Florey Neuroscience Institutes, University of Melbourne, Parkville, VIC, Australia.
J Cell Physiol. 2012 Mar;227(3):1199-211. doi: 10.1002/jcp.22838.
Inhibitors of Rho kinase (ROCK) have potential for management of neurological disorders by inhibition of glial scarring. Since astrocytes play key roles in brain physiology and pathology, we determined changes in the astrocytic transcriptome produced by the ROCK inhibitor Fasudil to obtain mechanistic insights into its beneficial action during brain injury. Cultured murine astrocytes were treated with Fasudil (100 µM) and morphological analyses revealed rapid stellation by 1 h and time-dependent (2-24 h) dissipation of F-actin-labelled stress fibres. Microarray analyses were performed on RNA and the time-course of global gene profiling (2, 6, 12 and 24 h) provided a comprehensive description of transcriptomic changes. Hierarchical clustering of differentially expressed genes and analysis for over-represented gene ontology groups using the DAVID database focused attention on Fasudil-induced changes to major biological processes regulating cellular shape and motility (actin cytoskeleton, axon guidance, transforming growth factor-β (TGFβ) signalling and tight junctions). Bioinformatic analyses of transcriptomic changes revealed how these biological processes contributed to changes in astrocytic motility and cytoskeletal reorganisation. Here genes associated with extracellular matrix were also involved, but unexpected was a subset of alterations (EAAT2, BDNF, anti-oxidant species, metabolic and signalling genes) indicative of adoption by astrocytes of a pro-survival phenotype. Expression profiles of key changes with Fasudil and another ROCK inhibitor Y27632 were validated by real-time PCR. Although effects of ROCK inhibition have been considered to be primarily cytoskeletal via reduction of glial scarring, we demonstrate additional advantageous actions likely to contribute to their ameliorative actions in brain injury.
Rho 激酶（ROCK）抑制剂通过抑制神经胶质瘢痕形成，具有治疗神经疾病的潜力。由于星形胶质细胞在大脑生理和病理中起着关键作用，我们确定了 ROCK 抑制剂法舒地尔对星形胶质细胞转录组的影响，以深入了解其在脑损伤过程中的有益作用。用 Fasudil（100µM）处理培养的鼠星形胶质细胞，形态分析显示 1 小时内迅速出现星状，F-肌动蛋白标记的应激纤维呈时间依赖性（2-24 小时）耗散。对 RNA 进行微阵列分析，并对全基因组基因表达谱的时间过程（2、6、12 和 24 小时）进行分析，全面描述了转录组的变化。差异表达基因的层次聚类和使用 DAVID 数据库对过表达基因本体组的分析，将重点放在 Fasudil 诱导的调节细胞形状和运动的主要生物学过程（肌动蛋白细胞骨架、轴突导向、转化生长因子-β（TGFβ）信号和紧密连接）的变化上。转录组变化的生物信息学分析揭示了这些生物学过程如何导致星形胶质细胞运动和细胞骨架重排的变化。在这里，与细胞外基质相关的基因也参与其中，但出乎意料的是，一组改变（EAAT2、BDNF、抗氧化物质、代谢和信号基因）表明星形胶质细胞采用了一种促生存表型。用 Fasudil 和另一种 ROCK 抑制剂 Y27632 验证了关键变化的表达谱，实时 PCR 验证了关键变化的表达谱。尽管 ROCK 抑制的作用主要被认为是通过减少神经胶质瘢痕形成来影响细胞骨架，但我们证明了其他有利的作用可能有助于其在脑损伤中的改善作用。
