For combination treatment, cells were pre-incubated with TSA for 1 h, followed by TSA plus cisplatin treatment. Rhodamine-phalloidin staining was used to label hair cells, and immunocytochemistry with an anti-neurofilament-200 antibody was applied to label spiral ganglion neurons (SGNs). Global expression profile microarray analysis was used to identify differentially expressed genes. Molecular function and signal pathway analysis were performed using a protein analysis through evolutionary relationships (PANTHER) classification system. Real-time quantitative PCR (qPCR) was carried out for data validation.

Results

Severe loss of hair cells and SGNs occurred after 48 h of cisplatin incubation, while TSA significantly increased the number of hair cells and SGNs in the combination treatment group (P < 0.05). Compared with control, expression of 71 genes were up-regulated and 383 genes were down-regulated upon cisplatin treatment. Addition of TSA induced the up-regulation of 1387 genes and down-regulation of 1226 genes as compared with cisplatin administration alone. After cisplatin treatment, we observed significant down-regulation of mRNA for several genes related to synaptic function genes, including Camk2a, Camk2b, Vglut1, Snap25 and Rab3b, whereas pretreatment with TSA elevated mRNA levels of these genes. TSA greatly decreased expression of genes related to the calcium signaling pathway (Capn1 and Capn2) and apoptosis signaling pathway (Tnfrsf1a and Tp53), while addition of TSA significantly reduced levels of Tnfrsf1a and Tp53 compared with cisplatin alone (P < 0.01).

Conclusions

Our results suggested that TSA might protect against cisplatin-induced ototoxicity via mediating expression of genes responsible for regulating apoptosis, intracellular calcium homeostasis, neurotransmitter synthesis and release, and synaptic plasticity.

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