丙泊酚后处理对离体培养胎鼠海马神经元凋亡及cPKCγ/GAP-43信号通路的影响

Effects of propofol post-treatment on apoptosis of and cPKCγ/GAP-43 signaling pathway in fetal rat hippocampal neurons in vitro

摘要:

目的 探究丙泊酚后处理对离体胎鼠海马神经元凋亡及经典型蛋白激酶Cγ（cPKCγ）/生长相关蛋白-43（GAP-43）信号通路的影响。方法 取培养7 d的胎鼠海马神经元，随机分为对照组、缺氧组、丙泊酚组。缺氧组和丙泊酚组于90% NO₂+10% CO₂的无氧培养箱中缺氧处理30 min。丙泊酚组在缺氧处理后立即加入终浓度为50 μmol/L的丙泊酚新培养基，对照组和缺氧组换等体积的新培养基孵育2 h。采用MTT法检测神经元存活率，流式细胞仪检测神经元凋亡率，比色法检测神经元超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量，实时荧光定量聚合酶链反应、Western blotting检测cPKCγ、GAP-43 mRNA和蛋白的表达。结果 免疫组织化学染色结果显示，培养7 d的海马神经元胞质饱满，状态良好。与对照组比较，缺氧组和丙泊酚组海马神经元存活率、SOD活性降低（P <0.05），凋亡率升高（P <0.05），MDA含量增加（P <0.05）；与缺氧组相比，丙泊酚组海马神经元存活率、SOD活性升高（P <0.05），凋亡率降低（P <0.05），MDA含量减少（P <0.05）。与对照组比较，缺氧组和丙泊酚组海马神经元cPKCγ、GAP-43 mRNA和蛋白相对表达量降低（P <0.05）；与缺氧组比较，丙泊酚组海马神经元cPKCγ、GAP-43 mRNA和蛋白相对表达量升高（P <0.05）。结论 缺氧的胎鼠海马神经元经丙泊酚后处理后可以增加神经元抗氧化应激水平，降低神经元凋亡率，其保护作用可能与激活cPKCγ/GAP-43信号通路有关。

Abstract:

Objective To investigate the effects of propofol post-treatment on apoptosis of and classical protein kinase C γ (cPKCγ)/growth associated protein-43 (GAP-43) signaling pathway in fetal rat hippocampal neurons in vitro.Methods Fetal rat hippocampal neurons cultured for seven days were randomly divided into control group, hypoxia group and propofol group. The neurons in the hypoxia group and propofol group were treated with hypoxia in the anaerobic incubator containing 90% nitrogen dioxide and 10% carbon dioxide for half an hour. The neurons in the propofol group were cultured in the medium containing propofol at a final concentration of 50 μmol/L after hypoxia treatment, while the neurons in the control group and hypoxia group were cultured in medium without propofol. All the neurons were cultured for two hours. MTT assay was used to detect the survival rate of neurons. The apoptosis rate of neurons was detected by flow cytometry. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in neurons were detected via colorimetry. The mRNA and protein expressions of cPKCγ and GAP-43 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively.Results Immunohistochemical results showed that the hippocampal neurons cultured for 7 days were in good conditions. The survival rate of hippocampal neurons and the SOD activity were lower, and the apoptosis rate and the MDA content were higher in the hypoxia group and the propofol group than those in the control group (P < 0.05). Compared with the hypoxia group, the survival rate of hippocampal neurons and the SOD activity were higher, and the apoptosis rate and the MDA content were lower (P < 0.05). The mRNA and protein expressions of cPKCγ and GAP-43 in the hippocampal neurons were decreased in the hypoxia group and propofol group compared with the control group (P < 0.05), while they were higher in the propofol group than those in the hypoxia group (P < 0.05).Conclusions Propofol post-treatment enhances the anti-oxidative defenses of fetal rat hippocampal neurons treated with hypoxia, and reduces the apoptosis rate of the neurons. The neuronal protective effect may be related to the activation of cPKCγ/GAP-43 signaling pathway.