Acute lung injury (ALI) arises from sepsis or bacterial infection, which are life-threatening respiratory disorders that cause the leading cause of death worldwide. 5-Methoxyflavone, a methylated flavonoid, is gaining increased attention for its various health benefits. In the current study, we investigated the potential effects of 5-methoxyflavone against LPS-mediated ALI and elucidated the corresponding possible mechanism. A mouse model with ALI was established by intratracheal instillation of LPS, and lung pathological changes, signaling pathway related proteins and apoptosis in lung tissues were estimated by H&E staining, immunofluorescence and TUNEL assay, respectively. Cell viability was evaluated by MTT assay; protein levels of pro-inflammatory mediators were measured by ELISA assay; levels of ROS and M1 macrophage polarization were assayed by flow cytometry; the expression of Nrf2 signaling, NOX4/TLR4 axis and P-STAT1 were detected by western blotting. Our results showed that 5-methoxyflavone treatment inhibited LPS-induced expression of NOX4 and TLR4 as well as the activation of downstream signaling (NF-κB and P38 MAPK), which was accompanied by markedly decreased ROS levels and pro-inflammatory cytokines (IL-6, TNF-α, MCP-1, and IL-8) in BEAS-2B cells. Moreover, we revealed that these effects of 5-methoxyflavone were related to its Nrf2 activating property, and blockade of Nrf2 prevented its inhibitory effects on NOX4/TLR4/NF-κB/P38 MAPK signaling, thus abrogating the anti-inflammatory effects of 5-methoxyflavone. Besides, the Nrf2 activating property of 5-methoxyflavone in RAW264.7 cells led to inhibition of LPS/IFN-γ-mediated STAT1 signaling, resulting in suppression of LPS/IFN-γ-induced M1 macrophage polarization and the repolarization of M2 macrophages to M1. In a mouse model of LPS-induced ALI, 5-methoxyflavone administration ameliorated LPS-mediated lung pathological changes, the increased lung index (lung/body weight ratio), and epithelial cell apoptosis. Meanwhile, we found 5-methoxyflavone effectively suppressed the hyperactive signaling pathways and the production of excessive pro-inflammatory mediators. Moreover, 5-methoxyflavone reduced LPS-mediated M1 macrophage polarization associated with elevated P-STAT1 activation in the lung tissues. In addition, 5-methoxyflavone improved the survival of LPS-challenged mice. These results indicated that 5-methoxyflavone might be suitable for the development of a novel drug for ALI therapeutic.

中文翻译：

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5-甲氧基黄酮通过促进 Nrf2 介导的抑制支气管上皮细胞中的 NOX4/TLR4 轴和巨噬细胞中的 M1 极化来减轻 LPS 介导的肺损伤

急性肺损伤 (ALI) 由败血症或细菌感染引起，这是危及生命的呼吸系统疾病，是全球死亡的主要原因。5-甲氧基黄酮是一种甲基化类黄酮，因其多种健康益处而受到越来越多的关注。在目前的研究中，我们研究了 5-甲氧基黄酮对 LPS 介导的 ALI 的潜在影响，并阐明了相应的可能机制。通过气管内滴注LPS建立ALI小鼠模型，分别通过H&E染色、免疫荧光和TUNEL检测评估肺组织病理变化、信号通路相关蛋白和肺组织凋亡。通过MTT测定评估细胞活力；通过 ELISA 测定法测量促炎介质的蛋白质水平；通过流式细胞术检测 ROS 和 M1 巨噬细胞极化水平；Western blotting检测Nrf2信号、NOX4/TLR4轴和P-STAT1的表达。我们的结果表明，5-甲氧基黄酮处理抑制了 LPS 诱导的 NOX4 和 TLR4 表达以及下游信号（NF-κB 和 P38 MAPK）的激活，同时伴随着 ROS 水平和促炎细胞因子（IL- 6，BEAS-2B 细胞中的 TNF-α、MCP-1 和 IL-8）。此外，我们发现 5-甲氧基黄酮的这些作用与其 Nrf2 激活特性有关，阻断 Nrf2 可阻止其对 NOX4/TLR4/NF-κB/P38 MAPK 信号传导的抑制作用，从而消除 5-甲氧基黄酮的抗炎作用甲氧基黄酮。此外，RAW264 中 5-甲氧基黄酮的 Nrf2 激活特性。7 细胞导致抑制 LPS/IFN-γ 介导的 STAT1 信号传导，从而抑制 LPS/IFN-γ 诱导的 M1 巨噬细胞极化和 M2 巨噬细胞复极化为 M1。在 LPS 诱导的 ALI 小鼠模型中，给予 5-甲氧基黄酮可改善 LPS 介导的肺病理变化、肺指数（肺/体重比）增加和上皮细胞凋亡。同时，我们发现 5-甲氧基黄酮有效抑制过度活跃的信号通路和过量促炎介质的产生。此外，5-甲氧基黄酮减少了与肺组织中 P-STAT1 激活升高相关的 LPS 介导的 M1 巨噬细胞极化。此外，5-甲氧基黄酮提高了 LPS 攻击小鼠的存活率。