基于Toll样受体4/核因子-κB信号通路探讨逆灸调控阿尔茨海默病大鼠小胶质细胞极化的机制  

Mechanisms of moxibustion preconditioning underlying improving learning-memory ability by regulating polarization of microglia via TLR4/NF-κB signaling pathway in AD rats

作　　者：宋宗胜 李真 王玉 李梦醒[1] 刘箐 史可鉴 姚晓雯 丁慧 李斯亮 唐巍[1] SONG Zong-sheng;LI Zhen;WANG Yu;LI Meng-xing;LIU Qing;SHI Ke-jian;YAO Xiao-wen;DING Hui;LI Si-liang;TANG Wei(College of Acupuncture-moxibustion,Anhui University of Chinese Medicine,Hefei 230031,China;School of Physical Education,Anqing Normal University,Anqing 246133,Anhui Province;Nursing School of Anhui SanlianCollege,Hefei 231602)

机构地区：[1]安徽中医药大学针灸推拿学院,合肥230031 [2]安庆师范大学体育学院,安徽安庆246133 [3]安徽三联学院护理学院,合肥231602

出　　处：《针刺研究》2023年第6期525-532,共8页Acupuncture Research

基　　金：国家自然科学基金项目(No.81874500);安徽高校自然科学研究项目(No.KJ2020A0377)。

摘　　要：目的:观察逆灸对阿尔茨海默病(AD)大鼠学习记忆能力、Toll样受体4(TLR4)/核因子-κB(NF-κB)信号通路相关蛋白及小胶质细胞的影响,探讨逆灸防治AD的作用机制。方法:SD大鼠随机分为正常组、假手术组、模型组和逆灸组,每组9只。逆灸组予“百会”“肾俞”“足三里”逆灸干预,每穴15min,1次/d,6d为1个疗程,共3个疗程。逆灸结束后于大鼠双侧海马注射β-淀粉样蛋白(Aβ)分子片段Aβ25-35聚集液制备AD大鼠模型。Morris水迷宫实验检测大鼠空间学习记忆能力,透射电镜观察大鼠海马组织神经元超微结构,HE染色法观察大鼠海马组织病理学变化,Westernblot法检测大鼠海马组织TLR4、NF-κBp65蛋白表达量,免疫荧光染色法检测大鼠海马CA1区离子钙结合配体分子-1(Iba-1)、分化簇80(CD80)、分化簇206(CD206)阳性表达,ELISA法检测大鼠海马组织炎性因子白细胞介素(IL)-1β、肿瘤坏死因子-α(TNF-α)及IL-10的含量。结果:与假手术组比较,模型组大鼠逃避潜伏期延长(P<0.01),穿越原平台次数减少(P<0.01);海马神经元肿胀变形,细胞核形态改变,大量细胞膜破裂,线粒体数量减少,内质网扩张,结构改变,甚至见基质有空泡,核质不易区分,细胞间隙增大;海马组织中TLR4、NF-κBp65蛋白表达量升高(P<0.01);海马CA1区Iba-1、CD80阳性表达升高(P<0.01),CD206阳性表达降低(P<0.01);海马组织中IL-1β、TNF-α含量升高(P<0.01),IL-10含量降低(P<0.01)。与模型组比较,逆灸组逃避潜伏期缩短(P<0.01),穿越原平台次数增加(P<0.01);神经元水肿程度减轻,细胞核清晰可见,核糖体、线粒体数量增多,内质网扩张程度减轻;海马组织中TLR4、NF-κBp65蛋白表达量降低(P<0.05);海马CA1区Iba-1、CD80阳性表达降低(P<0.01),CD206阳性表达升高(P<0.01);海马组织中IL-1β、TNF-α含量降低(P<0.01),IL-10含量升高(P<0.01)。结论:逆灸“百会”“肾俞”“足三里”可抑制AD大鼠小胶质细胞Objective To observe the effect of moxibustion preconditioning on learning-memory ability,Toll like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)signal pathway related proteins and microglia in rats with Alzheimer's disease(AD),so as to explore its possible mechanisms underlying improvement of AD.Methods Male SD rats were randomly divided into normal,sham operation,AD model and pre-moxibustion groups,with 9 rats in each group.Moxibustion was applied to“Baihui”(GV20),“Shenshu”(BL23)and“Zusanli”(ST36)for 15 min,once daily,6 days as a course of treatment for 3 courses.At the end of moxibustion,the AD model was established by injection of Aβ_(25-35)aggregation solution into the bilateral hippocampus.The sham operation group was only injected with the same amount of 0.9%Nacl solution.The spatial learning-memory ability of rats was detected by Morris water maze test,the ultrastructure of hippocampal neurons was observed by transmission electron microscope(TEM).The histopathological changes of hippocampus tissue were observed by HE staining,and the protein expression levels of TLR4 and NF-κB p65 in the hippocampus detected by Western blot,and the positive expressions of Iba-1,CD80 and CD206 in the hippocampal CA1 region were detected by immunofluorescence labeling.The contents of inflammatory factors IL-1β,TNF-αand IL-10 in the hippocampus were measured by ELISA.Results Compared with the sham operation group,the escape latency was significantly increased(P<0.01),and the number of platform quadrant crossing times was decreased(P<0.01)in the model group.In comparison with the model group,the increased escape latency and the decreased platform quadrant crossing times were reversed in the pre-moxibustion group(P<0.01).TEM and light microscope observation showed loose arrangement of cells,enlarged cell space,degeneration,swelling and deformation of hippocampal neurons,rupture of membranes of a large number of cells,reduction of mitochondria,dilation of endoplasmic reticulum,and matrix vacuoles,uneven distri

关 键 词：阿尔茨海默病 逆灸 学习记忆能力 TOLL样受体4 核因子-κB 小胶质细胞 炎性因子 

分 类 号：R245.81[医药卫生—针灸推拿学]