Department of Ophthalmology, Shanghai Jiaotong University Affiliated Shanghai First People's Hospital, Wujin Road 85, Shanghai 200080, China.
Neuroscience. 2012 Nov 8;224:282-93. doi: 10.1016/j.neuroscience.2012.07.056. Epub 2012 Aug 4.
Valproate (VPA) is an anticonvulsant and mood-stabilizing drug. It is a broad-spectrum histone deacetylase inhibitor with neuroprotective effects. We investigated whether VPA reduces retinal neuronal death induced by optic nerve crush (ONC). To evaluate further VPA-mediated neuroprotection on retinal ganglion cells (RGCs), another histone deacetylase (HDAC) inhibitor, sodium butyrate (SB) was compared with VPA. Adult male Wistar rats were subjected to ONC injury. VPA and SB were administered subcutaneously 1 day prior to ONC until sacrifice 14 days later. RGC density was counted using hematoxylin and eosin (H&E) staining of the retinal section and retrograde labeling with FluoroGold. Retinal function was evaluated by electroretinography (ERG) after ONC. Immunofluorescence of activated caspase-3 in ganglion cell layer (GCL) and the detection of bcl-2 mRNA expression in the retina were used to evaluate apoptosis of retinal cells. In addition, brain-derived neurotrophic factor (BDNF) in retinas was measured using an enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Western blot was used to analyze histone H3 acetylation, the protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) phosphorylation levels, and tropomyosin-related kinase B (TrkB) levels. The transcriptional activation of the BDNF gene was analyzed by measuring the levels of acetylation or methylation of histone H3 using chromatin immunoprecipitation assay. The RGC density in the VPA and SB treated-groups were significantly higher as compared with those of the corresponding vehicle group following ONC. VPA and SB suppressed reductions in a- and b-wave amplitudes of the ERG and attenuated the activation of caspase-3 in the RGCs, which was accompanied by upregulation in Akt and Erk phosphorylation in the retina. Furthermore, VPA upregulated levels of bcl-2, BDNF, TrkB in the retina post-injury. VPA and SB treatment resulted in the hyperacetylation of histone H3K14, attenuated histone H3K9 hypermethylation in the BDNF promoter, and promoted transcriptional activity. These results demonstrate that VPA appears to protect RGCs from ONC by inhibiting neuronal apoptosis possibly via the activation of BDNF-TrkB signaling and HDAC inhibition.
丙戊酸（VPA）是一种抗惊厥和稳定情绪的药物。它是一种具有神经保护作用的广谱组蛋白去乙酰化酶抑制剂。我们研究了 VPA 是否可以减少视神经挤压（ONC）引起的视网膜神经元死亡。为了进一步评估 VPA 介导的对视网膜神经节细胞（RGC）的神经保护作用，我们比较了另一种组蛋白去乙酰化酶（HDAC）抑制剂丁酸钠（SB）与 VPA。成年雄性 Wistar 大鼠接受 ONC 损伤。在 ONC 前 1 天皮下给予 VPA 和 SB，并在 14 天后处死。通过视网膜切片的苏木精和伊红（H＆E）染色和荧光金逆行标记来计数 RGC 密度。在 ONC 后通过视网膜电图（ERG）评估视网膜功能。用免疫荧光法检测 GCL 中活化的半胱天冬酶-3，并检测视网膜中 bcl-2 mRNA 的表达，以评估视网膜细胞的凋亡。此外，使用酶联免疫吸附试验（ELISA）和实时聚合酶链反应（PCR）测量视网膜中的脑源性神经营养因子（BDNF）。Western blot 用于分析组蛋白 H3 乙酰化、蛋白激酶 B（Akt）和细胞外信号调节激酶（Erk）磷酸化水平以及原肌球蛋白相关激酶 B（TrkB）水平。通过使用染色质免疫沉淀测定测量组蛋白 H3 的乙酰化或甲基化水平来分析 BDNF 基因的转录激活。与相应的载体组相比，ONC 后 VPA 和 SB 处理组的 RGC 密度明显更高。VPA 和 SB 抑制了 ERG 的 a-和 b-波幅度的降低，并减轻了 RGC 中半胱天冬酶-3 的激活，同时伴随着视网膜中 Akt 和 Erk 磷酸化的增加。此外，VPA 上调了损伤后视网膜中的 bcl-2、BDNF、TrkB 水平。VPA 和 SB 治疗导致组蛋白 H3K14 乙酰化增加，减弱了 BDNF 启动子中组蛋白 H3K9 的过度甲基化，并促进了转录活性。这些结果表明，VPA 通过抑制神经元凋亡似乎可以保护 RGC 免受 ONC 的侵害，可能是通过激活 BDNF-TrkB 信号和 HDAC 抑制。
