Alpinumisoflavone attenuates lipopolysaccharide-induced acute lung injury by regulating the effects of anti-oxidation and anti-inflammation both in vitro and in vivo

Abstract

Alpinumisoflavone (AIF) is a plant-derived pyranoisoflavone that exhibits a number of pharmacological activities, but the protective effects of AIF against pulmonary inflammation are still unknown. This study aimed to investigate the anti-inflammatory effects and possible molecular mechanisms of AIF in both lipopolysaccharide (LPS)-stimulated macrophages and mice. The results revealed that AIF dramatically suppressed the production of pro-inflammatory mediators [including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, IL-17, intercellular adhesion molecule-1 (ICAM-1), and nitric oxide (NO)] and increased the levels of anti-oxidative enzymes [including catalase (CAT), heme oxygenase-1 (HO-1), glutathione peroxidase (GPx), and superoxide dismutase (SOD)] both in vitro and in vivo. Additionally, pre-treatment with AIF could not only significantly prevent histopathological changes and neutrophil infiltration but also decreased the expression levels of nuclear factor-kappa B (NF-κB), mitogen-activated protein kinases (MAPKs), and the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, as well as IL-17 production in LPS-induced lung tissues. The anti-inflammatory effects of AIF were mediated by up-regulating anti-oxidative enzymes and suppressing the NF-κB, MAPK, NLRP3 inflammasome and IL-17 signaling pathways. This is the first study to reveal that AIF has a protective effect against LPS-induced lung injury in mice.

Fig. 1. Effects of AIF regarding the inflammatory responses in LPS-induced RAW264.7 cells. (A) The chemical structure of alpinumisoflavone (AIF). (B) Effect of AIF on cell viability were measured using MTT assay. Levels of (C) NO, (D) TNF-α, (E) IL-6, and (F) IL-1β in culture supernatants were quantitated using ELISA. (G) The protein expression of ICAM-1 were measured using western blot. All values represent mean ± S.D. of three or more replicates. ^###p < 0.001 compared with normal group; *p < 0.05, **p < 0.01 and ***p < 0.001 compared with LPS treated alone group, respectively.

Fig. 2. Effects of AIF regarding the activation of NF-κB, MAPKs, and NLRP3 inflammasome in LPS-induced RAW264.7 cells. (A) Effect of AIF on IKK, IκB-α phosphorylation and NF-κB activation were measured using western blot. (B) Effect of AIF on NF-κB nuclear translocation was detected by immunofluorescence assay using an antibody for p65 subunit. (C) Effect of AIF on MAPKs activation were measured using western blot. (D) The protein expression of NLRP3, caspase-1, and IL-1β were measured using western blot. All values represent mean ± S.D. of three or more replicates. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 compared with normal group, respectively; *p < 0.05, **p < 0.01 and ***p < 0.001 compared with LPS treated alone group, respectively.

Fig. 3. Effects of AIF regarding the activation of IL-17 signaling pathway in LPS-induced RAW264.7 cells. (A) The protein expression of Act1, Hsp90, and TRAF6 were measured using western blot. (B) Levels of IL-17 in culture supernatants were quantitated using ELISA. All values represent mean ± S.D. of three or more replicates. ^##p < 0.01 and ^###p < 0.001 compared with normal group, respectively; *p < 0.05, **p < 0.01 and ***p < 0.001 compared with LPS treated alone group, respectively.

Fig. 4. Effects of AIF regarding the expression of TLR-4, iNOS, COX-2, and anti-oxidant enzymes in LPS-induced RAW264.7 cells. (A) The protein expression of TLR-4, iNOS and COX-2 were measured using western blot. (B) The protein expression of anti-oxidative enzymes (CAT, HO-1, GPx, and SOD) were measured using western blot. (C) Levels of ROS production in RAW264.7 cells were determined using ELISA. All values represent mean ± S.D. of three or more replicates. ^#p < 0.05 and ^###p < 0.001 compared with normal group, respectively; *p < 0.05, **p < 0.01 and ***p < 0.001 compared with LPS treated alone group, respectively.

Fig. 5. Effects of AIF regarding the inflammatory response in LPS-stimulated ALI mice. (A) Pathological examinations on lung tissues in LPS-induced ALI mice. Lung sections were stained with hematoxylin and eosin (H&E). Original magnification: 200×. Scale bar represents 100 μm. Effects of AIF on the (B) wet-to-dry weight (W/D) ratio and (C) MPO activity in lung tissues of LPS-induced ALI. BALF was collected to measure the (D) protein contents using Bradford assay, and the levels of (E) TNF-α, (F) IL-6, and (G) IL-1β using ELISA. (H) The protein expression of ICAM-1 in lung tissues were measured using western blot. All values represent mean ± S.D. of three or more replicates. ^##p < 0.01 and ^###p < 0.001 compared with normal group, respectively; *p < 0.05, **p < 0.01 and ***p < 0.001 compared with LPS treated alone group, respectively.

Fig. 6. Effects of AIF regarding the activation of NF-κB, MAPKs, and NLRP3 inflammasome in ALI mice. Effect of AIF on (A) IκB-α phosphorylation, NF-κB activation, (B) MAPKs (ERK, p38, and JNK) activation, (C) NLRP3, caspase-1, and IL-1β in lung tissues were measured using western blot. All values represent mean ± S.D. of three or more replicates. ^##p < 0.01 and ^###p < 0.001 compared with normal group, respectively; *p < 0.05, **p < 0.01 and ***p < 0.001 compared with LPS treated alone group, respectively.

Fig. 7. Effects of AIF regarding the activation of IL-17 signaling pathway in ALI mice. (A) The protein expression of Act1, Hsp90, and TRAF6 in the lung tissues were measured by western blot analysis. (B) Levels of IL-17 in the BALF were quantitated using ELISA. All values represent mean ± S.D. of three or more replicates. ^##p < 0.01 and ^###p < 0.001 compared with normal group, respectively; *p < 0.05, **p < 0.01 and ***p < 0.001 compared with LPS treated alone group, respectively.

Fig. 8. Effects of AIF regarding the expression of TLR-4, iNOS, COX-2, and anti-oxidant enzymes in ALI mice. (A) The protein expression of TLR-4, iNOS, COX-2, and (B) anti-oxidative enzymes (CAT, HO-1, GPx, and SOD) in the lung tissues were measured by western blot. All values represent mean ± S.D. of three or more replicates. ^###p < 0.001 compared with normal group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with LPS treated alone group, respectively.

Fig. 9. Scheme of the mechanisms in the protective effect of alpinumisoflavone on LPS-induced ALI.