Luo Tianfei, Liu Guiying, Ma Hongxi, Lu Bin, Xu Haiyang, Wang Yujing, Wu Jiang, Ge Pengfei, Liang Jianmin
Department of Neurology, First Hospital of Jilin University, Changchun 130021, China.
Department of Pediatrics, Anzhen Hospital of Capital University of Medical Sciences, Beijing 10029, China.
Int J Mol Sci. 2014 Sep 1;15(9):15426-42. doi: 10.3390/ijms150915426.
Lethal autophagy is a pathway leading to neuronal death caused by transient global ischemia. In this study, we examined the effect of Ginsenoside Rb1 (GRb1) on ischemia/reperfusion-induced autophagic neuronal death and investigated the role of PI3K/Akt. Ischemic neuronal death in vitro was induced by using oxygen glucose deprivation (OGD) in SH-SY5Y cells, and transient global ischemia was produced by using two vessels occlusion in rats. Cellular viability of SH-SY5Y cells was assessed by MTT assay, and CA1 neuronal death was evaluated by Hematoxylin-eosin staining. Autophagic vacuoles were detected by using both fluorescent microscopy in combination with acridine orange (AO) and Monodansylcadaverine (MDC) staining and transmission electronic microscopy. Protein levels of LC3II, Beclin1, total Akt and phosphor-Akt at Ser473 were examined by western blotting analysis. GRb1 inhibited both OGD and transient ischemia-induced neuronal death and mitigated OGD-induced autophagic vacuoles in SH-SY5Y cells. By contrast, PI3K inhibitor LY294002 counteracted the protection of GRb1 against neuronal death caused by either OGD or transient ischemia. LY294002 not only mitigated the up-regulated protein level of phosphor Akt at Ser473 caused by GRb1, but also reversed the inhibitory effect of GRb1 on OGD and transient ischemia-induced elevation in protein levels of LC3II and Beclin1.
致死性自噬是一种由短暂性全脑缺血导致神经元死亡的途径。在本研究中，我们检测了人参皂苷Rb1（GRb1）对缺血/再灌注诱导的自噬性神经元死亡的影响，并研究了PI3K/Akt的作用。在SH-SY5Y细胞中采用氧糖剥夺（OGD）诱导体外缺血性神经元死亡，在大鼠中采用双侧颈总动脉结扎法造成短暂性全脑缺血。通过MTT法评估SH-SY5Y细胞的细胞活力，通过苏木精-伊红染色评估CA1区神经元死亡情况。使用荧光显微镜结合吖啶橙（AO）和单丹磺酰尸胺（MDC）染色以及透射电子显微镜检测自噬空泡。通过蛋白质印迹分析检测LC3II、Beclin1、总Akt和Ser473位点磷酸化Akt的蛋白水平。GRb1抑制OGD和短暂性缺血诱导的神经元死亡，并减轻OGD诱导的SH-SY5Y细胞中的自噬空泡。相比之下，PI3K抑制剂LY294002抵消了GRb1对OGD或短暂性缺血所致神经元死亡的保护作用。LY294002不仅减轻了GRb1引起的Ser473位点磷酸化Akt蛋白水平的上调，还逆转了GRb1对OGD和短暂性缺血诱导的LC3II和Beclin1蛋白水平升高的抑制作用。
