NADPH对KA介导的神经兴奋性毒性的保护作用及其机制
首发时间:2019-04-30
摘要:目的:研究还原型烟酰胺腺嘌呤二核苷酸磷酸(Reduced Nicotinamide Adenine Dinucleotide Phosphate, NADPH)在海人藻酸(Kainic acid, KA)介导的兴奋性毒性中的作用及其分子调控机制。方法:采用KA处理体外培养的大鼠原代皮层神经元,建立神经兴奋性毒性体外模型;KA处理细胞之前,用NADPH预处理,建立NADPH处理组。通过CCK-8法检测KA模型组和NADPH处理组原代神经元的存活率,观察KA对原代神经元的损伤作用以及NADPH对KA介导的兴奋性毒性损伤的保护作用;通过超氧化物阴离子荧光探针(Dihydroethidium, DHE)标记,检测KA模型组和NADPH处理组的原代神经元活性氧水平的变化;通过Western Blot检测KA模型组和NADPH处理组的自噬相关蛋白水平的变化,包括LC3和p62。结果:本实验成功建立了大鼠神经兴奋性毒性的体外模型。CCK-8结果显示,KA介导的兴奋性毒性引起神经元损伤,并且具有一定的时间依赖性,NADPH能显著增加神经元的存活率。DHE结果显示,KA引起细胞内ROS水平显著增加,给予NADPH能有效降低ROS水平。Western Blot结果显示,KA引起p62蛋白表达下调和LC3-II/LC3-I蛋白表达上调,NADPH可抑制KA引起的以上蛋白的表达变化。结论:在KA介导的神经兴奋性毒性体外模型中,NADPH通过抑制细胞内的氧化应激和自噬,从而发挥保护神经元的作用。
关键词: 药理学 NADPH Kainic Acid 神经兴奋性毒性 自噬
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The protective effect of NADPH on KA-induced excitotoxicity and its mechanisms
Abstract:Aim: To investigate the effects and mechanisms of NADPH on KA-induced excitotoxicity and its mechanisms in cultured primary cortical neurons.Methods: In vitro, the cytotoxicity of KA and neuroprotective effects of NADPH was assessed by CCK-8 to evaluate the damage of KA on primary neurons and the protective effect of NADPH. DHE fluorescence probe was used to detect the intracellular ROS levels. The changes of protein expression levels of LC3-II/LC3-I and p62protein levels were observed by Western blot analysis.Results: In this study, we have successfully established the in vitro excitoxic model by KA. CCK-8 results showed that KA decreased the cell viability in a time-dependent manner while NADPH could significantly help neurons to survive. KA increased the intracellular levels of ROS, but pretreatment of NADPH effectively reversed it. NADPH inhibited KA-induced down-regulation of p62 and up-regulation of ratio of LC3-II/LC3-Iin primary neurons by Western blot analysis.Conclusions: Our data provide a possible mechanism that NADPH ameliorated KA-induced excitotoxicity by blocking autophagy event in the primary cortical neurons.
Keywords: Pharmacology NADPH Kainic Acid Excitotoxicity Autophagy
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