Department of Geriatrics, First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116011, China.
Chin Med J (Engl). 2013 Nov;126(21):4132-8.
Transient sublethal ischemia is known as ischemic preconditioning, which enables cells and tissues to survive subsequent prolonged lethal ischemic injury. Ischemic preconditioning exerts neuroprotection through phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Cbl-b belongs to the Casitas B-lineage lymphoma (Cbl) family, and it can regulate the cell signal transduction.The roles of ubiquitin ligase Cbl-b and PI3K/Akt pathway and the relationship between them in oxygen-glucose deprivation preconditioning (OGDPC) in PC12 cells were investigated in the present study.
Oxygen and glucose deprivation (OGD) model in PC12 cells was used in the present study. The 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nuclear staining with Hoechst 33258, and Western blotting were applied to explore the roles of Cbl-b and PI3K/Akt pathway and the relationship between them in OGDPC in PC12 cells.
Cell viability was significantly changed by OGD and OGDPC. OGD significantly decreased cell viability compared with the control group (P < 0.05), and preconditioning could rescue this damage was demonstrated by the increase of cell viability (P < 0.05). The expression of Cbl-b was significantly increased after OGD treatment. However, the activation of Akt and GSK3β was greatly inhibited. Preconditioning could inhibit the increase of Cbl-b caused by OGD and increase the activation of Akt and GSK3β. LY294002, a specific inhibitor of PI3K, could effectively inhibit the increase of Akt and GSK3β after preconditioning treatment. It partly inhibited the decrease of Cbl-b expression after preconditioning treatment.
Ubiquitin ligase Cbl-b and PI3K/Akt pathway are differently involved in OGDPC in PC12 cells.
短暂的亚致死性缺血被称为缺血预处理，它使细胞和组织能够在随后的长时间致死性缺血损伤中存活。缺血预处理通过磷脂酰肌醇 3-激酶 (PI3K)/Akt 途径发挥神经保护作用。Cbl-b 属于 Casitas B-细胞淋巴瘤 (Cbl) 家族，可调节细胞信号转导。本研究旨在探讨泛素连接酶 Cbl-b 和 PI3K/Akt 途径及其在 PC12 细胞氧葡萄糖剥夺预处理 (OGDPC) 中的作用及相互关系。
本研究采用 PC12 细胞氧葡萄糖剥夺 (OGD) 模型。应用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐 (MTT) 比色法、Hoechst 33258 核染色和 Western blot 法，探讨 Cbl-b 和 PI3K/Akt 途径及其在 OGDPC 中的作用及相互关系。
OGD 和 OGDPC 显著改变细胞活力。与对照组相比，OGD 显著降低细胞活力 (P<0.05)，而细胞活力的增加证明预处理可以挽救这种损伤 (P<0.05)。OGD 处理后 Cbl-b 的表达明显增加。然而，Akt 和 GSK3β 的激活受到很大抑制。预处理可抑制 OGD 引起的 Cbl-b 增加，并增加 Akt 和 GSK3β 的激活。PI3K 的特异性抑制剂 LY294002 可有效抑制预处理后 Akt 和 GSK3β 的增加。它部分抑制了预处理后 Cbl-b 表达的下降。
泛素连接酶 Cbl-b 和 PI3K/Akt 途径在 PC12 细胞的 OGDPC 中不同程度地参与。
