Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Anhui Medical University, #218 Jixi Road, Hefei, 230022 Anhui, China.
Center Lab of The First Affiliated Hospital of Anhui Medical University, #218 Jixi Road, Hefei, 230022 Anhui, China.
Biomed Pharmacother. 2020 Dec;132:110827. doi: 10.1016/j.biopha.2020.110827. Epub 2020 Oct 13.
Oxidative stress plays a critical role in pulmonary fibrosis after acute lung injury (ALI), and epithelial-mesenchymal transition (EMT) events are involved in this process. The purpose of this study was to investigate the protective effects of melatonin, a natural antioxidant, on lipopolysaccharide (LPS)-induced EMT in human alveolar epithelial cells.
Human type II alveolar epithelial cell-derived A549 cells were incubated with LPS and melatonin alone or in combination for up to 24 h. The morphological changes of the treated cells were evaluated as well as indexes of oxidative stress. EMT-related proteins and the Nrf2 signaling pathway were detected by western blot analysis and immunofluorescence staining, respectively. To further investigate the underlying mechanisms, the effects of melatonin on cells transfected Nrf2 short hairpin RNA (shRNA) and the PI3K / GSK-3β signaling pathway were evaluated.
Treatment with melatonin upregulated Nrf2 expression, inhibited LPS-induced cell morphological change, reversed the expressions of EMT-related proteins, and reduced reactive oxygen species (ROS) production in A549 cells, as well as the levels of malondialdehyde (MDA) and anti-oxidative enzymes. Yet, the effects of melatonin were almost completely abolished in cells transfected Nrf2 shRNA. Furthermore, the data demonstrated that melatonin could activate the PI3K/AKT signaling pathway, resulting in phosphorylation of GSK-3β (Ser9) and upregulation of the Nrf2 protein in A549 cells, which ultimately attenuated LPS-induced EMT.
The present study is the first to demonstrate that melatonin can protect human alveolar epithelial cells against oxidative stress by effectively inhibiting LPS-induced EMT, which was mostly dependent on upregulation of the Nrf2 pathway via the PI3K/GSK-3β axis. Further studies are warranted to investigate the role of melatonin for the treatment of oxidative stress-associated diseases, as well as pulmonary fibrosis after ALI.
氧化应激在急性肺损伤（ALI）后肺纤维化中起关键作用，上皮-间充质转化（EMT）事件参与了这一过程。本研究旨在探讨天然抗氧化剂褪黑素对脂多糖（LPS）诱导的人肺泡上皮细胞 EMT 的保护作用。
将人 II 型肺泡上皮细胞衍生的 A549 细胞与 LPS 和褪黑素单独或联合孵育长达 24 小时。评估处理细胞的形态变化以及氧化应激指标。通过 Western blot 分析和免疫荧光染色分别检测 EMT 相关蛋白和 Nrf2 信号通路。为了进一步探讨潜在机制，评估了褪黑素对转染 Nrf2 短发夹 RNA（shRNA）的细胞和 PI3K/GSK-3β 信号通路的影响。
褪黑素处理上调了 Nrf2 的表达，抑制了 LPS 诱导的细胞形态变化，逆转了 EMT 相关蛋白的表达，并减少了 A549 细胞中活性氧（ROS）的产生以及丙二醛（MDA）和抗氧化酶的水平。然而，在转染 Nrf2 shRNA 的细胞中，褪黑素的作用几乎完全被消除。此外，数据表明褪黑素可以激活 PI3K/AKT 信号通路，导致 GSK-3β（Ser9）磷酸化和 A549 细胞中 Nrf2 蛋白上调，最终减轻 LPS 诱导的 EMT。
本研究首次表明，褪黑素通过有效抑制 LPS 诱导的 EMT 来保护人肺泡上皮细胞免受氧化应激，这主要依赖于通过 PI3K/GSK-3β 轴上调 Nrf2 通路。需要进一步研究褪黑素在治疗与氧化应激相关疾病以及 ALI 后肺纤维化中的作用。
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