Liu Ya, Li Junfa, Yang Jing, Ji Fang, Bu Xiangning, Zhang Nan, Zhang Bingxi
Department of Anesthesiology, Capital Medical University Affiliated Beijing Tongren Hospital, Beijing 100730, China.
Neurosci Lett. 2008 Oct 17;444(1):87-91. doi: 10.1016/j.neulet.2008.08.014. Epub 2008 Aug 8.
We previously reported that novel protein kinase C (nPKC) epsilon and N-methyl-d-aspartic acid (NMDA) receptors participated in morphine preconditioning (MP)-induced neuroprotection. In this study, we used Western blot analysis, 2,3,5-triphenyltetrazolium chloride (TTC) staining and lactate dehydrogenase (LDH) leakage assay to determine the involvement of conventional PKC isoforms (cPKC) in MP-induced neuroprotection against oxygen-glucose deprivation (OGD). Hippocampus slices (400-microm thickness) from healthy male BALB/c mice exposed to OGD for 5-45 min to mimic mild, moderate and severe ischemia in the presence of MP pretreatment. We found that OGD-induced damage in neuronal cell survival rate and LDH leakage could be improved by MP pretreatment (3 microM) within 20 min of OGD, which was abolished by concomitant incubation with non-selective opioid receptor antagonist naloxone (Nal, 50 microM). The results of Western blot analysis showed that only cPKCgamma membrane translocation, not alpha, betaI and betaII, increased under the condition of OGD 10 min and 2h reperfusion (OGD/2h), and this increment of cPKCgamma membrane translocation was inhibited by MP pretreatment. To further elucidate the role of cPKCgamma in MP-induced neuroprotection, we found that cPKCgamma membrane translocation inhibitor, Go6983 (6 nM) did not affect MP-induced neuroprotection while Go6983 alone exhibited a significant inhibition on OGD-induced increment in LDH leakage and decrease in cell survival rate. These phenomena were defined by the results that Go6983 could restore OGD-induced cPKCgamma membrane translocation, but had no further effect on MP-induced inhibition of cPKCgamma membrane translocation. These results demonstrated that MP can reduce OGD-induced neuronal injuries, and the down-regulation of cPKCgamma membrane translocation might be involved in the neuroprotection.
我们先前报道过，新型蛋白激酶C（nPKC）ε和N-甲基-D-天冬氨酸（NMDA）受体参与了吗啡预处理（MP）诱导的神经保护作用。在本研究中，我们使用蛋白质免疫印迹分析、2,3,5-三苯基氯化四氮唑（TTC）染色和乳酸脱氢酶（LDH）释放检测，来确定传统蛋白激酶C亚型（cPKC）在MP诱导的针对氧糖剥夺（OGD）的神经保护中的作用。从健康雄性BALB/c小鼠获取厚度为400微米的海马切片，在MP预处理的情况下，使其暴露于OGD 5至45分钟，以模拟轻度、中度和重度缺血。我们发现，在OGD 20分钟内，MP预处理（3微摩尔）可改善OGD诱导的神经元细胞存活率损伤和LDH释放，而与非选择性阿片受体拮抗剂纳洛酮（Nal，50微摩尔）共同孵育可消除这种作用。蛋白质免疫印迹分析结果显示，在OGD 10分钟和再灌注2小时（OGD/2h）的条件下，只有cPKCγ发生膜转位，而α、βI和βII未发生，并且MP预处理可抑制cPKCγ膜转位的增加。为进一步阐明cPKCγ在MP诱导的神经保护中的作用，我们发现cPKCγ膜转位抑制剂Go6983（6纳摩尔）不影响MP诱导的神经保护，而单独使用Go6983对OGD诱导的LDH释放增加和细胞存活率降低具有显著抑制作用。这些现象由以下结果确定：Go6983可恢复OGD诱导的cPKCγ膜转位，但对MP诱导的cPKCγ膜转位抑制没有进一步影响。这些结果表明，MP可减轻OGD诱导的神经元损伤，cPKCγ膜转位的下调可能参与了神经保护作用。
