5-Methoxyflavone alleviates LPS-mediated lung injury by promoting Nrf2-mediated the suppression of NOX4/TLR4 axis in bronchial epithelial cells and M1 polarization in macrophages

Background: 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.

Methods: 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.

Results: 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.

Conclusion: These results indicated that 5-methoxyflavone might be suitable for the development of a novel drug for ALI therapeutic.

5-Methoxyflavone; Acute lung injury; LPS; Methylated flavonoid; Nrf2.