山豆根激活小胶质细胞炎症诱导神经毒性的机制研究  

Mechanism of activation of microglia inflammation induced neurotoxicity by Sophorae tonkinensis

作　　者：周宏 陈硕 李博野 杨晋宁 韩韫聪 彭博[3] 胡秦[1] ZHOU Hong;CHEN Shuo;LI Boye;YANG Jinning;HAN Yuncong;PENG Bo;HU Qin(College of Chemistry and Life Science,Beijing University of Technology,Beijing 100124,China;Civil Aviation Medicine Center,Civil Aviation Administration of China,Beijing 100123,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]北京工业大学化学与生命科学学院,北京100124 [2]中国民用航空局民用航空医学中心,北京100123 [3]中国中医科学院中药研究所,北京100700

出　　处：《药物评价研究》2025年第1期100-109,共10页Drug Evaluation Research

基　　金：新疆维吾尔自治区重大科技专项(2023A02010-3)。

摘　　要：目的探讨高剂量的山豆根Sophorae tonkinensis提取物(STE)活化小胶质细胞,诱导小鼠神经炎症的神经毒性机制。方法采用山豆根提取物(STE,10、15 g·kg^(-1))ig给予小鼠,处理14 d后,分离小鼠中脑皮层,采用Label-free蛋白质组学技术进行脑差异蛋白表达分析、基因本体(GO)富集分析和京都基因和基因组百科全书(KEGG)通路分析。体外实验采用STE处理小鼠小胶质细胞BV2细胞,采用流式细胞术检测细胞表面CD80、CD86和CD206表达,酶联免疫吸附测定(ELISA)检测细胞上清中肿瘤坏死因子-α(TNF-α)和白细胞介素(IL)-6释放,实时荧光定量PCR(qRT-PCR)检测TNF-α、IL-6、IL-1β和IL-4基因表达。采用qRT-PCR、Western blotting和免疫荧光染色检测核因子-κB(NF-κB)p65的基因表达、蛋白磷酸化和核易位。结果对小鼠中脑皮层的蛋白质组学分析结果显示,与对照组相比,10 g·kg^(-1) STE诱导了109个差异表达蛋白,15 g·kg^(-1) STE诱导了52个差异表达蛋白,2组共同表达的差异表达蛋白20个。GO和KEGG分析结果显示2个剂量的STE均参与了化学突触信号传递、神经退行性疾病、神经内分泌信号通路和细胞因子调控、炎症调节、抗感染免疫等炎性免疫反应。体外细胞实验结果表明,与对照组比较,高质量浓度STE(800μg·mL^(-1))显著上调BV2细胞表面CD80、CD86表达(P<0.001),显著促进TNF-α、IL-6的释放和TNF-α、IL-6、IL-1β基因表达(P<0.01、0.001);高质量浓度STE对NF-κB p65蛋白的磷酸化和核易位有一定的促进作用,但无显著性差异。结论高剂量的STE可诱导神经炎症反应,其机制与诱导小胶质细胞向M1炎症表型极化和炎症细胞因子释放有关。Objective To explore the neurotoxic mechanism of microglia activation and neuroinflammation in mice induced by high dose of Sophorae tonkinensis extract(STE).Methods Mice were ig administered with STE(10 and 15 g·kg^(-1)).After 14 days of treatment,the mesencephalic cortex of the mice was isolated.Label-free proteomics technology was used to analyze brain differential protein expression,GO functional enrichment and KEGG pathway analysis.In vitro experiments,mouse microglia BV2 cells were treated with STE.The expressions of CD80,CD86 and CD206 on the cell surface were detected by flow cytometry,and the release of TNF-αand IL-6 in the supernatant was detected by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect TNF-α,IL-6,IL-1βand IL-4 gene expression.The gene expression,protein phosphorylation and nuclear translocation of NF-κB p65 were detected by qRT-PCR,Western blotting and immunofluorescence staining.ResultsProteomic analysis of mouse mesencephalic cortex showed that 109 differently expressed proteins were induced by 10 g·kg^(-1) and 52 differently expressed proteins were induced by 15 g·kg^(-1),and 20 different proteins were co-expressed by the two groups compared with the normal control group.The results of GO and KEGG analysis showed that two doses of extracts were involved in chemical synaptic signaling,neurodegenerative diseases,neuroendocrine signaling pathways and cytokine regulation,inflammation regulation,anti-infection immunity and other inflammatory immune responses.The results of in vitro cell experiment showed that high concentration of STE(800μg·mL^(-1))significantly up-regulated the expression of CD80 and CD86 on the surface of BV2 cells(P<0.001),promoted the release of TNF-αand IL-6 and the gene expression of TNF-α,IL-6 and IL-1β(P<0.01 and 0.001).High dose extract of STE had a certain promoting effect on the phosphorylation and nuclear translocation of NF-κB p65 protein,but there was no significant difference.Conclusion Hig

关 键 词：山豆根 小胶质细胞 神经炎症 神经毒性 M1炎症表型极化 苦参碱 

分 类 号：R965.3[医药卫生—药理学]