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巨噬细胞在PM2.5暴露肺泡气血屏障损伤过程中的作用研究
Role of macrophages in pulmonary blood-air barrier impairment induced by PM2.5 exposure
- 陆军军医大学学报 2024年46卷第8期 页码：849-858
- 作者机构：
1.100124 北京，北京工业大学化学与生命科学学
2.100029 北京，中日友好医院检验科
3.100068 北京，北京食品科学研究院中国肉类食品综合研究中心
4.102600 北京，北京科兴中维生物技术有限公司
- 作者简介：
[周志祥, E-mail: zhouzhixiang@bjut.edu.cnZHOU Zhixiang, E-mail: zhouzhixiang@bjut.edu.cn
- 基金信息：国家自然科学基金面上项目(42077399)
DOI：10.16016/j.2097-0927.202402002
中图分类号： R122.7;R322.35;R392.1收稿：2024-02-01，
修回：2024-02-20，
纸质出版：2024-04-30
- 稿件说明：
移动端阅览
姚梦菲, 王国镇, 侯潇楠, 等. 巨噬细胞在PM2.5暴露肺泡气血屏障损伤过程中的作用研究[J]. 陆军军医大学学报, 2024,46(8):849-858.
Mengfei YAO, Guozhen WANG, Xiaonan HOU, et al. Role of macrophages in pulmonary blood-air barrier impairment induced by PM2.5 exposure[J]. Journal of Army Medical University, 2024, 46(8): 849-858.
姚梦菲, 王国镇, 侯潇楠, 等. 巨噬细胞在PM2.5暴露肺泡气血屏障损伤过程中的作用研究[J]. 陆军军医大学学报, 2024,46(8):849-858. DOI： 10.16016/j.2097-0927.202402002.
Mengfei YAO, Guozhen WANG, Xiaonan HOU, et al. Role of macrophages in pulmonary blood-air barrier impairment induced by PM2.5 exposure[J]. Journal of Army Medical University, 2024, 46(8): 849-858. DOI： 10.16016/j.2097-0927.202402002.
摘要
目的
2
探究巨噬细胞在细颗粒物(fine particulate matter，PM
2.5
)暴露肺泡气血屏障损伤过程中的作用。
方法
2
将18只10周龄、体质量24~27 g的雄性BALB/C小鼠随机分为(
n
=6)：对照组(气管滴注生理盐水)、PM
2.5
低剂量组与PM
2.5
高剂量组(在实验第1、4、7天气管滴注PM
2.5
样品悬液，染毒浓度分别为1.8和16.2 mg/kg体质量)，末次染毒24 h后检测PM
2.5
暴露后小鼠肺组织病理改变、支气管肺泡灌洗液(bronchoalveolar lavage fluid，BALF)中总蛋白(total protein，TP)、乳酸脱氢酶(lactate dehydrogenase，LDH)和碱性磷酸酶(a
lkaline phosphatase，AKP)释放水平以及肺组织中F4/80蛋白表达水平，观察小鼠肺泡气血屏障的损伤效应及肺组织中巨噬细胞的浸润情况。以肺泡上皮细胞A549和血管内皮细胞EA.hy926构建体外肺泡气血屏障模型，结合THP-1巨噬细胞模型，将PM
2.5
上清、巨噬细胞培养上清和PM
2.5
-巨噬细胞培养上清分别与屏障模型孵育24 h，通过检测屏障模型跨膜电阻值(transepithelial electrical resistance，TEER)、荧光素钠透过率和屏障细胞LDH释放情况，确认PM
2.5
暴露诱导屏障受损过程中巨噬细胞的促进作用；实时荧光定量PCR(quantitative real-time PCR，RT-qPCR)和酶联免疫吸附试验(enzyme-linked immunosorbent assay，ELISA)检测PM
2.5
暴露后巨噬细胞炎性细胞因子肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)、白细胞介素-1β(interleukin-1β，IL-1β)、IL-6和IL-8的表达情况，探究巨噬细胞在PM
2.5
暴露中炎性应激效应。
结果
2
PM
2.5
暴露可引起小鼠肺组织损伤，且随染毒剂量增加，BALF中TP、LDH、AKP含量及肺组织中巨噬细胞数量随之增加，PM
2.5
高剂量组与对照组相比具有显著差异(
P
<
0.01)。体外肺泡气血屏障模型暴露结果显示：与150、300 μg/mL PM
2.5
上清或巨噬细胞培养上清单独作用于屏障模型相比，将150、300 μg/mL PM
2.5
-巨噬细胞培养上清暴露于屏障后，屏障TEER显著降低(
P
<
0.01)，透过率显著增加(
P
<
0.01)，且300 μg/mL PM
2.5
-巨噬细胞上清处理的组别可以明显提高上皮和内皮屏障细胞LDH的释放(
P
<
0.01)。150、300 μg/mL PM
2.5
刺激后巨噬细胞中TNF-α、IL-1β、IL-6、IL-8表达显著增加(
P
<
0.01)。
结论
2
巨噬细胞促进PM
2.5
引起的肺泡气血屏障功能损伤。
Abstract
Objective
2
To investigate the role of macrophages in the process of fine particulate matter (PM
2.5
) exposure induced damage to pulmonary blood-air barrier.
