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| Emodin Inhibits Lipopolysaccharide-Induced Inflammation by Activating Autophagy in RAW 264.7 Cells |
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| KeyWord:emodin, lipopolysaccharide, inflammation, autophagy |
| Author Name | Affiliation | E-mail | | TU Yan-jie, TAN Bo, JIANG Lei | | | | YANG Ai-dong | 1. Research Centre on Application of Classical Prescriptions, Basic Medical College, Shanghai University of Traditional Chinese Medicine, Shanghai (201203), China
2. Department of Febrile Disease, Basic Medical College, Shanghai University of Traditional Chinese Medicine, Shanghai (201203), China | aidongy@126.com |
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| Abstract: |
| Objective: To investigate the effects of emodin on inflammation and autophagy in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and reveal its underlying mechanism. Methods: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was conducted to find the appropriate dose for emodin. RAW264.7 cells pretreated with different concentrations (0–50 μ mol/L) of emodin or vehicle for 2 h prior to exposure to LPS for 16 h. Cell morphology was examined and propidium iodide staining was used to examine cell cycle. Expressions of inflammation-related proteins [nuclear factor-kappaB (NF-κ B) and I-kappaB (Iκ B)α ] and autophagy-related proteins [light chain (LC)3, P62/sequestosome 1, mammalian target of rapamycin (mTOR), and p-mTOR] were examined using Western blot analysis. Expression of inflammation-related cytokines including tumor necrosis factor (TNF)-α , interleukin (IL)-1β and IL-6 were detected by enzyme-linked immunosorbent assay. Autophagy was examined with LC3B fluorescence intensity and aggregation. The effect of emodin on autophagy was conducted with an autophagy inhibitor, 3-methyladenine (3-MA). Results: The expression of NF-κ B in LPS-induced cells was significantly increased (P<0.01) and simultaneously Iκ Bα decreased compared with the normal cell (P<0.05). The expressions of TNF-α , IL-1β , and IL-6 proteins in the LPS-induced RAW264.7 cells were significantly higher than in the normal cell (P<0.05 or P<0.01). LPS increased the percentage of cells in the G0/G1 phase, which was recovered by emodin at different doses (12.5, 25, and 50μ mol/L, P<0.05 or P<0.01). The medium dose (25 μ ml/L) emodin decreased the expressions of NF-κ B, P62 and p-mTOR (P<0.01) and increased Iκ Bα expression, LC3B Ⅱ /Ⅰ ratio as well as LC3B fluorescence intensity (P<0.05 or P<0.01). Meanwhile, the enhanced autophagic effects of emodin, such as the increment of LC3BⅡ / ratio and the decrement of P62 expression, were suppressed by autophagy inhibitor 3-MA. Conclusion: Emodin could inhibit inflammation of mice RAW264.7 macrophages induced by LPS, possibly through activating autophagy. |
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