吸入暴露柴油机尾气通过激活神经胶质细胞引发炎症反应导致小鼠嗅球损伤
Diesel exhaust inhalation exposure induced toxicity on olfactory bulb in mice through inflammatory response mediated by activating glial cells
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摘要:
背景 空气污染与精神类疾病的发生发展有一定关系，嗅球损伤可能是这类疾病的潜在早期症状和标志，而柴油机尾气（DE）作为空气污染的主要来源之一，对嗅球的毒效应及潜在机制还有待阐明。
目的 探究DE吸入暴露对小鼠嗅球的毒性效应及机制。
方法 将40只C57BL/6小鼠随机分为4组进行全身性吸入暴露DE：对照组（洁净空气）、低剂量暴露组（750 μg·m−3）、中剂量暴露组（1500 μg·m−3）、高剂量暴露组（3000 μg·m−3），每天1 h，连续暴露28 d后处死。采用HE染色观察小鼠嗅球组织病理学改变，TUNEL荧光染色观察嗅球神经元的凋亡情况，通过京都基因与基因组百科全书（KEGG）法分析通路的富集情况。随后，采用实时荧光定量PCR（qPCR）检测炎症因子肿瘤坏死因子（TNF）-α、白介素-6（IL-6）的表达水平，通过免疫荧光染色实验观察嗅球内小胶质细胞和星形胶质细胞的激活情况。
结果 HE染色显示，小鼠经全身吸入暴露染毒DE后，随着暴露剂量的增加，暴露组小鼠嗅球组织突触小球层周围的球周细胞减少，颗粒细胞层中颗粒细胞排列紊乱。TUNEL染色发现暴露组小鼠嗅球内TUNEL阳性细胞增多，神经元凋亡数量增加，与对照组相比差异有统计学意义（
P ＜0.05）。KEGG通路富集分析发现DE暴露导致嗅球组织TNF炎症通路被显著富集。qPCR结果显示，相比对照组，高剂量暴露组IL-6相对表达量增加了340%，TNF-α相对表达量增加了67%，差异具有统计学意义（P ＜0.05）。免疫荧光结果显示，与对照组相比，DE高剂量暴露组小鼠嗅球组织中激活的小胶质细胞和星形胶质细胞数量显著增加，高剂量暴露组离子钙结合衔接分子1（IBA-1）相对荧光表达量增加120%，颗粒细胞层神经胶质纤维酸性蛋白（GFAP）相对荧光表达量增加400%，突触小球层GFAP荧光表达量增加240%，差异具有统计学意义（P ＜0.05）。结论 吸入暴露DE会激活嗅球中的神经胶质细胞包括小胶质细胞和星形胶质细胞，引发炎症通路释放炎症因子TNF-α、IL-6造成炎症反应，导致嗅球神经元凋亡。
Abstract:Background Air pollution is related to the occurrence and development of mental diseases. Olfactory bulb damage might be the potential prodromal symptom and sign of these diseases. The toxicity of diesel exhaust (DE), one of the main sources of air pollution, on olfactory bulb and the underlying mechanisms remain to be elucidated.
Objective To explore the toxicity of DE on mouse olfactory bulb and underlying mechanisms.
Methods A total of 40 C57BL/6 mice were randomly divided into four groups for exposure to DE by systemic inhalation: control group (filtered air), low exposure group (750 μg·m−3 DE), medium exposure group (1500 μg·m−3 DE), and high exposure group (3000 μg·m−3 DE). The mouse inhalation exposure to DE was performed 1 h per day for 28 d. HE staining was performed to observe pathological changes in mouse olfactory bulb tissue. TUNEL assay was used to observe apop-tosis in olfactory bulb. Kyoto Encyclopedia of Genes and Genomes (KEGG) was exhibited to explore potential mechanisms of olfactory bulb damage associated with DE. Quantitative real-time PCR (qPCR) was used to determine mRNA expression levels of inflammatory factors including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Immunofluorescence staining was conducted to observed the microglia and astrocyte activation in olfactory bulb.
Results The HE staining results showed that the number of periglomerular cells in the glomerular layer of olfactory bulb decreased in a dose-dependent manner, and the cells in the granule cell layer of olfactory bulb became disordered after DE exposure. The TUNEL staining showed that TUNEL positive cells in olfactory bulb tissue and neuronal apoptosis increased in the exposed groups compared with the control group (
P ＜0.05). The KEGG pathway analysis showed that DE associated with significant enrichment of TNF signaling pathway in olfactory bulb tissue. The qPCR results showed that the TNF-α relative expression level significantly increased by 67% and the IL-6 relative expression level by 340% in the DE high exposure dose group compared with the control group (P ＜0.05). According to the immunofluorescence staining results, the numbers of activated microglia and astrocytes in olfactory bulb tissue significantly increased in the DE high exposure group, the relative fluorescence intensity of ionized calcium binding adaptor molecule 1 (IBA-1) increased by 120%, the granule cell layer relative fluorescence intensity of glial fibrillary acidic protein (GFAP) increased by 400%, and the glomerular layer relative fluorescence intensity of GFAP increased by 240% than those in the control group (P ＜0.05).Conclusion Inhalation exposure to DE can lead to glial cell activation including microglia and astrocytes in olfactory bulb tissue by activating inflammatory pathways and releasing inflammatory factors TNF-α and IL-6, leading to neuronal apoptosis in olfactory bulb tissue.
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