文献类型：期刊文献

中文题名：黑逍遥散调控NOX2/ROS/NF-κB信号通路干预AD模型小鼠小胶质细胞极化

英文题名：Heixiaoyao Powder interferes with microglia polarization in AD model mice by regulating NOX2/ROS/NF-κB signaling pathway

作者：李明成[1];周君[1];胡韵韵[1];孟志鹏[1];吕育洁[1];王虎平[1,2]

第一作者：李明成

机构：[1]甘肃中医药大学,甘肃兰州730000;[2]甘肃省中医方药挖掘与创新转化重点实验室,甘肃省中药新产品创制工程实验室,甘肃兰州730000

第一机构：甘肃中医药大学

年份：2023

卷号：48

期号：15

起止页码：4027

中文期刊名：中国中药杂志

外文期刊名：China Journal of Chinese Materia Medica

收录：CSTPCD;;Scopus;北大核心:【北大核心2020】；CSCD:【CSCD2023_2024】；PubMed;

基金：国家自然科学基金项目(81960828);兰州市科技发展指导性计划项目(2020-ZD-53)。

语种：中文

中文关键词：阿尔茨海默病;黑逍遥散;小胶质细胞极化;免疫炎症;NOX2/ROS/NF-κB信号通路

外文关键词：Alzheimer′s disease;Heixiaoyao Powder;microglia polarization;immune inflammation;NOX2/ROS/NF-κB signaling pathway

