Department of Otolaryngology-Head and Neck Surgery, First Hospital of Jilin University, Changchun 130021, China.
Neurotoxicology. 2013 Jul;37:51-62. doi: 10.1016/j.neuro.2013.03.007. Epub 2013 Apr 1.
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.
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 1h, 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.
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).
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.
尽管组蛋白去乙酰化酶（HDACs）抑制剂已被证明可预防顺铂诱导的听力损失，但其中的机制仍知之甚少。本研究旨在探讨特异性 HDACs 抑制剂曲古抑菌素 A（TSA）对顺铂耳毒性的保护作用，并确定该过程中涉及的差异表达基因。
从 3 日龄新生 Wistar 大鼠的耳蜗底膜中分离出基底膜。对器官型培养物进行 150μM 顺铂或 200nM TSA 处理。对于联合治疗，细胞先用 TSA 孵育 1h，再用 TSA 加顺铂处理。用罗丹明鬼笔环肽染色标记毛细胞，用抗神经丝-200 抗体免疫细胞化学标记螺旋神经节神经元（SGNs）。采用通过进化关系进行蛋白质分析（PANTHER）分类系统进行全基因组表达谱微阵列分析，以鉴定差异表达基因。采用实时定量 PCR（qPCR）进行数据验证。
顺铂孵育 48 小时后，毛细胞和 SGN 大量丢失，而 TSA 联合治疗组中毛细胞和 SGN 的数量显著增加（P<0.05）。与对照组相比，顺铂处理后有 71 个基因上调，383 个基因下调。与单独给予顺铂相比，加入 TSA 后诱导了 1387 个基因上调和 1226 个基因下调。顺铂处理后，我们观察到几个与突触功能基因相关的基因的 mRNA 表达显著下调，包括 Camk2a、Camk2b、Vglut1、Snap25 和 Rab3b，而 TSA 预处理可上调这些基因的 mRNA 水平。TSA 大大降低了钙信号通路（Capn1 和 Capn2）和细胞凋亡信号通路（Tnfrsf1a 和 Tp53）相关基因的表达，而与单独给予顺铂相比，加入 TSA 后 Tnfrsf1a 和 Tp53 的水平显著降低（P<0.01）。
我们的结果表明，TSA 可能通过调节凋亡、细胞内钙稳态、神经递质合成和释放以及突触可塑性相关基因的表达来保护顺铂诱导的耳毒性。
