摘要
目的观察钠-钾-氯共转运体(Na+-K+-2Cl-cotransporter 1,NKCC1)的特异性抑制剂布美他尼(Bumetanide)对LPS(脂多糖)诱导的小胶质细胞活化及炎性因子分泌的影响。方法差速贴壁法培养大鼠原代小胶质细胞,纯化的小胶质细胞分为对照组,LPS组(1.0μg/ml)和布美他尼干预组(NKCC1抑制剂,10μM),在干预1、3、6和12 h固定细胞爬片,免疫荧光双标显示小胶质细胞活化形态;各时间点收取上层培养基,用ELISA法测定各组培养基中TNF-α和IL-1β的水平。结果 LPS干预后小胶质细胞活化,体积增大,布美他尼干预后小胶质细胞活化受到抑制。小胶质细胞分泌的TNF-α和IL-1β在LPS干预1 h时开始明显增加,TNF-α的分泌在6 h达到最高峰;IL-1β的分泌在3 h时达到最高峰(P<0.05)。布美他尼干预后与LPS组比较,TNF-α和IL-1β分泌明显减少,其中在3、6、12 h时TNF-α分泌显著降低(P<0.05),而IL-1β的分泌在1、3、6、12 h时明显降低(P<0.05)。结论布美他尼可减少LPS诱导的小胶质细胞活化及炎性因子分泌增加,提示NKCC1通路在小胶质细胞活化及炎性因子分泌中发挥了重要作用。
Objective To observe the effect of Bumetanide （NKCC1 inhibitor）on activation and inflam-matory secretion of microglia indueced by LPS.Methods Primary cultured microglias were randomly divided into three groups：control group,LPS group and LPS plus Bumetanide group.At different time points （1 h,3 h,6 h,12 h）,cell climbing piece and medium were harvested for further use.Immunofluorescence was used to detect cell morphology and activation.ELISA was used to detect the concentration of TNF-αand IL-1βin the medium.Results Compared with the control group,the concentration of TNF-αand IL-1βin LPS group were began significantly increased at 1 h.The concentration of IL-1βreached peak lever at 3 h and the TNF-αreached peak lever at 6 h （P 〈0.05）.And added with Bumetanide,TNF-αand IL-1βsecretion were reduced.TNF-αsecretion at 3 h,6 h,12 h and IL-1βsecretion at 1 h,3 h,6 h,12 h were reduced statistically difference （P 〈0.05）.Conclusions Bumetanide can reduce activation and inflammatory secretion of microglia caused by LPS in vitro.Our results suggested that NKCC1 was involved in microglia activation and inflammatory secretion.
出处
《卒中与神经疾病》
2015年第1期6-8,64,共4页
Stroke and Nervous Diseases
基金
国家自然科学基金项目(81030021
81000521)
