Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, South Korea.
Department of Physiology and Pathophysiology, Peking University School and Hospital of Stomatology, Beijing, China.
Am J Physiol Cell Physiol. 2021 Mar 1;320(3):C448-C461. doi: 10.1152/ajpcell.00295.2020. Epub 2021 Jan 20.
Gram-negative bacterial lipopolysaccharide (LPS) increases the susceptibility of cells to pathogenic diseases, including inflammatory diseases and septic syndrome. In our experiments, we examined whether LPS induces epithelial barrier disruption in secretory epithelia and further investigated its underlying mechanism. The activities of Ca-activated Cl channels (CACC) and epithelial Na channels (ENaC) were monitored with a short-circuit current using an Ussing chamber. Epithelial membrane integrity was estimated via transepithelial electrical resistance and paracellular permeability assays. We found that the apical application of LPS evoked short-circuit current () through the activation of CACC and ENaC. Although LPS disrupted epithelial barrier integrity, this was restored with the inhibition of CACC and ENaC, indicating the role of CACC and ENaC in the regulation of paracellular pathways. We confirmed that LPS, CACC, or ENaC activation evoked apical membrane depolarization. The exposure to a high-K buffer increased paracellular permeability. LPS induced the rapid redistribution of zonula occludens-1 (ZO-1) and reduced the expression levels of ZO-1 in tight junctions through apical membrane depolarization and tyrosine phosphorylation. However, the LPS-induced epithelial barrier disruption and degradation of ZO-1 were largely recovered by blocking CACC and ENaC. Furthermore, although LPS-impaired epithelial barrier became vulnerable to secondary bacterial infections, this vulnerability was prevented by inhibiting CACC and ENaC. We concluded that LPS induces the disruption of epithelial barrier integrity through the activation of CACC and ENaC, resulting in apical membrane depolarization and the subsequent tyrosine phosphorylation of ZO-1.
革兰氏阴性菌脂多糖 (LPS) 增加了细胞对包括炎症性疾病和败血症综合征在内的病原体疾病的易感性。在我们的实验中，我们研究了 LPS 是否会诱导分泌上皮细胞的上皮屏障破坏，并进一步研究了其潜在机制。使用 Ussing 室通过短路电流监测钙激活氯通道 (CACC) 和上皮钠通道 (ENaC) 的活性。通过跨上皮电阻和旁道通透性测定来评估上皮膜完整性。我们发现，LPS 的顶端应用通过激活 CACC 和 ENaC 引起短路电流 ()。尽管 LPS 破坏了上皮屏障完整性，但通过抑制 CACC 和 ENaC 可恢复其完整性，表明 CACC 和 ENaC 在调节旁道途径中发挥作用。我们证实 LPS、CACC 或 ENaC 的激活会引起顶端膜去极化。暴露于高钾缓冲液会增加旁道通透性。LPS 通过顶端膜去极化和酪氨酸磷酸化诱导紧密连接中封闭蛋白-1 (ZO-1) 的快速重分布，并降低 ZO-1 的表达水平。然而，通过阻断 CACC 和 ENaC，LPS 诱导的上皮屏障破坏和 ZO-1 降解得到了很大的恢复。此外，尽管 LPS 破坏的上皮屏障易受二次细菌感染，但通过抑制 CACC 和 ENaC 可以防止这种易感性。我们的结论是，LPS 通过激活 CACC 和 ENaC 诱导上皮屏障完整性的破坏，导致顶端膜去极化和随后的 ZO-1 酪氨酸磷酸化。
