Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, South Korea.
Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, South Korea; Sensory Organ Research Institute, Seoul National University Medical Research Center, Seoul, Republic of Korea.
Environ Pollut. 2019 May;248:736-742. doi: 10.1016/j.envpol.2019.02.082. Epub 2019 Feb 26.
Tight junctions (TJs) in the epithelium play a critical role in the formation of a paracellular epithelial barrier against the extracellular environment. Diesel exhaust particles (DEPs) disrupt the epithelial barrier. The aim of this study was to investigate how DEPs disrupt the epithelial barrier and whether Toll-like receptor 4 (TLR4) is involved in DEP-induced epithelial barrier dysfunction in primary human nasal epithelial (PHNE) cells.
PHNE cells were cultured at an air-liquid interface (ALI) to create a fully differentiated in vivo-like model of the epithelium and then exposed to DEPs (particulate matter <4 μm) or lipopolysaccharide (LPS) alone (mono-exposure) and DEPs plus LPS (co-exposure) at the apical side of the PHNE. TJ formation and integrity were monitored by measuring transepithelial electric resistance (TEER) and fluorescently labeled dextran permeability. The expression of TJ proteins was assessed by confocal microscopy and a biochemical assay.
PHNE cell viability was reduced in a time- and dose-dependent manner following DEP exposure. TEER was significantly decreased at ALI day 20 but not at day 12 following DEP exposure. The dextran permeability of the PHNE was significantly increased at both ALI day 12 and day 20 following DEP exposure. The increased dextran permeability recovered to that of the control following co-exposure to DEPs plus LPS. In the presence of DEPs, the membrane expression of myosin light chain kinase (MLCK) was dramatically increased, and the expression of occludin, ZO1, claudin-1, and E-cadherin was significantly decreased. Co-exposure to DEPs plus LPS significantly reduced membrane MLCK, claudin-1, and E-cadherin but increased occludin and ZO1 expression at ALI day 12.
The activation of TLR4 by LPS inhibits MLCK trafficking to the plasma membrane, and this increased during DEP exposure, resulting in increased occludin expression at the plasma membrane that partially recovered TJ barrier dysfunction following DEP exposure.
上皮细胞中的紧密连接（TJ）在上皮细胞形成针对细胞外环境的细胞旁上皮屏障中起着关键作用。柴油废气颗粒（DEP）会破坏上皮屏障。本研究旨在探讨 DEP 如何破坏上皮屏障，以及 Toll 样受体 4（TLR4）是否参与 DEP 诱导的原代人鼻上皮（PHNE）细胞上皮屏障功能障碍。
将 PHNE 细胞在气液界面（ALI）培养以创建类似于体内的上皮完全分化模型，然后将其暴露于 DEP（颗粒<4μm）或脂多糖（LPS）单独（单暴露）和 DEP 加 LPS（共暴露）于 PHNE 的顶侧。通过测量跨上皮电阻（TEER）和荧光标记的葡聚糖通透性来监测 TJ 形成和完整性。通过共聚焦显微镜和生化测定评估 TJ 蛋白的表达。
DEP 暴露后，PHNE 细胞活力呈时间和剂量依赖性降低。DEP 暴露后 ALI 第 20 天的 TEER 显著降低，但第 12 天没有降低。DEP 暴露后，PHNE 的葡聚糖通透性在 ALI 第 12 天和第 20 天都显著增加。DEP 加 LPS 共暴露后，葡聚糖通透性恢复至对照水平。在 DEP 存在的情况下，肌球蛋白轻链激酶（MLCK）的膜表达显著增加，而紧密连接蛋白 1、ZO1、claudin-1 和 E-钙黏蛋白的表达明显降低。DEP 加 LPS 共暴露后 ALI 第 12 天，膜 MLCK、claudin-1 和 E-钙黏蛋白显著减少，但 occludin 和 ZO1 的表达增加。
LPS 激活 TLR4 抑制 MLCK 向质膜的转运，而在 DEP 暴露时增加，导致质膜上 occludin 表达增加，部分恢复 DEP 暴露后 TJ 屏障功能障碍。
