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（800 μg·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-α and 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-1β and 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. Results Proteomic 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 (800 μg·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-α and 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 High dose of STE extract could induce neuroinflammatory response, the mechanism of which was related to inducing microglia to polarize to M1 inflammatory phenotype and inflammatory cytokine release.

R965.3

新疆自治区重大科技专项（2023A02010-3）