Regan R F, Kumar N, Gao F, Guo Y
Department of Surgery, Thomas Jefferson University, 1020 Sansom Street, 239 Thompson Building, Philadelphia, PA 19107, USA.
Neuroscience. 2002;113(4):985-94. doi: 10.1016/s0306-4522(02)00243-9.
Hemin is released from hemoglobin after CNS hemorrhage and may contribute to its cytotoxic effect. In a prior study, we demonstrated that heme oxygenase-1 induction protected murine cortical astrocytes from hemoglobin toxicity. Since heme metabolism releases iron, this observation suggested that these cells are able to effectively sequester and detoxify free iron. In this study, we tested the hypotheses that astrocytes increased ferritin synthesis after exposure to heme-bound iron, and that this induction protected cells from subsequent exposure to toxic concentrations of hemin. Incubation with low micromolar concentrations of hemin, hemoglobin, or ferrous sulfate increased ferritin expression, as detected on immunoblots stained with a polyclonal antibody that was raised against horse spleen ferritin. Time course studies demonstrated an increase in ferritin levels within 2 h. Weak and scattered cellular staining was detected by immunohistochemistry in control, untreated cultures, while diffuse immunoreactivity was observed in cultures exposed to heme-bound iron. An enhanced ferritin band was detected on immunoblots from cultures that were treated with purified apoferritin, consistent with astrocytic ferritin uptake. Immunoreactivity after apoferritin treatment was not altered by concomitant treatment with cycloheximide. Pretreatment with apoferritin protected astrocytes from hemin toxicity in a concentration-dependent fashion between 1 and 4 mg/ml. At the highest concentration, cell death due to a 6-h exposure to 30 microM hemin was decreased by about 85%. A protective effect was also produced by induction of endogenous ferritin with nontoxic concentrations of ferrous sulfate, hemoglobin, or hemin. These results suggest that cortical astrocytes respond to exogenous heme-bound or free iron by rapidly increasing ferritin synthesis. The combined action of heme oxygenase-1 and ferritin may be a primary astrocytic defense against heme-mediated injury.
中枢神经系统出血后，血红素从血红蛋白中释放出来，可能导致其细胞毒性作用。在先前的一项研究中，我们证明血红素加氧酶-1的诱导可保护小鼠皮质星形胶质细胞免受血红蛋白毒性的影响。由于血红素代谢会释放铁，这一观察结果表明这些细胞能够有效螯合和解毒游离铁。在本研究中，我们检验了以下假设：星形胶质细胞在暴露于血红素结合铁后会增加铁蛋白的合成，并且这种诱导作用可保护细胞免受随后暴露于有毒浓度血红素的影响。用低微摩尔浓度的血红素、血红蛋白或硫酸亚铁孵育可增加铁蛋白表达，这在使用针对马脾铁蛋白的多克隆抗体染色的免疫印迹上得以检测。时间进程研究表明，2小时内铁蛋白水平会升高。在对照未处理的培养物中，免疫组织化学检测到微弱且分散的细胞染色，而在暴露于血红素结合铁的培养物中观察到弥漫性免疫反应性。在用纯化的脱铁铁蛋白处理的培养物的免疫印迹上检测到增强的铁蛋白条带，这与星形胶质细胞摄取铁蛋白一致。脱铁铁蛋白处理后的免疫反应性不会因同时用环己酰亚胺处理而改变。用脱铁铁蛋白预处理可在1至4毫克/毫升的浓度范围内以浓度依赖的方式保护星形胶质细胞免受血红素毒性的影响。在最高浓度下，6小时暴露于30微摩尔血红素导致的细胞死亡减少了约85%。用无毒浓度的硫酸亚铁、血红蛋白或血红素诱导内源性铁蛋白也产生了保护作用。这些结果表明，皮质星形胶质细胞通过快速增加铁蛋白合成来对外源性血红素结合铁或游离铁做出反应。血红素加氧酶-1和铁蛋白的联合作用可能是星形胶质细胞对血红素介导损伤的主要防御机制。
