体积调节性氯离子通道阻滞剂NPPB对低渗条件下小胶质细胞活化的影响  

The effect of NPPB on microglia activation induced by hypotonic solutions in vitro

作　　者：刘阳[1] 骆翔[1] 王伟[1] 喻志源 Liu Yang;Luo Xiang;Wang Wei(Department of Neurology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030)

机构地区：[1]华中科技大学同济医院学院附属同济医院神经内科,武汉430030

出　　处：《卒中与神经疾病》2020年第3期287-292,共6页Stroke and Nervous Diseases

基　　金：湖北省自然科学基金(2017CFB705、2019CFB678);国家自然科学基金(No.81501020)。

摘　　要：目的探讨体积调节性氯离子通道(volume regulated anion channel, VRAC)阻滞剂NPPB(5-nitro-2-3-phenylpropylamino benzoic acid)对低渗状态下小胶质细胞活化的影响及机制。方法采用小胶质细胞系BV2细胞,分为对照组、低渗组、低渗+NPPB干预组,低渗+U0126组,通过LDH试剂盒检测NPPB对BV2细胞的毒性作用;采用免疫荧光技术和FITC标记鬼笔环肽染色检测BV2细胞形态变化及细胞面积;western blot检测BV2细胞MAPK信号通路的变化。结果 50μmol和100μmol的NPPB对BV2细胞无明显毒性作用。与对照组比较,低渗干预后BV2细胞迅速由静息状态下的分支状转变为活化的阿米巴状,IBA1表达显著增加,细胞面积显著增大;NPPB显著逆转低渗条件下BV2细胞形态变化,减少IBA1表达及细胞面积(P<0.05);与对照组比较,低渗干预显著增加p-Erk蛋白表达(P<0.05),而Erk、p-Jnk及p-p38蛋白表达水平无明显差异(P>0.05),NPPB显著降低BV2细胞p-Erk蛋白表达水平(P<0.05);Erk选择性抑制剂U0126干预后低渗状态下BV2细胞的面积显著减小(P<0.05)。结论低渗条件可诱导BV2细胞活化,VRAC阻滞剂NPPB可抑制低渗诱导的BV2细胞活化,Erk信号通路可能参与了VRAC介导的BV2细胞活化。Objective To investigate the effect of volume regulated anion channel inhibitor NPPB on microglia activation and its downstream mechanism induced by hypotonic solutions in vitro.Methods Microglia cell line BV2 cells were randomly divided into different groups:control group, hopotonic group, hopotonic+NPPB group and hopotonic +U0126 group.LDH assay was used to evaluate the cytotoxicity of NPPB on BV2. Microglia morphology change and IBA1 expression were investigated by immunofluorescence staining. Fluorescein phalloidin was used to quantify cell area. Western blot was used to analyze the key proteins expression level in MAPK signaling trasduction pathway. Results LDH cytotoxicity test indicated that there was no obvious toxic effect on BV2 cells under 50 μmol and 100 μmol NPPB. BV2 cells were activated and displayed round or amoeboid shape in hypotonic group, as compared with resting BV2 cells with small and branch-like shape. In parallel with morphological activation, cell size and immunoreactivity of IBA1 increased in hypotonic group. After NPPB treatment, prevention of morphology changes induced by hypotonic solution was observed. Meanwhile, compared with hypotonic treatment group, average cell size decreased and IBA1 expression reduced in NPPB treatment group. Compared with control group, the expression level of p-Erk increased remarkably(P<0.05), while the expression levels of Erk, p-Jnk and p-p38 were not statistically different in hypotonic group(P>0.05). The expression level of p-Erk in hypotonic group was attenuated in 100μmol NPPB group significantly(P<0.05)and pretreatment with Erk selective inhibitor U0126 reduced the cell size under hypotonic condition.Conclusion VRAC inhibitor NPPB inhibited microglia activation and Erk signaling might be the underlying downstream mechanism.

关 键 词：体积调节性氯离子通道 小胶质细胞 低渗 NPPB 

分 类 号：R742[医药卫生—神经病学与精神病学]