摘要：基于氧化酶2(NADPH oxidase 2,NOX2)/活性氧簇(reactive oxygen species,ROS)/核转录因子κB(nuclear factor kappaB,NF-κB)信号通路探究黑逍遥散对APP/PS1双转基因小鼠小胶质细胞(microglia,MG)极化的影响及作用机制。4月龄雄性APP/PS1双转基因小鼠50只,随机分为模型组、MCC950组(10 mg·kg-1)及黑逍遥散低、中、高剂量组(6.45、12.89、25.78 g·kg-1),同月龄、同系种雄性C57BL/6J小鼠30只,随机分为空白组、空白灌胃干预组和空白腹腔注射组,各组给药干预90 d。Morris水迷宫检测学习认知能力,尼氏染色和透射电镜观察海马神经元病理形态和超微结构,免疫荧光检测小胶质细胞M1型标志物CD16/32+/Iba-1^(+)、M2型标志物CD206^(+)/Iba-1^(+)的阳性表达及海马ROS表达情况,比色法检测海马丙二醛(malondialdehyde,MDA)和超氧化物歧化酶(superoxide dismutase,SOD)含量,酶联免疫吸附法检测海马白细胞介素-6(interleukin-6,IL-6)、白细胞介素-8(interleukin-8,IL-8)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)炎症因子含量,蛋白免疫印迹检测海马β-淀粉样蛋白(β-amyloid protein,Aβ)、Iba-1、CD16/32、CD206、NOX2、NF-κB、p-NF-κB、核因子κB抑制蛋白α(NF-κB inhibitor alpha,IκBα)和p-IKBα蛋白表达。结果显示,与空白组比较,模型组小鼠目标象限运动距离、逃避潜伏期显著延长(P<0.01),目标象限滞留时间及百分比显著缩短(P<0.01);神经元细胞排列紊乱,出现肿胀、核消失或偏置,细胞数量明显减少,尼氏小体溶解甚至消失,胞质呈透亮区域;细胞膜出现缺损、皱缩,形态异常,胞质中少见细胞器,线粒体明显肿大,数量减少;小胶质细胞M1型标志物CD16/32+/Iba-1^(+)显著升高(P<0.01),M2型标志物CD206^(+)/Iba-1^(+)显著降低(P<0.01),ROS活性和MDA含量显著升高(P<0.01),SOD水平显著降低(P<0.01),炎症因子IL-6、IL-8、TNF-α含量显著升高(P<0.01);Aβ、CD16/32、Iba-1、NOX2、NF-κB和IKBα蛋白表达及磷酸化显著升高(P<0.01),CD206显著降低(P<0.01)。空白组与空白灌胃干预组、空白腹腔注射组差异无统计学意义。黑逍遥散干预后,各剂量组小鼠目标象限运动距离和逃避潜伏期显著缩短(P<0.01),目标象限滞留时间及百分比显著延长(P<0.01);细胞数量明显增多,排列较为整齐,肿胀明显缓解,尼氏小体增加;细胞形态较为规则,胞膜较为完整,线粒体肿胀明显缓解,但仍有部分内质网轻度扩张;M1型标志物CD16/32+/Iba-1^(+)显著降低(P<0.05或P<0.01),M2型标志物CD206^(+)/Iba-1^(+)显著升高(P<0.01),ROS活性、MDA含量显著降低(P<0.01),SOD水平显著升高(P<0.01),炎症因子IL-6、IL-8、TNF-α含量显著降低(P<0.01);Aβ、CD16/32、Iba-1、NOX2、NF-κB、IKBα蛋白及其磷酸化显著降低(P<0.01),CD206显著升高(P<0.01)。综上所述,黑逍遥散能够缓解神经元损伤,提高APP/PS1小鼠学习记忆能力,作用机制可能与抑制NOX2/ROS/NF-κB信号通路,调节MG极化,增加M2型表达,抑制M1型表达,减少炎症因子释放有关。
The effect and mechanism of Heixiaoyao Powder on the polarization of microglia(MG)in APP/PS1 double transgenic mice were explored based on NADPH oxidase 2(NOX2)/reactive oxygen species(ROS)/nuclear factor kappaB(NF-κB)signaling pathway.Fifty 4-month-old male APP/PS1 mice were randomly divided into a model group,an MCC950 group(10 mg·kg^(-1)),and low-,medium-,and high-dose Heixiaoyao Powder groups(6.45,12.89,and 25.78 g·kg^(-1)).Thirty male C57BL/6J mice of the same age and strain were randomly divided into a blank group,a blank+intragastric intervention group,and a blank+intraperitoneal injection group.Drug intervention lasted 90 days.Morris water maze test was used to detect learning and cognitive ability.Nissl staining and transmission electron microscopy were used to observe the pathological morphology and ultrastructure of hippocampal neurons.Immunofluorescence was used to detect the positive expression of M1-type marker CD16/32^(+)/Iba-1^(+),M2-type marker CD206^(+)/Iba-1^(+)of MG and the expression of hippocampal ROS.The colorimetric method was used to detect the content of malondialdehyde(MDA)and superoxide dismutase(SOD)in the hippocampus.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory factors,including interleukin-6(IL-6),interleukin-8(IL-8),and tumor necrosis factor-α(TNF-α),in the hippocampus.Western blot was used to detect the protein expression ofβ-amyloid protein(Aβ),Iba-1,CD16/32,CD206,NOX2,NF-κB,p-NF-κB,NF-κB inhibitor alpha(IκBα),and p-IKBαin the hippocampus.The results showed that as compared with the blank group,the model group showed prolonged target quadrant movement distance and escape latency(P<0.01),shortened target quadrant retention time and percentage(P<0.01),disorganized neuronal cells with swelling,nuclear disappearance or bias,reduced number of cells,dissolved or absent Nissl bodies,and a clear area in the cytoplasm,damaged and shrunk cell membrane with abnormal cell morphology,few organelles in the cytoplasm,reduced and swollen mitochondria,increased MG M1-type marker CD16/32^(+)/Iba-1^(+)(P<0.01),decreased M2-type marker CD206^(+)/Iba-1^(+)(P<0.01),increased ROS activity and MDA content(P<0.01),decreased SOD level(P<0.01),elevated inflammatory factors IL-6,IL-8,and TNF-α(P<0.01),up-regulated protein expression and phosphorylation of Aβ,CD16/32,Iba-1,NOX2,NF-κB,and IKBα(P<0.01),and down-regulated CD206(P<0.01).There was no statistically significant difference between the blank group,the blank+intragastric intervention group,and the blank+intraperitoneal injection group.After the intervention of Heixiaoyao Powder,the Heixiaoyao Powder groups showed shortened target quadrant movement distance and escape latency(P<0.01),prolonged target quadrant retention time and percentage(P<0.01),increased and neatly arranged cells with relieved swelling,increased Nissl bodies,regular cell morphology,and intact cell membrane,relieved swelling of mitochondria,slightly expanded endoplasmic reticulum,decreased CD16/32^(+)/Iba-1^(+)(P<0.05 or P<0.01),increased CD206^(+)/Iba-1^(+)(P<0.01),decreased ROS activity and MDA content(P<0.01),increased SOD level(P<0.01),decreased content of inflammatory factors IL-6,IL-8,and TNF-α(P<0.01),down-regulated protein expression and phosphorylation of Aβ,CD16/32,Iba-1,NOX2,NF-κB,and IKBα(P<0.01),and up-regulated CD206(P<0.01).In conclusion,Heixiaoyao Powder can alleviate neuronal damage and improve the learning and memory abilities of APP/PS1 mice.The mechanism of action may be related to the inhibition of NOX2/ROS/NF-κB signaling pathway,regulating the polarization of MG,increasing the expression of M2 type,inhibiting the expression of M1 type,and reducing the release of inflammatory factor.