基于淫羊藿次苷Ⅱ调节细胞自噬抗脑缺血再灌注损伤的机制研究

The mechanism of cerebral ischemia-reperfusion (I/R) is very complicated, and effective drugs for the treatment of I/R is still unvailable. Recently, some studies suggest that excessive autophagy is involved in I/R. Therefore, inhibition of autophagy is important to the treatment of I/R. Icariside II is one of the major components of traditional Chinese medicine Epimedium, and our previous research has demonstrated that icariside II inhibits the middle cerebral artery occlusion in rats may be through regulated autophagy. However, whether the agent can ameliorate I/R via autophagy remains still unclear. The present study aims to investigate the possible intervention effect of icariside II on I/R pathogenesis in rats model, then, the underlying molecular mechanism of action of icariside II against I/R including the expression of LC3-I, LC3-II, Beclin 1 and the change of ROS/GSK-3β/mitochondrial signal pathway is further detected in primary cultured rat cortical neurons injury subjected to oxygen-glucose deprivation and reperfusion with the help of all kinds of tool agents and RNA interference technique. Rapamycin, an autophagy inducer, and 3-Methyladenine, an autophagy inhibitor, are utilized as positive and negative control, respectively. The effects and the molecular mechanisms of icariside II on autophagy were investigated. This project will provide pharmacological basis for application of icariside II into the treatment of I/R.

脑缺血再灌注损伤（I/R）作用机制复杂，目前尚无理想的治疗药物。近年来，自噬增强被认为是加重I/R的重要原因，因此，抑制自噬是治疗I/R的策略之一。淫羊藿次苷Ⅱ是中药淫羊藿的主要成分之一，前期研究发现淫羊藿次苷Ⅱ可能通过抑制自噬减轻线栓法致大脑中动脉阻塞的大鼠急性缺血性脑卒中，但其是否能够通过自噬途径抗I/R尚不清楚。因此，本项目拟首先通过局灶性脑缺血再灌注损伤大鼠模型，研究淫羊藿次苷Ⅱ对I/R发病可能的干预作用；在此基础上，通过原代培养脑皮质神经元氧糖剥夺/再灌注损伤模型，借助各种工具药、免疫印迹法和RNA干扰等技术考察自噬相关蛋白LC3-I、LC3-II和Beclin 1的表达以及ROS/GSK-3β/线粒体信号通路的变化。本研究以自噬诱导剂雷帕霉素和自噬抑制剂3-甲基腺嘌呤为正反对照，从调控自噬的角度探讨淫羊藿次苷Ⅱ对I/R的作用机制，旨在为淫羊藿次苷Ⅱ用于I/R的治疗提供实验依据。

脑缺血再灌注损伤（I/R）作用机制复杂，目前尚无理想的治疗药物。近年来，自噬增强被认为是加重I/R的重要原因，因此，抑制自噬是治疗I/R的策略之一。淫羊藿次苷Ⅱ是中药淫羊藿的主要成分之一，前期研究发现淫羊藿次苷Ⅱ可能通过抑制自噬减轻线栓法致大脑中动脉阻塞（middle cerebral artery occlusion, MCAO）的大鼠急性缺血性脑卒中，但其是否能够通过自噬途径抗I/R尚不清楚。本研究结果发现：（1）淫羊藿次苷Ⅱ不仅能够在体内发挥抗MCAO导致的I/R的作用，在体外还可以抗氧糖剥夺/复氧复糖（oxygen-glucose deprivation and reoxygenation, OGD/R）诱导的大鼠原代脑皮质神经元损伤。其神经保护作用可能与调控PKG/GSK-3β/自噬信号途径发挥作用。值得注意的是，通过计算机模拟技术进一步表明淫羊藿次苷Ⅱ可以直接与PDE5和GSK-3β结合并且PKG和GSK-3β存在蛋白质间相互作用。（2）ICS II对过氧化氢诱导的PC12细胞氧化损伤具有保护作用，其机制可能与调控ROS/GSK-3β/线粒体途径，进而抑制细胞过度自噬有关。（3）ICS II作为PDE5抑制剂能够抗OGD/R诱导的原代海马神经元凋亡，其作用机制可能与调控BDNF/TrkB/CREB通路有关。综上所述，淫羊藿次苷Ⅱ能够通过调控PKG/GSK-3β/自噬信号途径发挥抗脑缺血再灌注损伤作用。本研究为将淫羊藿次苷Ⅱ开发成为新型PDE5抑制剂在临床用于缺血性脑卒中的治疗提供了药理学依据。