【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 100 μmol/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-1β in 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-α and IL-1β were 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-α and 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 100 μmol/L. Treatment with LPS/Aβ1-42 upregulated the expression of Iba1，NO production，and levels of pro-inflammatory factors IL-6， TNF - α， and IL-1β in BV2 cells， while Osthole significantly inhibited the expression of these LPS/Aβ1-42-induced indicators. Meanwhile，Osthole attenuated the damage of BV2-CM on Na2 cells. The molecular docking results indicated a good binding affinity between Osthole and PKM2. Treatment with Osthole can down-regulated the levels of lactate，Pan-kla and PKM2-kla in the AD cell model. Conclusion Osthole can improve the condition of AD and reduce neuroinflammation by inhibiting the lactylation of PKM2.

R285.5

武汉市医学科研项目（编号：WZ19C29）