College of Life Sciences, Zhejiang Chinese Medical University, No.548, Binjiang District, Hangzhou, 310053, Zhejiang, People's Republic of China.
College of Medical Technology, Zhejiang Chinese Medical University, No.548, Binjiang District, Hangzhou, 310053, Zhejiang, People's Republic of China.
Eur J Pharmacol. 2021 Apr 5;896:173931. doi: 10.1016/j.ejphar.2021.173931. Epub 2021 Feb 4.
Accumulating studies suggest that fine particulate matter (PM) pollutants in the air are easily enter into alveoli and even the bloodstream, resulting in an inflammatory response that not only triggers respiratory disorders but also causes permanent damage to various organs. Recent findings suggest that coelonin and militarine enriched in orchids can inhibit inflammation-induced injury against respiratory diseases. Here, we evaluated the anti-inflammatory properties of coelonin and militarine and examined their underlying molecular mechanisms in A549 cells exposed to PM. PM induced significant intracellular reactive oxidative stress accumulation at a concentration of 250 μg/ml, as determined using the dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay. Cell viability was assessed via the MTS assay to determine the concentrations of compounds appropriate for use in subsequent experiments. Data from the enzyme-linked immunosorbent assay (ELISA) showed that both coelonin (10 and 20 μg/ml) and militarine (5 and 10 μg/ml) mitigated PM-induced inflammation by reducing the generation of inflammatory factors, including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Quantitative real-time PCR (qRT-PCR) analysis revealed a remarkable decrease in IL-6, TNF-α, cyclooxygenase-2 (COX-2) and interleukin-1β (IL-1β) mRNA levels in the coelonin and militarine-pretreated groups. In Western blot analysis, expression of inhibitor of NF-κB (IκBα) protein in the coelonin and militarine pretreatment groups was significantly increased compared with the PM (only) treatment group (P < 0.05), concomitant with a significant decrease in phospho-IκB kinase β/IκB kinase β (p-IKKβ/IKKβ), phospho-nuclear factor of kappa B p65/nuclear factor of kappa B p65 (p-NF-κBp65/NF-κBp65) and COX-2 proteins (P < 0.05). Both coelonin and militarine inhibited migration and inflammation by suppressing PM-induced IKK phosphorylation, and followed by IκBα protein degradation and NF-κB activation. Our collective data strongly supported the utility of coelonin and militarine as novel sources for development of treatments for PM-induced lung diseases.
越来越多的研究表明，空气中的细颗粒物（PM）污染物很容易进入肺泡甚至血液，引发炎症反应，不仅引发呼吸道疾病，还会对各种器官造成永久性损伤。最近的研究结果表明，兰花中的 Coelonin 和 Militarine 可以抑制炎症引起的呼吸道疾病损伤。在这里，我们评估了 Coelonin 和 Militarine 的抗炎特性，并研究了它们在 PM 暴露的 A549 细胞中的潜在分子机制。PM 诱导的细胞内活性氧应激积累在 250μg/ml 的浓度下使用二氯二氢荧光素二乙酸酯 (DCFH-DA) 荧光测定法确定。通过 MTS 测定法评估细胞活力，以确定适用于后续实验的化合物浓度。酶联免疫吸附试验 (ELISA) 的数据表明，Coelonin（10 和 20μg/ml）和 Militarine（5 和 10μg/ml）均通过减少炎症因子（包括白细胞介素-6 (IL-6) 和肿瘤坏死因子-α (TNF-α)）的产生来减轻 PM 诱导的炎症。定量实时 PCR (qRT-PCR) 分析显示，在 Coelonin 和 Militarine 预处理组中，IL-6、TNF-α、环氧化酶-2 (COX-2) 和白细胞介素-1β (IL-1β) mRNA 水平显著降低。在 Western blot 分析中，与仅用 PM（PM 处理组）处理相比，Coelonin 和 Militarine 预处理组的抑制剂核因子-κB (IκBα) 蛋白表达显著增加（P<0.05），同时磷酸化 IκB 激酶β/ IκB 激酶β（p-IKKβ/IKKβ）、磷酸化核因子κB p65/核因子κB p65（p-NF-κBp65/NF-κBp65）和 COX-2 蛋白显著降低（P<0.05）。Coelonin 和 Militarine 通过抑制 PM 诱导的 IKK 磷酸化，随后 IκBα 蛋白降解和 NF-κB 激活，抑制迁移和炎症。我们的综合数据有力地支持了 Coelonin 和 Militarine 作为开发治疗 PM 诱导的肺部疾病的新型药物的新来源的应用。
