Apigenin-7-glycoside prevents LPS-induced acute lung injury via downregulation of oxidative enzyme expression and protein activation through inhibition of MAPK phosphorylation

Abstract

Apigenin-7-glycoside (AP7Glu) with multiple biological activities is a flavonoid that is currently prescribed to treat inflammatory diseases such as upper respiratory infections. Recently, several studies have shown that its anti-inflammatory activities have been strongly linked to the inhibition of secretion of pro-inflammatory proteins, such as inducible nitric oxide synthase (iNOs) and cyclooxygenase-2 (COX-2) induced through phosphorylation nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK) pathways. Additionally, inflammation, which can decrease the activities of antioxidative enzymes (AOEs) is also observed in these studies. At the same time, flavonoids are reported to promote the activities of heme oxygenase-1 (HO-1) decreased by LPS. The purpose of this study was to assess these theories in a series of experiments on the suppressive effects of AP7Glu based on LPS-induced nitric oxide production in RAW264.7 macrophages in vitro and acute lung injury in mice in vivo. After six hours of lipopolysaccharide (LPS) stimulation, pulmonary pathological, myeloperoxidase (MPO) activity, total polymorphonuclear leukocytes (PMN) cells, cytokines in bronchoalveolar lavage fluid (BALF) and AOEs, are all affected and changed. Meanwhile, our data revealed that AP7Glu not only did significantly inhibit the LPS-enhanced inflammatory activity in lung, but also exhibited anti-inflammatory effect through the MAPK and inhibitor NF-κB (IκB) pathways.

Figure 1

AP7Glu inhibited lipopolysaccharide (LPS)-induced cell inflammation in RAW 264.7 cells. Raw cells were pre-treated with different concentrations of AP7Glu from 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16 μM, or 0 μM (referred as (−)) for 1 h prior to the addition of 100 ng/mL LPS for 24 h. (A) The structure of AP7Glu is shown; (B) the percentage of cell viability was determined by ELISA; (C) The supernatants were harvested and NO production was quantified using ELISA. The data were presented as mean ± SD for the three different experiments performed in triplicate. ^### compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests). ** p < 0.01, and *** p < 0.001 were compared with LPS-alone group.

Figure 1

AP7Glu inhibited lipopolysaccharide (LPS)-induced cell inflammation in RAW 264.7 cells. Raw cells were pre-treated with different concentrations of AP7Glu from 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16 μM, or 0 μM (referred as (−)) for 1 h prior to the addition of 100 ng/mL LPS for 24 h. (A) The structure of AP7Glu is shown; (B) the percentage of cell viability was determined by ELISA; (C) The supernatants were harvested and NO production was quantified using ELISA. The data were presented as mean ± SD for the three different experiments performed in triplicate. ^### compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests). ** p < 0.01, and *** p < 0.001 were compared with LPS-alone group.

Figure 2

AP7Glu attenuated pulmonary inflammation in vivo. Seventy-two hours after LPS injection with or without AP7Glu pretreatments, mice were exsanguinated and their left lower lungs were fixed. Then, tissue sections were stained with hematoxylin and eosin (H&E). The figure demonstrates a representative view (×200) from each group; each bar represents the mean ± SD of 6 mice. (A) Control; (B) LPS; (C) LPS + Dex; (D) LPS + AP7Glu-H; (E) LPS + AP7Glu-M; (F) LPS + AP7Glu-L. The infiltrating neutrophils were more abundant in (B) LPS group as shown by arrows.

Figure 2

AP7Glu attenuated pulmonary inflammation in vivo. Seventy-two hours after LPS injection with or without AP7Glu pretreatments, mice were exsanguinated and their left lower lungs were fixed. Then, tissue sections were stained with hematoxylin and eosin (H&E). The figure demonstrates a representative view (×200) from each group; each bar represents the mean ± SD of 6 mice. (A) Control; (B) LPS; (C) LPS + Dex; (D) LPS + AP7Glu-H; (E) LPS + AP7Glu-M; (F) LPS + AP7Glu-L. The infiltrating neutrophils were more abundant in (B) LPS group as shown by arrows.

Figure 3

AP7Glu improved pulmonary edema in vivo. Seventy-two hours after LPS injection with or without AP7Glu pretreatments, mice were exsanguinated and their right lower lungs were obtained. (A) The right lower lungs were used to assess wet to dry (W/D) ratio of lung; (B) Severity of lung injury was analyzed by the lung injury scoring system. Each value represents as mean ± SD of 6 mice. ^### compared with sample of control group. ** p < 0.01, and *** p < 0.001 were compared with LPS-alone group.

Figure 4

AP7Glu reduced cellular counts (A); and total protein (B) in BALF. Six hours after LPS injection with or without AP7Glu pretreatments, mice were sacrificed and their lungs were lavaged. Cells in the BALF were collected and cytospin preparations were made. Total cells, and total proteins in BALF were analyzed. Each value represents as mean ± SD of 6 mice. ^### compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range test). ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 5

AP7Glu down regulated TNF-α, IL-6, and IL-1β in BALF. Six hours after LPS injection with or without AP7Glu pre-treatments, mice were sacrificed, their lungs were lavaged and the BALF were collected. TNF-α, IL-6 and IL-1β were detected by ELISA. Data represents mean ± SD of 6 mice. ^### compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range tests). ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 6

AP7Glu reduced (A) Myeloperoxidase activity (MPO) in vivo; (B) Antioxidative enzyme activation presented in western blotting. The antioxidative enzymes are represented SOD, catalase, and GPx which were performed at 6 h after LPS challenge. MPO activity was detected by ELISA reader described in Materials and Methods; its activity reflects the neutrophil infiltration in the lungs. Data represents mean ± SD of 6 mice. ^### compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range tests). ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 7

AP7Glu inhibited iNOs and COX-2 expression in lung. Seventy-two hours after LPS injection with or without AP7Glu pre-treatments, mice were exsanguinated and their lungs were removed. A representative Western blot from two separate experiments is shown and its relative protein levels were calculated with reference to a LPS-stimulated culture. Data represents mean ± SD of 6 mice. ^### compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range tests). *** p < 0.001, were compared with LPS-alone group.

Figure 8

Effects of AP7Glu on LPS-induced (A) MAPK (B) IκBα, (C) NF-κB phosphorylation and non-phosphorylation protein expressions in ALI mice. Mice were pretreated with different concentrations of AP7Glu for 1 h and stimulated with LPS. Western blotting was performed using an antibody specific for the detection of IκBα phosphorylated, NF-κB nuclear and cytosol, and three forms of MAPK molecules, ERK, p38, and JNK. The fold change in protein expression between the treated and control groups was calculated. A representative Western blot from two separate experiments is shown. Data represents mean ± SD of six mice. ^### compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range tests). *** and *** p < 0.001, were compared with LPS-alone group.

Figure 9

Effects of AP7Glu on LPS-induced HO-1 expression in lung. Tissue suspensions were prepared and subjected to Western blotting by using an antibody specific for HO-1, and β-actin was used as an internal control. The fold change in HO-1 expression between the treated and the control groups was calculated. Data represents mean ± SD of 6 mice. ^### compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range tests). *** p < 0.001, were compared with LPS-alone group.