Alliin Attenuated RANKL-Induced Osteoclastogenesis by Scavenging Reactive Oxygen Species through Inhibiting Nox1
<p>Alliin had no cytotoxicity during RANKL-induced osteoclastogenesis. (<b>A</b>) The chemical formula of alliin; (<b>B</b>) CCK-8 analysis of cell viability of RAW264.7 cells treated with various concentrations of alliin for 48 h; and (<b>C</b>) CCK-8 analysis of cell viability of RAW264.7 cells treated with various concentrations of alliin for 72 h. Data in the figures represent the mean ± SD. N.S. represents the difference was not statistically significant. It was based on one way ANOVA.</p> "> Figure 2
<p>Alliin had an inhibitory effect on TRAP-positive cell formation. (<b>A</b>) Representative light microscope images of RAW264.7 cells cultured in a 96-well plate in the presence of RANKL (50 ng/mL) and M-CSF (50 ng/mL) with different concentration of alliin for 72 h were stained for TRAP (<b>red</b>). Scale bar represents 200 µm. The black arrows pointed out the representative TRAP (+) cells; and (<b>B</b>) quantitative analysis shows the number of TRAP (+) cells with more than three nuclei in each well (96-well plate). Data in the figures represent the mean ± SD. ** (<span class="html-italic">p</span>-value < 0.01) based on one way ANOVA.</p> "> Figure 3
<p>Alliin inhibited RANKL-induced osteoclast fusion and differentiation in a dose-dependent manner. (<b>A</b>) RAW264.7 cells were pretreated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for about 72 h along with a range of alliin concentrations (0, 1, 10 µg/mL), following focal and adhesion staining, and finally photographed. The nuclei were stained for double immunofluorescence microscopy by DAPI (<b>blue</b>) and Vinculin monoclonal antibody (<b>red</b>). Each experiment was performed thrice. Scale bar was at 200 µm; (<b>B</b>) the quantitative test for the TRAP (+) cells having multiple nuclei in each well of 96-well plate; and (<b>C</b>) the total RNA extracted from RAW264.7 cells during RANKL-induced osteoclastogenesis treated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for 72 h with varying doses of alliin (0, 1, 10, 100 µg/mL). Relative mRNA expression levels of NFATc1, c-Fos, MMP-9, CD9, DC-STAMP, OC-STAMP, TRAP, and RANK against GAPDH are shown. Data in the figures represent the averages ± SD. * (<span class="html-italic">p</span>-value < 0.05); ** (<span class="html-italic">p</span>-value < 0.01) or *** (<span class="html-italic">p</span>-value < 0.001) based on one way ANOVA.</p> "> Figure 4
<p>Alliin inhibited osteoclastic RANKL-induced bone resorption. (<b>A</b>) RAW264.7 cells cultured on bovine slices, were pretreated with RANKL and M-CSF (50 ng/mL each) for five days, along with a range of alliin concentrations (0, 1, 10 µg/mL), following focal and adhesion staining, and finally photographed. Experiments were done in triplicate. Scale bar represents 400 µm; (<b>B</b>) quantification of the bone resorption area on the bone slices; (<b>C</b>) representative images of RAW264.7 cells cultured on osteo assay surface 96-well plates treated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for five days with varying concentrations of alliin (0, 1, 10 µg/mL) followed removal of osteoclasts. Each experiment was performed thrice. The resorption area can be measured by 400 µm bars; and (<b>D</b>) measurement of the bone resorption area at the osteo surface. Data represented here at respective places is the averages ± SD. ** (<span class="html-italic">p</span>-value < 0.01) based on one way ANOVA.</p> "> Figure 5
<p>Alliin scavenged ROS production in a dose-dependent way. (<b>A</b>) ROS detection by fluorescent probe DCFH-DA. Scale bar represents 200 µm; and (<b>B</b>) quantitative analysis of ROS (+) cells in each well (96-well plate). Data in the figures represent the mean ± SD. ** (<span class="html-italic">p</span>-value < 0.01) and *** (<span class="html-italic">p</span>-value < 0.001) based on one way ANOVA.</p> "> Figure 6
<p>Alliin inhibited RANKL-induced osteoclast differentiation and fusion through inhibiting Nox1 activity and blocking the c-Fos-NFATc1 signaling pathway. Total protein was extracted from RAW264.7 cells during RANKL-induced osteoclastogenesis treated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for 72 h with varying doses of alliin (0 µg/mL, 1 µg/mL, 10 µg/mL). (<b>A</b>) Representative Western blot images of c-Fos, NFATc1, Nox1, and β-actin from RAW264.7 cells in different groups; (<b>B</b>) relative intensity of expression level of c-FOS, NFATc1, and Nox1 against β-actin; and (<b>C</b>) relative mRNA expression levels of Nox1 against GAPDH. Data in the figures represent the mean ± SD. * (<span class="html-italic">p</span>-value < 0.05); ** (<span class="html-italic">p</span>-value < 0.01) based on one way ANOVA.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Alliin Has No Cytotoxicity on RAW264.7 Cells
2.2. Inhibitory Effect of Alliin on Osteoclast Differentiation and Fusion in RANKL-Induced Osteoclastogenesis
2.3. Alliin Inhibits RANKL-Induced Osteoclastic Bone Resorption
2.4. The Inhibitory Effect of ROS Level and NADPH Oxidase Expression by Alliin during the Osteoclastic Differentiation of RAW264.7 Cells
2.5. Alliin Inhibits RANKL-Induced Osteoclast Specific Transcription Factor Translation and Protein Expression
3. Discussion
4. Materials and Methods
4.1. Reagents and Antibodies
4.2. Totoxity Evaluation of Alliin on RANKL-Induced Osteoclastogenesis
4.3. Tartrate-Resistant Acid Phosphatase (TRAP) Staining Assay
4.4. Actin Cytoskeleton and Focal Adhesion Staining
4.5. Resorption Pit Assay
4.6. Determination of Intracellular ROS
4.7. RNA Extraction and Quantitative PCR Assay
4.8. Western Blotting
4.9 Statistical Analysis
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Chen, Y.; Sun, J.; Dou, C.; Li, N.; Kang, F.; Wang, Y.; Cao, Z.; Yang, X.; Dong, S. Alliin Attenuated RANKL-Induced Osteoclastogenesis by Scavenging Reactive Oxygen Species through Inhibiting Nox1. Int. J. Mol. Sci. 2016, 17, 1516. https://doi.org/10.3390/ijms17091516
Chen Y, Sun J, Dou C, Li N, Kang F, Wang Y, Cao Z, Yang X, Dong S. Alliin Attenuated RANKL-Induced Osteoclastogenesis by Scavenging Reactive Oxygen Species through Inhibiting Nox1. International Journal of Molecular Sciences. 2016; 17(9):1516. https://doi.org/10.3390/ijms17091516
Chicago/Turabian StyleChen, Yueqi, Jingjing Sun, Ce Dou, Nan Li, Fei Kang, Yuan Wang, Zhen Cao, Xiaochao Yang, and Shiwu Dong. 2016. "Alliin Attenuated RANKL-Induced Osteoclastogenesis by Scavenging Reactive Oxygen Species through Inhibiting Nox1" International Journal of Molecular Sciences 17, no. 9: 1516. https://doi.org/10.3390/ijms17091516