褪黑素通过NF-κB通路抑制脑室周边白质内星形胶质细胞活化的机制研究  

Melatonin inhibits astrocyte activation in periventricular white matter via NF-κB pathway

作　　者：江书奇 周秋萍 王慧芳 李倩[2] 刘楠 邓医宇 Jiang Shuqi;Zhou Qiuping;Wang Huifang;Li Qian;Liu Nan;Deng Yiyu(School of Medicine,South China University of Technology,Guangzhou 510006,China;Department of Emergency and Critical Care Medicine,Guangdong Provincial People's Hospital,Guangzhou 510080,China)

机构地区：[1]华南理工大学医学院,广州510006 [2]广东省人民医院急危重症医学部,广州510080

出　　处：《中华急诊医学杂志》2021年第2期173-178,共6页Chinese Journal of Emergency Medicine

基　　金：国家自然科学基金(82072230,81471237);广东省自然科学基金(2019A1515010206);广州市科技计划项目(202002030094)。

摘　　要：目的探讨褪黑素(melatonin,MEL)对脂多糖(lipopolysaccharides,LPS)诱导的SD幼鼠大脑星形胶质细胞活化及远期焦虑样行为的影响。方法将新生鼠随机(随机数字法)分为对照组(CON)组、LPS组、LPS+MEL组。LPS组:腹腔注射P1d幼鼠LPS(1 mg/kg)制作脓毒症模型,CON组仅注射等体积生理盐水,LPS+MEL组于建模0.5 h后注射MEL溶液10 mg/kg。在各组注射后不同时间点分为3 d、7 d、28 d三个亚组。免疫荧光染色和Western Blot法检测三组3 d、7 d后胼胝体内GFAP、TNF-α的表达变化;采用旷场实验检测28 d动物焦虑样行为。细胞实验以LPS(1μg/mL)诱导原代星形胶质细胞为细胞模型,随机分为对照组、LPS组、LPS+MEL组和LPS+MEL+褪黑素受体抑制剂(Luzindole,LUZ)组,采用免疫荧光观察各组GFAP、TNF-α表达情况,Western Blot法检测GFAP、TNF-α、p-NF-κBp65、NF-κBp65蛋白表达,荧光定量PCR检测对照组、LPS组、LPS+MEL组神经营养因子GDNF、BDNF mRNA的表达。计量资料多组间比较采用单因素及双因素方差分析,以P<0.05为差异有统计学意义。结果与CON组相比,LPS组中央区活动时间、中央路程/总路程比例下降,MEL干预可明显改善LPS组大鼠焦虑样行为;与LPS组相比,MEL组可降低LPS注射后3 d、7 d胼胝体内GFAP、TNF-α的表达(P<0.05)。与LPS刺激组相比,MEL可降低星形胶质细胞GFAP、TNF-α、p-NF-κBp65的表达上调(P<0.05),该效应可被LUZ所阻断(P<0.05)。此外,与LPS组相比,MEL预处理原代星形胶质细胞可逆转LPS诱发的GDNF、BDNF表达下调(P<0.05)。结论褪黑素可改善LPS诱导的脓毒症新生鼠远期焦虑样行为,其作用机制可能是通过NF-κB途径抑制星形胶质细胞活化及炎症反应有关。Objective To investigate the effect of melatonin(MEL)infl uence on lipopolysaccharide(LPS)-induced long-term anxiety-like behavior and activation of astrocytes in septic neonatal rats.Methods Sprague-Dawley rats were randomly(random number)assigned to the control group,LPS group and LPS+MEL group.Sepsis model was intraperitoneally injected with LPS(1 mg/kg),and neonatal rats in the MEL group were administered with MEL(10 mg/kg)30 min after LPS injection.At different time points after injection,rats in each group were divided into three subgroups:3 d,7 d and 28 d.The expression of GFAP and TNF-αin the corpus callosum was detected by immunofl uorescence staining and Western blot.Open-fi eld test was applied to observe anxiety-like behaviors.In vitro,cultured neonatal SD rat astrocytes were divided into the control group,LPS group,LPS+MEL group,and LPS+MEL+luzindole group.Immunofl uorescence staining was used to observe the expression of GFAP and TNF-α.Expression of GFAP,TNF-α,p-NF-κBp65,NF-κBp65 protein in astrocytes were assessed by Western blot.RT-qPCR was used to investigate the mRNA expression of GDNF and BDNF.One-way ANOVA and two-way ANOVA were used for comparison of multiple groups of variables.A P<0.05 was considered statistically signifi cant.Results LPS reduced the duration of movement in the central area and distance in the central area/total distance in open-fi eld test,while melatonin evidently reversed the LPS-induced anxiety-like behavior.Compared with the LPS group,the expressions of GFAP and TNF-αwere signifi cantly decreased in the corpus callosum at 3 d and 7 d in the MEL group(P<0.05).Compared with the LPS group,MEL could signifi cantly decrease the expression of GFAP,TNF-αand p-NF-κBp65 in astrocytes(P<0.05),which could be blocked by Luzindole.In addition,compared with the LPS group,MEL pretreatment could reverse the down regulation of GDNF and BDNF induced by LPS(P<0.05).Conclusions MEL can relieve LPS-induced long-term anxiety-like behavior in septic neonatal rats.The mechanism may be

关 键 词：褪黑素 脓毒症 星形胶质细胞 脂多糖 炎症因子 神经营养因子 NF-ΚB 焦虑 

分 类 号：R722.1[医药卫生—儿科]