Zhou Feng, Gomi Masanori, Fujimoto Motoaki, Hayase Makoto, Marumo Takeshi, Masutani Hiroshi, Yodoi Junji, Hashimoto Nobuo, Nozaki Kazuhiko, Takagi Yasushi
Department of Neurosurgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Brain Res. 2009 May 26;1272:62-70. doi: 10.1016/j.brainres.2009.03.023. Epub 2009 Mar 25.
Thioredoxin (Trx) is a 12-kDa protein ubiquitously expressed in all living cells that fulfills a variety of biological functions related to cell proliferation and apoptosis. It is characterized by the highly conserved reduction/oxidation (redox)-active site sequence Trp-Cys-Gly-Pro-Cys-Lys. Trx acts as a powerful antioxidant and plays an important role in maintaining critical protein thiols in the reduced state. Moreover, it has been shown to scavenge reactive oxygen species (ROS) and to protect against oxidative stress. We have reported that Trx-1 protects against neuronal damage during focal ischemia. However, the mechanisms underlying this protective effect and the effect of Trx-1 on neuronal apoptosis during ischemia have not been fully clarified. In this study, we analyzed the effect of Trx-1 overexpression against neuronal degeneration after a short duration of transient brain ischemia. Mild focal ischemia was reported to induce neuronal death through apoptosis. We employed Fluorojade-B staining to detect neuronal degeneration. In Trx transgenic mice, a smaller number of Fluorojade-B-positive neurons were detected after ischemia-reperfusion than in wild-type mice. In addition, we detected cleaved caspase-3- and TUNEL-positive cells, which indicated caspase-dependent apoptosis. Fewer caspase-3- and TUNEL-positive neurons were detected after ischemia-reperfusion in Trx transgenic mice than in wild-type mice. Furthermore, Akt signaling was reported to play a role in neuronal survival in Trx-1 overexpressing mice. After ischemia-reperfusion, Western blot and immunohistochemical analysis indicated that phosphorylation of Akt was enhanced in Trx transgenic mice after ischemia-reperfusion. Intraventricular injection of LY294002,which is a phosphoinositide 3-kinase (PI3K), vanished the neuroprotective effect in Trx-1 transgenic mice. These results indicate that Trx-1 overexpression protects neurons from apoptosis after ischemia-reperfusion.
硫氧还蛋白（Trx）是一种12千道尔顿的蛋白质，在所有活细胞中普遍表达，具有多种与细胞增殖和凋亡相关的生物学功能。其特征在于高度保守的还原/氧化（redox）活性位点序列Trp-Cys-Gly-Pro-Cys-Lys。Trx作为一种强大的抗氧化剂，在维持关键蛋白巯基处于还原状态方面发挥着重要作用。此外，它已被证明能够清除活性氧（ROS）并抵御氧化应激。我们曾报道Trx-1可在局灶性缺血期间保护神经元免受损伤。然而，这种保护作用的潜在机制以及Trx-1在缺血期间对神经元凋亡的影响尚未完全阐明。在本研究中，我们分析了短暂性脑缺血后Trx-1过表达对神经元变性的影响。据报道，轻度局灶性缺血可通过凋亡诱导神经元死亡。我们采用Fluorojade-B染色来检测神经元变性。在Trx转基因小鼠中，缺血再灌注后检测到的Fluorojade-B阳性神经元数量比野生型小鼠少。此外，我们检测到了裂解的caspase-3和TUNEL阳性细胞，这表明存在caspase依赖性凋亡。缺血再灌注后，Trx转基因小鼠中检测到的caspase-3和TUNEL阳性神经元比野生型小鼠少。此外，据报道Akt信号通路在Trx-1过表达小鼠的神经元存活中发挥作用。缺血再灌注后，蛋白质免疫印迹和免疫组织化学分析表明，Trx转基因小鼠缺血再灌注后Akt的磷酸化增强。脑室内注射磷脂酰肌醇3激酶（PI3K）抑制剂LY294002消除了Trx-1转基因小鼠的神经保护作用。这些结果表明，Trx-1过表达可保护神经元免受缺血再灌注后的凋亡。
