Kesherwani Varun, Tarang Shikha, Barnes Robert, Agrawal Sandeep K
Division of Neurosurgery, Department of Surgery, University of Nebraska Medical Center, Omaha, NE 68198-7690, USA.
Department of Oral Biology, Creighton University School of Dentistry, Omaha, NE 68178, USA.
Neurosci Lett. 2014 Jul 25;576:45-50. doi: 10.1016/j.neulet.2014.05.053. Epub 2014 Jun 4.
Fasudil (HA-1077), a specific Rho kinase II (ROCKII) inhibitor, is in clinical trials for recovery from spinal cord injury (SCI). The primary role of Fasudil is in axonal regeneration, as it inhibits ROCKII, the key signaling molecule involved in collapse of axon growth cone. Astrogliosis, due to the activation of astrocytes is an indicator of CNS injury. In early stages of injury, GFAP expression increases, helping to restore the integrity of the CNS. An increase in GFAP expression is also a marker of astrogliosis. Thus, reducing GFAP and hence astrogliosis at later stages of SCI is important for neuroregeneration and functional recovery. CoCl2 was used to induce hypoxic injury in astrocytic cell lines A172 (24h) and in spinal cord dorsal column white matter (8h). Several different techniques were used to study the changes in GFAP expression such as real-time PCR, western blotting and immunofluorescence staining with confocal microscopy. Hypoxia increased the expression of GFAP in A172 cells and in the spinal cord dorsal column after CoCl2 (100μM) treatment for 24h and 8h, respectively. We observed 11 folds increase in protein expression in A172 cells (24h) and 4.5 folds in spinal cord dorsal column (8h). The RNA expression was increased 3 folds in A172 cells after 24h of treatment and 4 folds in spinal cord dorsal column after 8h of treatment with 100μM CoCl2. Treatment with fasudil (20μM) significantly reduces the expression of GFAP in A172 cells and in spinal cord dorsal column. Fasudil also decreased activation of NF-κB in A172 cells after hypoxic injury. In the present study, we observed that fasudil reduces the expression of GFAP (consequently, astrogliosis) after hypoxic injury to A172 cells and spinal cord dorsal column. Our studies demonstrate that fasudil also plays a role in GFAP expression by reducing NF-κB activation at the injury site which could further help in axonal regeneration.
法舒地尔（HA - 1077）是一种特异性的Rho激酶II（ROCKII）抑制剂，正在进行用于脊髓损伤（SCI）恢复的临床试验。法舒地尔的主要作用在于轴突再生，因为它能抑制ROCKII，而ROCKII是参与轴突生长锥塌陷的关键信号分子。由于星形胶质细胞活化导致的星形胶质细胞增生是中枢神经系统损伤的一个指标。在损伤早期，胶质纤维酸性蛋白（GFAP）表达增加，有助于恢复中枢神经系统的完整性。GFAP表达增加也是星形胶质细胞增生的一个标志。因此，在脊髓损伤后期降低GFAP从而减轻星形胶质细胞增生对于神经再生和功能恢复很重要。用氯化钴（CoCl2）诱导星形胶质细胞系A172（24小时）和脊髓背柱白质（8小时）发生缺氧损伤。使用了几种不同技术来研究GFAP表达的变化，如实时聚合酶链反应（PCR）、蛋白质免疫印迹法以及共聚焦显微镜免疫荧光染色。缺氧分别在CoCl2（100μM）处理24小时和8小时后增加了A172细胞和脊髓背柱中GFAP的表达。我们观察到A172细胞（24小时）中蛋白质表达增加了11倍，脊髓背柱（8小时）中增加了4.5倍。在用100μM CoCl2处理24小时后，A172细胞中的RNA表达增加了3倍，处理8小时后脊髓背柱中的RNA表达增加了4倍。用法舒地尔（20μM）处理可显著降低A172细胞和脊髓背柱中GFAP的表达。法舒地尔还降低了缺氧损伤后A172细胞中核因子κB（NF - κB）的活化。在本研究中，我们观察到法舒地尔在A172细胞和脊髓背柱缺氧损伤后降低了GFAP的表达（从而减轻了星形胶质细胞增生）。我们的研究表明，法舒地尔还通过降低损伤部位的NF - κB活化在GFAP表达中发挥作用，这可能进一步有助于轴突再生。