Methods
2
Eighteen male BALB/C mice (aged of 10 weeks
weighing 24~27 g) were randomly divided into control group and low- and high-dose PM
2.5
exposure groups (receiving 1.8 and 16.2 mg/kg
respectively)
with 6 mice in each group. The control group received tracheal instillations of normal saline on days 1
4
and 7
whereas the exposure groups were administered corresponding dose of PM
2.5
exposure at the same time points. In 24 h after last exposure
pathological changes in the lung tissues were observed
and the contents of total protein (TP)
lac
tate dehydrogenase (LDH)
and alkaline phosphatase (AKP) in bronchoalveolar lavage fluid (BALF)
and F4/80 protein level in lung tissue were measured to evaluate the blood-air barrier damage and macrophage infiltration within the lung tissues. Additionally
an
in vitro
model of the blood-air barrier was established using A549 alveolar epithelial cells and EA.hy926 vascular endothelial cells. In combination with a THP-1 macrophage model
the supernatant PM
2.5
supernatant
macrophage supernatant
and PM
2.5
-macrophage supernatant were incubated with the barrier model for 24 h
respectively. Transmembrane electrical resistance (TEER)
sodium fluorescein permeability of the barrier model
and LDH release from the barrier cells were measured to ascertain the extent of macrophage-mediated enhancement in barrier damage induced by PM
2.5
exposure. Furthermore
the expression of inflammatory cytokines
such as TNF-α
IL-1β
IL-6
and IL-8 in the macrophages after PM
2.5
exposure was analyzed with quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA).
Results
2
PM
2.5
exposure induced lung tissue damage in mice in a dose-dependent manner
significantly elevated the contents of TP
LDH and AKP in the BALF and caused marked infiltration of macrophages into the lung tissue
especially the high-dose exposure when compared with the mice from the control group (
P
<
0.01).
In vitro
barrier model exposure experiments showed that in comparison with the treatment of 150 and 300 μg/mL PM
2.5
and macrophage supernatant
the same doses of PM
2.5
-macrophage supernatant resulted in notably decreased TEER and significantly enhanced permeability in the barrier model (
P
<
0.01)
and markedly increased LDH release from epithelial and endothelial barrier cells (
P
<
0.01). Additionally
the exposure of 150 and 300 μg/mL PM
2.5
led to a significant up-regulation of TNF-α
IL-1β
IL-6
and IL-8 in the macrophages (
P
<
0.01).
Conclusion
2
Macrophages deteriorate PM
2.5
-induced functional impairment of the pulmonary blood-air barrier.
关键词
Keywords
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