磷脂酰肌醇3-激酶通路对脂多糖预激动小胶质细胞高迁移率族蛋白1表达的影响  

Effects of phosphoinositide 3-kinase pathway on the expression of high mobility group box protein-1 from microglia with lipopolyssacride-pretreatment

作　　者：谢泽锋[1] 丁任重[1] 辛岗[2] 李苹[2] 陈城[2] 李康生[2] 苏芸 

机构地区：[1]汕头大学医学院第一附属医院心胸外科,515041 [2]汕头大学医学院微生物学与免疫学教研室,515041

出　　处：《国际免疫学杂志》2017年第3期233-237,共5页International Journal of Immunology

基　　金：国家自然科学基金项目（81001340、81471622）;广东省医学科研基金项目（A2015211）;广东省优秀青年教师培训计划（Yq2013079）;广东省高水平大学建设项目子项目（2015013）;汕头大学创新强校青年教师人才培育项目（2016）

摘　　要：目的 探讨磷酸肌醇-3激酶/蛋白激酶B（phosphoinositide 3-kinase/protein kinaseB,P13K/Akt）通路对脂多糖 （lipopolyssacride,LPS）预处理体外培养大脑皮质小胶质细胞高迁移率族蛋白1（high mobility group box-1protein 1,HMGB-1）表达的影响.方法 体外分离纯化小鼠大脑皮质小胶质细胞,纯度鉴定后按未刺激对照组、0.01μg/mL LPS单次刺激组（18 h）、1μg/mL LPS刺激组（24 h）、预处理组、PI3K/AKT通路抑制组（2 h）分别进行处理.收获各组细胞与培养上清,采用酶联免疫吸附试验（Enzyme linked immunosorbent assay,ELISA）法检测肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）分泌水平,免疫荧光法检测HMGB-1在细胞中的定位情况,Western blot 检测 HMGB-1和磷酸化Akt的表达水平.结果 PI3K/Akt通路抑制后,LPS预刺激小胶质细胞产生的TNF-α水平较1μg/mL单次刺激组、预刺激组水平降低（P=0.043;P=0.046）;磷酸化Akt表达降低,小胶质细胞HMGB-1表达以及向胞浆中转位呈现降低趋势.结论 PI3K/Akt通路在LPS预激诱导的HMGB-1活化中起正性调节作用,这些分子及调节通路的变化参与LPS耐受诱导的重新调配过程,从而共同影响小胶质细胞的最终效应.Objective To investigate effects of phosphoinositide 3-kinase/protein kinaseB （P13K/Akt） pathway on high mobility group box-1protein 1 （HMGB-1） in cortical microglia pretreated with lipopolyssacride （LPS） in vitro.Methods Mouse cortical microglia was isolated,purified and divided into five groups： unstimulated control group,0.01 μg/mL LPS stimulation group （18 h）,1 μg/mL LPS stimulation group （24 h）,pretreatment group,PI3K/AKT inhibitory group （2 h）.Cells and cultured supernatant were harvested.The secretion of tumor necrosis factor-α （TNF-α） was detected by enzyme-linked immunosorbent assay（ELISA）.Immunofluorescence was performed to measure HMGB-1 localization in cells.The expression levels of HMGB-1 and p-Akt were tested by western blot assay.Results After inhibition of PI3K/Akt pathway,the level of TNF-α was down-regulated in microglia pretreated with LPS,compared with 1 μg/mL single stimulation group and pretreatment group （P=0.043;P=0.046）.The expression of p-Akt and HMGB-1 was reduce.The function of HMGB-1 translocation was inhibited.Conclusion P13K/Akt pathway positive regulates HMGB-1 induced by LPS,is involved in the reallocation process of LPS tolerance induction,so as to influence the final effect of microglia.

关 键 词：小胶质细胞 高迁移率族蛋白1 脂多糖耐受 磷脂酰肌醇3-激酶 

分 类 号：R741[医药卫生—神经病学与精神病学]