Melatonin prevents LPS-induced epithelial-mesenchymal transition in human alveolar epithelial cells via the GSK-3β/Nrf2 pathway
Melatonin prevents LPS-induced epithelial-mesenchymal transition in human alveolar epithelial cells via the GSK-3β/Nrf2 pathway
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DOI：
10.1016/j.biopha.2020.110827
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发表时间：
2020-12-01
影响因子：
7.5
通讯作者：
Fei, Guanghe
中科院分区：
文献类型：
--
作者：
Ding, Zhenxing;Wu, Xu;Fei, Guanghe
背景：氧化应激在急性肺损伤(ALI)后肺纤维化中起重要作用，上皮-间充质转化(EMT)事件参与了这一过程。目的：探讨天然抗氧化剂褪黑素对内毒素诱导的人肺泡上皮细胞内皮细胞转化的保护作用。方法：将人肺泡上皮细胞株A549与内毒素和褪黑素单独或联合孵育24 h，观察细胞的形态变化和氧化应激指标。Western印迹分析和免疫荧光染色分别检测EMT相关蛋白和Nrf2信号通路。结果：褪黑素可上调Nrf2基因的表达，抑制脂多糖诱导的细胞形态改变，逆转EMT相关蛋白的表达，降低A549细胞中活性氧(ROS)的产生，降低丙二醛(MDA)和抗氧化酶的水平。然而，在转导Nrf2 shRNA的细胞中，褪黑素的作用几乎完全消失。此外，褪黑素还可以激活A549细胞中PI3K/AKT信号通路，导致GSK-3β(Ser9)的磷酸化和Nrf2蛋白的上调，从而最终减弱内毒素诱导的EMT。结论：本研究首次证明褪黑素通过有效抑制内毒素诱导的肺泡上皮细胞氧化应激，保护肺泡上皮细胞免受氧化应激的影响，这主要依赖于通过PI3K/GSK-3β轴上调Nrf2通路。有必要进一步研究褪黑素在治疗氧化应激相关疾病以及ALI后肺纤维化中的作用。
Background: 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.Methods: 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-3 beta signaling pathway were evaluated.Results: 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-3 beta (Ser9) and upregulation of the Nrf2 protein in A549 cells, which ultimately attenuated LPS-induced EMT.Conclusion: 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-3 beta 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.