丁苯酞对脑出血后小胶质细胞极化的影响及其机制  

Effects of butyphthalide on microglia polarization after intracerebral hemorrhage and the underlying mechanisms

作　　者：张伊柳 卢伟[1] 许念桂[1] ZHANG Yiliu;LUWei;XU Niangui(Department of Neurology,Second Xiangya Hospital,Central South University,Changsha 410011,China)

机构地区：[1]中南大学湘雅二医院神经内科,长沙410011

出　　处：《中南大学学报（医学版）》2022年第6期717-729,共13页Journal of Central South University ：Medical Science

基　　金：湖南省教育厅创新研发课题[(2021)23]。

摘　　要：目的:脑出血(intracerebral hemorrhage,ICH)具有较高的发病率、致残率及病死率,寻找新的有效的ICH治疗方法具有重要意义。本文旨在探讨丁苯酞(butyphthalide,NBP)对ICH后继发性损伤和小胶质细胞极化的作用和机制。方法:将48只健康雄性Sprague-Dawley大鼠随机分为6组,分别为假手术24 h组、假手术72 h组、ICH24 h组、ICH 72 h组、ICH+NBP 24 h组、ICH+NBP 72 h组。术后评估神经功能缺损,采用蛋白质印迹法检测各组Toll样受体4(Toll-like receptor 4,TLR4)/核因子-κB(nuclear factor-kappa B,NF-κB)、核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、水通道蛋白4(aquaporin-4,AQP4)、紧密连接蛋白、小胶质细胞不同表型的表达;采用ELISA检测炎症因子;采用免疫荧光染色检测小胶质细胞的极化情况。结果:1)与假手术组相比,ICH组中血肿周围脑组织TLR4(24 h:P<0.05;72 h:P<0.01)、NF-κB(均P<0.01)、Nrf2(均P<0.01)表达均增加,小胶质细胞激活增加(均P<0.01),血肿周围组织的促炎症因子IL-6(24 h:P<0.05;72 h:P<0.01)、TNF-α(均P<0.01)表达均增加,而抗炎因子IL-4表达均减少(均P<0.01),同时AQP4表达均增加(均P<0.01),紧密连接蛋白闭锁小带蛋白1(zonula occludens-1,ZO-1)与闭合蛋白(occludin)表达均下降(均P<0.01),大鼠的神经功能均受损(均P<0.01)。2)与ICH组相比,ICH+NBP组的血肿周围组织TLR4(均P<0.05)及NF-κB(均P<0.01)表达均下降,Nrf2表达上升(均P<0.05),M1型小胶质细胞标志物CD86表达均下降(均P<0.05),M2型小胶质细胞标志物CD206表达均上升(均P<0.01)。3)在炎症反应方面,与ICH组同时间点相比,ICH+NBP组IL-4表达均增加(24 h:P<0.05;72 h:P<0.01);ICH+NBP 72 h组IL-6较ICH 72 h组下降(P<0.01);ICH+NBP 24 h组的AQP4表达较ICH 24 h组明显下降(P<0.05),ICH+NBP 72 h组其表达与ICH 72 h组相比有下降趋势,但差异无明显统计学意义(P>0.05)。4)ICH+NBP组ZO-1表达较ICH组上升(24 h:P<0.05;72 h:P<0.01);与ICH组相比,ICH+NBP�Objective:Because intracerebral hemorrhage(ICH)has high morbidity,disability and mortality,it is significant to find new and effective treatments for ICH.This study aims to explore the effect of butyphthalide(NBP)on neuroinflammation secondary to ICH and microglia polarization.Methods:A total of 48 healthy male SD rats were randomly divided into 6 groups:a sham24 h group,a sham 72 h group,an ICH 24 h group,an ICH 72 h group,an ICH+NBP 24 h group,and an ICH+NBP 72 h group(8 rats per group).After operation,the neurological deficiencies were assessed based on improved Garcia scores and corner test.The expressions of Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB),nuclear factor erythroid 2-related factor 2(Nrf2),aquaporin-4(AQP4),zonula occludens-1(ZO-1),occludin,CD68,CD86,and CD206 were observed by Western blotting.Inflammatory cytokines were detected by ELISA.The immunofluorescence was to detect the polarization of microglia.Results:1)Compared with the sham groups,the expression of TLR4(24 h:P<0.05;72 h:P<0.01),NF-κB(both P<0.01)and Nrf2(both P<0.01)in the perihematoma of the ICH group was increased,leading to microglia activation(P<0.01).The expressions of IL-6(24 h:P<0.05;72 h:P<0.01)and TNF-α(both P<0.01),the pro-inflammatory cytokines were up-regulated,and the expression of anti-inflammatory cytokine IL-4 was downregulated(both P<0.01).Besides,the expression of AQP4 was enhanced(both P<0.01).The protein level of tightly connected proteins(including ZO-1,occludin)was decreased(all P<0.01).The neurological function of the rats in the ICH group was impaired in the 2time points(both P<0.01).2)Compared with the sham group at 24 h and 72 h after the intervention of NBP,the expressions of TLR4(both P<0.05)and NF-κB(both P<0.01)were significantly declined,and the expression of Nrf2 was further enhanced(both P<0.05)in the perihematoma of the ICH+NBP group.Furthermore,the expression of M1 microglia marker was inhibited(P<0.05),and the polarization of microglia to the M2 phenotype was promoted(P<0.01).3)In t

关 键 词：脑出血 Toll样受体4 核因子-ΚB 核因子E2相关因子2 小胶质细胞极化 

分 类 号：R743.34[医药卫生—神经病学与精神病学]