Abundant natural flavonoids can induce nuclear factor-erythroid 2 related factor 2 (Nrf2) and/or AMP-activated protein kinase (AMPK) activation, which play crucial roles in the amelioration of various inflammation- and oxidative stress-induced diseases, including acute lung injury (ALI). Xanthohumol (Xn), a principal prenylflavonoid, possesses anti-inflammation and anti-oxidant activities. However, whether Xn could protect from LPS-induced ALI through inducing AMPK/Nrf2 activation and its downstream signals, are still poorly elucidated. Accordingly, we focused on exploring the protective effect of Xn in the context of ALI and the involvement of underlying molecular mechanisms. Our findings indicated that Xn effectively alleviated lung injury by reduction of lung W/D ratio and protein levels, neutrophil infiltration, MDA and MPO formation, and SOD and GSH depletion. Meanwhile, Xn significantly lessened histopathological changes, reactive oxygen species (ROS) generation, several cytokines secretion, and iNOS and HMGB1 expression, and inhibited Txnip/NLRP3 inflammasome and NF-κB signaling pathway activation. Additionally, Xn evidently decreased t-BHP-stimulated cell apoptosis, ROS generation and GSH depletion but increased various anti-oxidative enzymes expression regulated by Keap1-Nrf2/ARE activation, which may be associated with AMPK and GSK3β phosphorylation. However, Xn-mediated inflammatory cytokines and ROS production, histopathological changes, Txnip/NLRP3 inflammasome and NF-κB signaling pathway in WT mice were remarkably abrogated in Nrf2-/- mice. Our experimental results firstly provided a support that Xn effectively protected LPS-induced ALI against oxidative stress and inflammation damage which are largely dependent upon upregulation of the Nrf2 pathway via activation of AMPK/GSK3β, thereby suppressing LPS-activated Txnip/NLRP3 inflammasome and NF-κB signaling pathway.

Background Oxidative stress and the resulting inflammation are essential pathological processes in acute lung injury (ALI). Nuclear factor erythroid 2-related factor 2 (Nrf2), a vital transcriptional factor, possesses antioxidative potential and has become a primary target to treat many diseases. Oridonin (Ori), isolated from the plant Rabdosia Rrubescens, is a natural substance that possesses antioxidative and anti-inflammatory effects. Our aim was to study whether the anti-inflammatory and antioxidant effects of Ori on LPS-induced ALI were mediated by Nrf2. Methods MTT assays, Western blotting analysis, a mouse model, and hematoxylin-eosin (H & E) staining were employed to explore the mechanisms by which Ori exerts a protective effect on LPS-induced lung injury in RAW264.7 cells and in a mouse model. Results Our results indicated that Ori increased the expression of Nrf2 and its downstream genes (HO-1, GCLM), which was mediated by the activation of Akt and MAPK. Additionally, Ori inhibited LPS-induced activation of the pro-inflammatory pathways NLRP3 inflammasome and NF-κB pathways. These two pathways were also proven to be Nrf2-independent by the use of a Nrf2 inhibitor. In keeping with these findings, Ori alleviated LPS-induced histopathological changes, the enhanced production of myeloperoxidase and malondialdehyde, and the depleted expression of GSH and superoxide dismutase in the lung tissue of mice. Furthermore, the expression of LPS-induced NLRP3 inflammasome and NF-κB pathways was more evident in Nrf2-deficient mice but could still be reversed by Ori. Conclusions Our results demonstrated that Ori exerted protective effects on LPS-induced ALI via Nrf2-independent anti-inflammatory and Nrf2-dependent antioxidative activities.

Oxidative damage and inflammation are closely associated with the pathogenesis of acute lung injury (ALI). Thus, we explored the protective effect of isovitexin (IV), a glycosylflavonoid, in the context of ALI. To accomplish this, we created in vitro and in vivo models by respectively exposing macrophages to lipopolysaccharide (LPS) and using LPS to induce ALI in mice. In vitro, our results showed that IV treatment reduced LPS-induced pro-inflammatory cytokine secretion, iNOS and COX-2 expression and decreased the generation of ROS. Consistent findings were obtained in vivo. Additionally, IV inhibited H2O2-induced cytotoxicity and apoptosis. However, these effects were partially reversed following the use of an HO-1 inhibitor in vitro. Further studies revealed that IV significantly inhibited MAPK phosphorylation, reduced NF-κB nuclear translocation, and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) expression in RAW 264.7 cells. In vivo, pretreatment with IV attenuated histopathological changes, infiltration of polymorphonuclear granulocytes and endothelial activation, decreased the expression of ICAM-1 and VCAM-1, reduced the levels of MPO and MDA, and increased the content of GSH and SOD in ALI. Furthermore, IV treatment effectively increased Nrf2 and HO-1 expression in lung tissues. Therefore, IV may offer a protective role against LPS-induced ALI by inhibiting MAPK and NF-κB and activating HO-1/Nrf2 pathways.

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Among the cellular response mechanisms, the nuclear factor erythroid-2 related factor 2 (Nrf2) pathway is considered a survival pathway that alleviates oxidative injury, while both the NOD-like receptor protein 3 (NLRP3) and NF-κB pathways are pro-inflammatory pathways that cause damage to cells. These pathways are implicated in the development and resolution of acute lung injury (ALI). Isoliquiritigenin (ISL), a flavonoid from the liquorice compound, is suggested to be a regulator of the above pathways, but the mechanisms of how the NLRP3/NF-κB pathway interacts with Nrf2 and its protective effects in ALI remain unknown. In the present study, ISL inhibited reactive oxygen species (ROS) generation and cytotoxicity induced by t-BHP and pro-inflammatory enzymes production induced by LPS in RAW 264.7 cells. Such cytoprotective effects coincided with the induction of AMP-activated protein kinase (AMPK)/Nrf2/antioxidant response element (ARE) signaling and the suppression of the NLRP3 and NF-κB pathways. Consistent with these findings, ISL treatment significantly alleviated lung injury in LPS-induced ALI mice, which was reflected by reductions in histopathological changes, pulmonary edema, and protein leakage. At the same time, the increased levels of inflammatory cell exudation and pro-inflammatory mediators, the enhanced production of ROS, myeloperoxidase, and malondialdehyde, and the depleted expression of GSH and superoxide dismutase induced by LPS were ameliorated by ISL. Furthermore, ISL notably activated AMPK/Nrf2/ARE signaling and inhibited LPS-induced NLRP3 and NF-κB activation in the lung. Moreover, although inhibition of the LPS-induced histopathological changes and ROS production were attenuated in Nrf2-deficient mice, the repression of the NLRP3 and NF-κB pathways by ISL was Nrf2-dependent and Nrf2-independent, respectively. In conclusion, our results are the first to highlight the beneficial role and relevant mechanisms of ISL in LPS-induced ALI and provide novel insight into its application.