Wu Jingxian, Chen Yanlin, Yu Shanshan, Li Lingyu, Zhao Xiujuan, Li Qiong, Zhao Jing, Zhao Yong
Department of Pathology, Chongqing Medical University, Chongqing 400016, PR China.
Department of Pathology, Chongqing Medical University, Chongqing 400016, PR China; The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China.
Brain Res Bull. 2017 Jun;132:99-108. doi: 10.1016/j.brainresbull.2017.05.012. Epub 2017 May 24.
As an endogenous antioxidant protein, Sulfiredoxin1 (Srxn1) can prevent cell oxidative stress damage. However, its role in cerebral ischemia/reperfusion (I/R) injury and the underlying signaling mechanisms remain largely unknown. Here, we explored effects of Srxn1 knockdown on oxidative stress using in vitro and in vivo I/R models and investigated related neuroprotective mechanisms. For in vitro studies, primary cortical neuronal cultures were transfected with an interfering lentivirus targeting Srxn1. Oxygen-glucose deprivation (OGD) was conducted after Srxn1 knockdown. MTS and lactate dehydrogenase assays indicated that knockdown of Srxn1 increased cell death and reduced cell viability. Similarly, superoxide dismutase (SOD) and reduced glutathionekits assays showed that knockdown of Srxn1 worsened oxidative stress injury. For in vivo studies, siRNA for Srxn1 or negative control siRNA was injected intracerebroventricularly 24h before middle cerebral artery occlusion (MCAO). Data shows silencing Srxn1 resulted in a significant increase in cerebral infarction, neurological deficits, histological injury, and oxidative stress injury 24h after ischemic stroke. Moreover, immunoblot analysis assessed the relationship between Srxn1 levels and Prdx1-4 as well as Prdx-SO3 activity both in vitro and in vivo models. We found that decreased Srxn1 reduced Prdx1-4 and enhanced Prdx-SO3 protein levels. In addition, knockdown of Nrf2 was performed; immunoblot analysis was used to measure Srxn1 and NQO1 protein levels. We further found that interference of Nrf2 reduced Srxn1 and NQO1 protein levels. In summary, Srxn1 can protect neurons from I/R oxidative stress injury and the mechanism involves Prdx activity. Srxn1, which might be downstream of Nrf2, can prevent cerebral ischemia reperfusion by reversing overoxidized Prdx and restoring antioxidant activity of Prdx.
作为一种内源性抗氧化蛋白，硫氧还蛋白1（Srxn1）可预防细胞氧化应激损伤。然而，其在脑缺血/再灌注（I/R）损伤中的作用及潜在信号机制仍不清楚。在此，我们利用体外和体内I/R模型探讨了Srxn1基因敲低对氧化应激的影响，并研究了相关的神经保护机制。体外研究中，用靶向Srxn1的干扰慢病毒转染原代皮质神经元培养物。Srxn1基因敲低后进行氧糖剥夺（OGD）。MTS和乳酸脱氢酶检测表明，Srxn1基因敲低会增加细胞死亡并降低细胞活力。同样，超氧化物歧化酶（SOD）和还原型谷胱甘肽试剂盒检测显示，Srxn1基因敲低会加重氧化应激损伤。体内研究中，在大脑中动脉闭塞（MCAO）前24小时脑室内注射Srxn1的siRNA或阴性对照siRNA。数据显示，缺血性卒中24小时后，沉默Srxn1会导致脑梗死、神经功能缺损、组织学损伤和氧化应激损伤显著增加。此外，免疫印迹分析评估了体外和体内模型中Srxn1水平与Prdx1 - 4以及Prdx - SO3活性之间的关系。我们发现，Srxn1水平降低会降低Prdx1 - 4并提高Prdx - SO3蛋白水平。此外，进行了Nrf2基因敲低；用免疫印迹分析测量Srxn1和NQO1蛋白水平。我们进一步发现，干扰Nrf2会降低Srxn1和NQO1蛋白水平。总之，Srxn1可保护神经元免受I/R氧化应激损伤，其机制涉及Prdx活性。Srxn1可能是Nrf2的下游分子，可通过逆转过度氧化的Prdx并恢复Prdx的抗氧化活性来预防脑缺血再灌注。
