肺炎是一种严重的、危及生命的肺部炎症,具有很高的发病率和死亡率。越来越多的证据表明,esculin,香豆素的衍生物,具有有效的抗炎作用。本研究旨在探讨七叶皂苷抗脂多糖(LPS)诱导的肺炎的药物作用和潜在机制。LPS刺激TC-1细胞以模拟体外炎症损伤模型。细胞活力,扩散,用MTT法测定细胞凋亡,5-乙炔基-2'-脱氧尿苷测定,和流式细胞术。使用酶联免疫吸附测定法分析白细胞介素1β和肿瘤坏死因子α水平。使用特殊的测定试剂盒检查活性氧和超氧化物歧化酶。使用流式细胞术检测巨噬细胞极化。实时定量聚合酶链反应检测丝裂原活化蛋白激酶14(MAPK14)水平。使用蛋白质印迹测定法测定MAPK14和泛素特异性蛋白酶7(USP7)蛋白水平。在Ubibrowser数据库预测之后,USP7和MAPK14之间的相互作用通过免疫共沉淀试验得到验证.在LPS攻击的ALI小鼠体内验证了esculin的生物学作用。这里,我们发现esculin显著缓解LPS诱导的TC-1细胞增殖抑制,和细胞凋亡,炎症反应,氧化应激,和M1型巨噬细胞极化促进。在LPS处理的TC-1细胞中MAPK14和USP7表达增强,部分被esculin治疗废除了。过表达MAPK14减弱了esculin对LPS触发的TC-1细胞损伤的抑制作用。在分子水平上,USP7与MAPK14相互作用并通过去除泛素来维持其稳定性。此外,esculin通过调节MAPK14抑制体内肺炎的进展。一起来看,esculin暴露可部分通过靶向USP7/MAPK14轴减轻LPS诱导的TC-1细胞损伤,提供更好地了解esculin在肺炎抗炎治疗中的作用。
Pneumonia is a serious and life-threatening lung inflammation with high morbidity and mortality. Accumulating evidence has suggested that esculin, a derivative of coumarin, possesses potent anti-inflammatory effects. This study is designed to explore the pharma role and underlying mechanism of esculin against lipopolysaccharides (LPS)-induced pneumonia. TC-1 cells were stimulated by LPS to mimic the inflammatory injury model in vitro. Cell viability, proliferation, and apoptosis were determined using MTT assay, 5-ethynyl-2\'-deoxyuridine assay, and flow cytometry. Interleukin-1β and tumor necrosis factor α levels were analyzed using an enzyme-linked immunosorbent assay. Reactive oxygen species and superoxide dismutase were examined using special assay kits. Macrophage polarization was detected using flow cytometry. Mitogen-activated protein kinase 14 (MAPK14) level was detected by real-time quantitative polymerase chain reaction. MAPK14 and ubiquitin-specific protease 7 (
USP7) protein levels were determined using western blot assay. After Ubibrowser database prediction, the interaction between
USP7 and MAPK14 was verified using a Co-immunoprecipitation assay. The biological role of esculin was verified in LPS-challenged ALI mice in vivo. Here, we found that esculin significantly relieved LPS-induced TC-1 cell proliferation inhibition, and apoptosis, inflammatory response, oxidative stress, and M1-type macrophage polarization promotion. MAPK14 and
USP7 expressions were enhanced in LPS-treated TC-1 cells, which was partly abolished by esculin treatment. Overexpressing MAPK14 attenuated the repression of esculin on LPS-triggered TC-1 cell injury. At the molecular level,
USP7 interacted with MAPK14 and maintained its stability by removing ubiquitin. Moreover, esculin repressed the progression of pneumonia in vivo by regulating MAPK14. Taken together, esculin exposure could mitigate LPS-induced TC-1 cell injury partly by targeting the
USP7/MAPK14 axis, providing a better understanding of the role of esculin in the anti-inflammatory therapeutics for pneumonia.