蛇床子素通过调控PKM2的乳酸化修饰减轻阿尔茨海默病神经炎症  

Osthole Reducing Neuroinflammation in Alzheimer's Disease by Regulating the Lactylation of PKM2

作　　者：宋欢[1] 夏丽秀 黄雨威[1] 胡媛媛[1] SONG Huan;XIA Lixiu;HUANG Yuwei;HU Yuanyuan(Dept.of Comprehensive Pharmacy,Wuhan Hospital of Traditional Chinese Medicine,Wuhan 430010 Hubei,China;Dept.of Geriatry,Hubei Provincial Hospital of Integrated Chinese and Western Medicine,Wuhan 430010 Hubei,China)

机构地区：[1]武汉市中医医院综合药学部,湖北武汉430010 [2]湖北省中西医结合医院老年病科,湖北武汉430010

出　　处：《广州中医药大学学报》2025年第3期732-740,共9页Journal of Guangzhou University of Traditional Chinese Medicine

基　　金：武汉市医学科研项目(编号:WZ19C29)。

摘　　要：【目的】探讨蛇床子素通过调控丙酮酸激酶M2(PKM2)的乳酸化修饰对阿尔兹海默病(AD)神经炎症的影响。【方法】(1)动物实验:将18只小鼠分为野生型(WT)组、APP/PS1组和APP/PS1+蛇床子素组。比较各组学习和记忆相关生物行为学指标。免疫组织化学法检测脑组织Iba1阳性表达,酶联免疫吸附分析(ELISA)检测脑组织白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α和IL-1β水平,Weatern Blot法检测脑组织Pan乳酸化修饰(Pan-kla)以及PKM2乳酸化修饰(PKM2-kla)水平。(2)细胞实验:使用LPS/Aβ1-42处理小胶质细胞(BV2细胞)构建体外AD模型,并使用蛇床子素处理。四甲基偶氮唑盐(MTT)法检测细胞活力,Weatern Blot法检测小胶质细胞激活标志物Iba1的表达,Griess试剂评估一氧化氮(NO)的产生,ELISA法检测细胞IL-6、TNF-α和IL-1β水平。将BV2细胞条件培养基(CM)与神经母细胞瘤细胞(Na2细胞)共培养,评估蛇床子素对Na2细胞的保护作用。(3)将蛇床子素与PKM2做分子对接,并进行实验验证。【结果】动物实验中,与WT组比较,APP/PS1组小鼠学习和记忆缺陷加重,加入蛇床子素处理则改善了APP/PS1组小鼠的学习和记忆缺陷。此外,与WT组比较,APP/PS1组小鼠脑组织Iba1阳性细胞增加,促炎因子IL-6、TNF-α和IL-1β水平升高,Pan-kla以及PKM2-kla水平升高,而蛇床子素处理则抑制了上述指标水平。细胞实验中,蛇床子素在浓度不超过100µmol/L时对BV2细胞活力无明显影响。LPS/Aβ1-42处理上调BV2中Iba1的表达、NO的产生以及促炎因子IL-6、TNF-α和IL-1β水平,而蛇床子素可显著抑制LPS/Aβ1-42诱导的上述指标的表达。同时,蛇床子素减弱BV2-CM对Na2细胞的损伤。分子对接结果显示,蛇床子素与PKM2结合良好。蛇床子素处理后下调AD细胞模型中乳酸、Pan-kla和PKM2-kla的水平。【结论】蛇床子素通过抑制PKM2的乳酸化修饰减轻AD神经炎症。Objective To explore the effect of Osthole on neuroinflammation in Alzheimer s disease(AD)by regulating the lactylation of pyruvate kinase M2(PKM2).Methods(1)Animal experiments:18 mice were divided into three groups,namely wild-type(WT)group,APP/PS1 group and APP/PS1+Osthole group.Learning-and memory-related biobehavioral indicators were compared among the three groups.Immunohistochemistry was used to detect the positive expression of Iba1 in brain tissue,enzyme-linked immunosorbent assay(ELISA)was employed to detect the levels of interleukin(IL)-6,tumor necrosis factor(TNF)-α,and IL-1βin brain tissue,and Western Blot was used to detect the protein expression levels of Pan lactylation(Pan-kla)and PKM2 lactylation(PKM2-kla)in brain tissue.(2)Cell experiments:an in vitro AD model was constructed by treated in mouse microglia(BV2 cells)with LPS/Aβ1-42,and followed by treatment with Osthole.Cell viability was detected by methyl thiazolyl tetrazolium(MTT),expression of Iba1(a marker of microglial activation)was detected by Western Blot,nitric oxide(NO)production was assessed by Griess reagent,and levels of IL-6,TNF-αand IL-1βwere detected by ELISA.BV2 cell-conditioned medium(CM)was co-cultured with neuroblastoma cells(Na2 cells)to assess the protective effect of Osthole on Na2 cells.(3)Molecular docking was performed between Osthole and PKM2,and experimental verification was conducted.Results In animal experiments,deficits of learning and memory in mice were aggravated in APP/PS1 group compared with that in WT group,which were improved upon treatment with Osthole.Furthermore,the APP/PS1 group mice showed an increase in Iba1 positive cells in brain tissue,an increase in the levels of pro-inflammatory factors IL-6,TNF-αand IL-1β,as well as an increase in the levels of Pan-kla and PKM2-kla compared with the WT group,while the above indexes were inhibited by the Osthole treatment.In cell experiments,Osthole had no significant effect on BV2 cell viability at concentrations up to 100µmol/L.Treatment with LPS/Aβ1-42

关 键 词：蛇床子素 阿尔茨海默病 神经炎症 PKM2 乳酸化 小鼠 BV2细胞 

分 类 号：R285.5[医药卫生—中药学]