大气细颗粒物诱导IL-17A表达增强香烟暴露小鼠气道炎症反应的机制

钟宇; 苏国媚; 熊志林; 黄彤; 罗朝乐; 赖天文

doi:10.16766/j.cnki.issn.1674-4152.001856

大气细颗粒物诱导IL-17A表达增强香烟暴露小鼠气道炎症反应的机制

doi: 10.16766/j.cnki.issn.1674-4152.001856

广东医科大学附属医院呼吸与危重症医学科，广东湛江 524001

基金项目:

广东省基础与应用基础研究项目 2018A0303130269

广东省基础与应用基础研究项目 2020B1515020004

详细信息

通讯作者:
赖天文，E-mail：laitianwen2011@163.com

中图分类号: R563
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- 被引次数: 0
出版历程
- 收稿日期: 2020-09-05
- 网络出版日期: 2022-02-16

PM-induced IL-17A expression enhances airway inflammation in cigarette-exposed mice

Department of Respiratory and Critical Care Medicine, the Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524001, China

摘要

摘要: 目的探讨大气细颗粒物(PM)增强香烟暴露小鼠炎症反应的机制。方法用野生型(WT)及IL-17A基因敲除(IL-17A^-/-)小鼠，按随机数字表法随机分为对照组、熏烟组、PM组、熏烟+PM组，每组6~8只。采用香烟烟雾暴露装置烟熏，气道滴注方法吸入PM，观察气道炎症反应，连续干预3个月后取检。用HE检测肺组织炎症浸润、用ELISA及RT-PCR检测肺组织炎症因子表达、免疫组化检测平滑肌表达、Masson染色观察胶原沉积及细胞流式检测分泌IL-17A的淋巴细胞类型。体外实验，用不同浓度的香烟提取物(CSE)和/或大气细微颗粒(PM)刺激人气道上皮(HBE)细胞，用IL-17A siRNA转染在HBE细胞中敲除IL-17A基因，用RT-PCR检测炎症因子表达。结果与对照组、熏烟组及PM组比较，熏烟联合PM组小鼠肺组织炎症因子(CXCL1、TFG-β1、IL-6及IL-17A)、胶原沉积及平滑肌表达明显增高。相反，IL-17A^-/-小鼠能缓解上述指标。流式细胞检测发现PM主要通过调控CD4⁺细胞促进IL-17A表达，增强熏烟诱导的炎症反应。在体外，CSE、PM分别干预HBE细胞均能诱导IL-6、IL-8表达，而CSE联合PM干预HBE细胞能进一步增加IL-6、IL-8表达。敲除HBE细胞IL-17A基因后，能缓解IL-6、IL-8表达。结论 PM能诱导IL-17A表达，加剧熏烟小鼠肺组织的炎症反应、胶原沉积及平滑肌增生，提示针对IL-17A信号通路靶向治疗可能对缓解PM导致的慢性阻塞性肺疾病急性加重有效。
- 大气细颗粒物 /
- 气道炎症 /
- 白介素17A
Abstract: Objective This study aimed to investigate the mechanisms of atmospheric fine particulate matter (PM), which enhanced the inflammatory response in cigarette-exposed mice. Methods Wild-type and IL-17A knockout (IL-17A^-/-) mice were randomly divided into the control group, smoked group, PM group and smoked+PM group according to the random number table method (6-8 mice/group). Mice were exposed to smoke using cigarette smoke exposure device. PM was inhaled by airway instillation, and airway inflammation was observed. After continuous intervention for 3 months, mice were sacrificed to measure airway inflammation. HE was used to detect inflammatory infiltration in lung tissue, and ELISA and RT-PCR were used to detect inflammatory cytokines in lung tissue. Smooth muscle expression was detected by immunohistochemistry, and collagen deposition was observed by Masson staining. In addition, IL-17A-secreting lymphocytes were detected by cell flow cytometry. In in vitro experiments, different concentrations of cigarette smoke extract (CSE) and/or PM were used to stimulate human airway epithelial (HBE) cells, and IL-17A siRNA was used to knock down IL-17A gene in HBE cells. RT-PCR detects the expression of inflammatory factors. Results Compared with the control, smoked and PM groups, lung tissue inflammatory factors (CXCL1, TFG-β1, IL-6 and IL-17A), collagen deposition and smooth muscle expression were significantly increased in the smoked+PM group. By contrast, IL-17A^-/- mice can alleviate the above-mentioned indicators. Flow cytometry found that PM promoted IL-17A expression by regulating CD4 cells and worsened the inflammatory response induced by smoking. In vitro, CSE or PM intervention in HBE cells can induce the secretion of IL-6 and IL-8, whereas CSE combined with PM can further increase the expression of IL-6 and IL-8. Knockout of IL-17A gene in HBE cells can alleviate the expression of IL-6 and IL-8. Conclusion PM can induce the expression of IL-17A and exacerbate the inflammation, collagen deposition and smooth muscle hyperplasia in the lung tissue of cigarette-exposed mice, suggesting that targeted therapy for the IL-17A signalling pathway may be effective in relieving acute exacerbation of COPD caused by PM.
- Particulate matter /
- Airway inflammation /
- IL-17A

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图 1 PM增强熏烟诱导的气道炎症反应

注：A为肺泡灌洗液细胞计数; B为肺泡灌洗液中性粒细胞计数; C为用Pro 6.1软件对炎症浸润性半定量统计分析。

下载: 全尺寸图片幻灯片

图 2 PM促进熏烟诱导的IL-17A表达

注：用流式细胞分析小鼠肺组织分泌IL-17A淋巴细胞亚群，利用流式分析软件FlowJo统计分析CD4⁺、CD8⁺及RORγt⁺T细胞中IL-17A表达。

下载: 全尺寸图片幻灯片

表 1 引物序列

项目	类别
IL-1β(鼠)	Forward Primer	GAAATGCCACCTTTTGACAGTG
	Reverse Primer	TGGATGCTCTCATCAGGACAG
IL-6(鼠)	Forward Primer	CTGCAAGAGACTTCCATCCAG
	Reverse Primer	AGTGGTATAGACAGGTCTGTTGG
IL-17A(鼠)	Forward Primer	TCAGCGTGTCCAAACACTGA
	Reverse Primer	CGCCAAGGGAGTTAAAGACTT
GAPDH(鼠)	Forward Primer	GGGTTCCTATAAATACGGACTGC
	Reverse Primer	CCATTTTGTCTACGGGACGA
IL-6(人)	Forward Primer	TGATGGATGCTACCAAACTGG
	Reverse Primer	TTCATGTACTCCAGGTAGCTATGG
IL-8(人)	Forward Primer	ACTGAGAGTGATTGAGAGTGGAC
	Reverse Primer	AACCCTCTGCACCCAGTTTTC
GAPDH(人)	Forward Primer	TGTTGCCATCAATGACCCCTT
	Reverse Primer	CTCCACGACGTACTCAGCG

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参考文献(15)

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