虾青素干预脂多糖介导原代小胶质细胞氧化应激反应的初步研究

Astaxanthin Modulates Oxidative Stress in Primary Microglia Mediated by Lipopolysaccharide

目的:诱导型一氧化氮合酶(Inducible Nitric Oxide Synthase,iNOS)生成增加,是检测原代小胶质细胞激活产生氧化应激反应的有效端点(endpoint)之一,也是阿尔茨海默病等神经变性疾病(neurodegenerative diseases)中发病机理构成之一。本研究拟探索天然强抗氧化剂虾青素(Astaxanthin,AST)是否对脂多糖(Lipopolysaccharide,LPS)介导Sprague Dawley(SD)大鼠原代小胶质细胞激活有抗氧化作用。方法:首先,采用Shake-off法分离原代小胶质细胞,用LPS介导原代小胶质细胞激活建立细胞氧化应激反应模型,经免疫荧光鉴定原代小胶质细胞,经免疫印迹法检测iNOS的表达。其次,应用该模型对AST是否在LPS介导原代小胶质细胞激活有调节作用进行初步探索。结果:使用CD11b特异性抗体确证了原代小胶质细胞,LPS介导原代小胶质细胞获得iNOS上调,与对照组相比,iNOS上调有差异显著性(P<0.05),提示原代小胶质细胞氧化应激反应模型成功建立;用(12.5,25和50μg/ml)三个浓度的AST预处理细胞24h后,使LPS分别介导原代小胶质细胞12h、24h或36h产生iNOS下调,36h下调最为明显,与LPS相比呈现差异显著性(P<0.05)。结论:LPS介导SD大鼠原代小胶质细胞出现iNOS上调的氧化应激反应;AST使LPS介导原代小胶质细胞激活产生的iNOS下调。本研究提示,AST对阿尔茨海默病等一类神经变性疾病中小胶质细胞激活的氧化应激反应有抗氧化作用,有可能成为临床上有价值的候选药物。

Objective:The increased production of Inducible Nitric Oxide Synthase(iNOS),one of the pathogenesis components of Alzheimer's disease and other neurodegenerative diseases,could be detected as one of endpoints in the oxidative-stress responses caused by the activation of primary microglia.Our aim was to explore the anti-oxidative effect of Astaxanthin(AST)on the primary microglia,derived from neonatal rats of Sprague Dawley(SD),mediated by Lipopolysaccharide(LPS).Methods:First,primary microglia were isolated from primary mixed glia by Shake-off method,and stimulated by LPS.The primary microglia were identified through immunofluorescence while iNOS was measured by Western Blotting analysis.Secondly,this model was used to preliminarily explore the action of AST in the activated primary microglia induced by LPS.Results:Primary microglial cultures were confirmed using a specific CD11b antibody.iNOS was upregulated in the cells induced by LPS.The upregulation of iNOS was significantly different to that in the control(P<0.05),which suggested that the activated microglial model with oxidative stress be successfully established.Furthermore,12.5,25 or 50μg/ml of AST were pre-treated to the microglia for 24h before the application of LPS,which led to iNOS down-regulation at 12h,24h or 36h,and the significant difference in the latter by comparing to that in LPS alone(P<0.05).Conclusion:LPS led to the oxidative stress like iNOS upregulation in rat primary microglia.AST could down-regulated the oxidative stress such as iNOS in the microglia induced by LPS.This study suggested that AST can be with an antioxidative effect on the microglial activation in Alzheimer's disease and other neurodegenerative diseases.It may potentially be a valuable drug candidate in the clinics.