目的 探討高劑量的山豆根Sophorae tonkinensis提取物（STE）活化小膠質(zhì)細(xì)胞，誘導(dǎo)小鼠神經(jīng)炎癥的神經(jīng)毒性機(jī)制。方法 采用山豆根提取物（STE，10、15 g·kg^-1）ig給予小鼠，處理14 d后，分離小鼠中腦皮層，采用Label-free蛋白質(zhì)組學(xué)技術(shù)進(jìn)行腦差異蛋白表達(dá)分析、基因本體（GO）富集分析和京都基因和基因組百科全書（KEGG）通路分析。體外實(shí)驗(yàn)采用STE處理小鼠小膠質(zhì)細(xì)胞BV2細(xì)胞，采用流式細(xì)胞術(shù)檢測細(xì)胞表面CD80、CD86和CD206表達(dá)，酶聯(lián)免疫吸附測定（ELISA）檢測細(xì)胞上清中腫瘤壞死因子-α（TNF-α）和白細(xì)胞介素（IL）-6釋放，實(shí)時(shí)熒光定量PCR（qRT-PCR）檢測TNF-α、IL-6、IL-1β和IL-4基因表達(dá)。采用qRT-PCR、Western blotting和免疫熒光染色檢測核因子-κB（NF-κB）p65的基因表達(dá)、蛋白磷酸化和核易位。結(jié)果 對(duì)小鼠中腦皮層的蛋白質(zhì)組學(xué)分析結(jié)果顯示，與對(duì)照組相比，10 g·kg^-1 STE誘導(dǎo)了109個(gè)差異表達(dá)蛋白，15 g·kg^-1 STE誘導(dǎo)了52個(gè)差異表達(dá)蛋白，2組共同表達(dá)的差異表達(dá)蛋白20個(gè)。GO和KEGG分析結(jié)果顯示2個(gè)劑量的STE均參與了化學(xué)突觸信號(hào)傳遞、神經(jīng)退行性疾病、神經(jīng)內(nèi)分泌信號(hào)通路和細(xì)胞因子調(diào)控、炎癥調(diào)節(jié)、抗感染免疫等炎性免疫反應(yīng)。體外細(xì)胞實(shí)驗(yàn)結(jié)果表明，與對(duì)照組比較，高質(zhì)量濃度STE（800 μg·mL^-1）顯著上調(diào)BV2細(xì)胞表面CD80、CD86表達(dá)（P＜0.001），顯著促進(jìn)TNF-α、IL-6的釋放和TNF-α、IL-6、IL-1β基因表達(dá)（P＜0.01、0.001）；高質(zhì)量濃度STE對(duì)NF-κB p65蛋白的磷酸化和核易位有一定的促進(jìn)作用，但無顯著性差異。結(jié)論 高劑量的STE可誘導(dǎo)神經(jīng)炎癥反應(yīng)，其機(jī)制與誘導(dǎo)小膠質(zhì)細(xì)胞向M1炎癥表型極化和炎癥細(xì)胞因子釋放有關(guān)。

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

新疆自治區(qū)重大科技專項(xiàng)（2023A02010-3）