Apigenin-7-Glycoside Prevents LPS-Induced Acute Lung Injury via Downregulation of Oxidative Enzyme Expression and Protein Activation through Inhibition of MAPK Phosphorylation
"> Figure 1
<p>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. (<b>A</b>) The structure of AP7Glu is shown; (<b>B</b>) the percentage of cell viability was determined by ELISA; (<b>C</b>) 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. <sup>###</sup> compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests). <b>**</b> <span class="html-italic">p</span> < 0.01, and <b>***</b> <span class="html-italic">p</span> < 0.001 were compared with LPS-alone group.</p> "> Figure 1 Cont.
<p>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. (<b>A</b>) The structure of AP7Glu is shown; (<b>B</b>) the percentage of cell viability was determined by ELISA; (<b>C</b>) 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. <sup>###</sup> compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests). <b>**</b> <span class="html-italic">p</span> < 0.01, and <b>***</b> <span class="html-italic">p</span> < 0.001 were compared with LPS-alone group.</p> "> Figure 2
<p>AP7Glu attenuated pulmonary inflammation <span class="html-italic">in vivo</span>. 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. (<b>A</b>) Control; (<b>B</b>) LPS; (<b>C</b>) LPS + Dex; (<b>D</b>) LPS + AP7Glu-H; (<b>E</b>) LPS + AP7Glu-M; (<b>F</b>) LPS + AP7Glu-L. The infiltrating neutrophils were more abundant in (<b>B</b>) LPS group as shown by arrows.</p> "> Figure 2 Cont.
<p>AP7Glu attenuated pulmonary inflammation <span class="html-italic">in vivo</span>. 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. (<b>A</b>) Control; (<b>B</b>) LPS; (<b>C</b>) LPS + Dex; (<b>D</b>) LPS + AP7Glu-H; (<b>E</b>) LPS + AP7Glu-M; (<b>F</b>) LPS + AP7Glu-L. The infiltrating neutrophils were more abundant in (<b>B</b>) LPS group as shown by arrows.</p> "> Figure 3
<p>AP7Glu improved pulmonary edema <span class="html-italic">in vivo</span>. Seventy-two hours after LPS injection with or without AP7Glu pretreatments, mice were exsanguinated and their right lower lungs were obtained. (<b>A</b>) The right lower lungs were used to assess wet to dry (W/D) ratio of lung; (<b>B</b>) Severity of lung injury was analyzed by the lung injury scoring system. Each value represents as mean ± SD of 6 mice. <sup>###</sup> compared with sample of control group. <b>**</b> <span class="html-italic">p</span> < 0.01, and <b>***</b> <span class="html-italic">p</span> < 0.001 were compared with LPS-alone group.</p> "> Figure 4
<p>AP7Glu reduced cellular counts (<b>A</b>); and total protein (<b>B</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. <sup>###</sup> compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range test). ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001, were compared with LPS-alone group.</p> "> Figure 5
<p>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. <sup>###</sup> compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range tests). <b>**</b> <span class="html-italic">p</span> < 0.01, and <b>***</b> <span class="html-italic">p</span> < 0.001, were compared with LPS-alone group.</p> "> Figure 6
<p>AP7Glu reduced (<b>A</b>) Myeloperoxidase activity (MPO) <span class="html-italic">in vivo</span>; (<b>B</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. <sup>###</sup> compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range tests). <b>**</b> <span class="html-italic">p</span> < 0.01, and <b>***</b> <span class="html-italic">p</span> < 0.001, were compared with LPS-alone group.</p> "> Figure 7
<p>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. <sup>###</sup> compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range tests). <b>***</b> <span class="html-italic">p</span> < 0.001, were compared with LPS-alone group.</p> "> Figure 8
<p>Effects of AP7Glu on LPS-induced (<b>A</b>) MAPK (<b>B</b>) IκBα, (<b>C</b>) 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. <sup>###</sup> compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range tests). <b>***</b> and *** <span class="html-italic">p</span> < 0.001, were compared with LPS-alone group.</p> "> Figure 9
<p>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. <sup>###</sup> compared with sample of control group. (One-way ANOVA followed by Scheffe’s multiple range tests). *** <span class="html-italic">p</span> < 0.001, were compared with LPS-alone group.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Cytotoxicity
2.2. AP7Glu Attenuates Pulmonary Inflammation in LPS-Induced Acute Lung Injury (ALI)
2.3. AP7Glu Attenuates Pulmonary Edema in LPS-Induced Acute Lung Injury (ALI)
2.4. AP7Glu Reduces Cellular Counts and Proteins in BALF
2.5. AP7Glu Downregulates TNF-α, IL-6, and IL-1β in BALF
2.6. Effects of AP7Glu on MPO Activity and Antioxidative Enzymes in LPS Induced ALI
2.7. Inhibition of LPS Induced iNOs and COX-2 Proteins by AP7Glu in Lung Tissue
2.8. Effects of AP7Glu on MAPK, IκB and NF-κB Activation in LPS Induced ALI
2.9. Effects of AP7Glu on HO-1 Expression in LPS-Induced ALI
3. Discussion
4. Experimental Section
4.1. Cell Culture
4.2. Cytotoxicity and NO Production
4.3. Animals
4.4. Model of LPS Induced ALI
4.5. Bronchoalveolar Lavage Fluid (BALF), Total Cell Count and Protein Analysis
4.6. TNF-α, IL-6, and IL-1β Cytokines in BALF
4.7. Myeloperoxidase (MPO) Activity Assay
4.8. Lung Wet/Dry Weight Ratio
4.9. H&E Staining
4.10. Western Blot Analysis of Lung Tissue
4.11. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Li, K.-C.; Ho, Y.-L.; Hsieh, W.-T.; Huang, S.-S.; Chang, Y.-S.; Huang, G.-J. Apigenin-7-Glycoside Prevents LPS-Induced Acute Lung Injury via Downregulation of Oxidative Enzyme Expression and Protein Activation through Inhibition of MAPK Phosphorylation. Int. J. Mol. Sci. 2015, 16, 1736-1754. https://doi.org/10.3390/ijms16011736
Li K-C, Ho Y-L, Hsieh W-T, Huang S-S, Chang Y-S, Huang G-J. Apigenin-7-Glycoside Prevents LPS-Induced Acute Lung Injury via Downregulation of Oxidative Enzyme Expression and Protein Activation through Inhibition of MAPK Phosphorylation. International Journal of Molecular Sciences. 2015; 16(1):1736-1754. https://doi.org/10.3390/ijms16011736
Chicago/Turabian StyleLi, Kun-Cheng, Yu-Ling Ho, Wen-Tsong Hsieh, Shyh-Shyun Huang, Yuan-Shiun Chang, and Guan-Jhong Huang. 2015. "Apigenin-7-Glycoside Prevents LPS-Induced Acute Lung Injury via Downregulation of Oxidative Enzyme Expression and Protein Activation through Inhibition of MAPK Phosphorylation" International Journal of Molecular Sciences 16, no. 1: 1736-1754. https://doi.org/10.3390/ijms16011736