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Emodin Inhibits Lipopolysaccharide-Induced Inflammation by Activating Autophagy in RAW 264.7 Cells
Yan-Jie Tu1,2, Bo Tan3, Lei Jiang1,2
1Research Centre on Application of Classical Prescriptions, Basic Medical College, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
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 μmol/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 (I κ 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 I κ B α decreased compared with the normal cell (P<0.05). The expressions of TNF-α, IL-β, 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 I κ B α 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 LC3B II/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.
Insights
Emodin effectively reduces inflammation in macrophages by activating autophagy. This natural compound may offer therapeutic benefits by modulating key inflammatory and autophagy pathways.
Area of Science:
- Cell Biology
- Immunology
- Pharmacology
Background:
- Lipopolysaccharide (LPS) induces inflammation and alters cell cycle in RAW 264.7 macrophages.
- Inflammation is characterized by increased NF-κB and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6).
- Autophagy, involving proteins like LC3B and P62, plays a role in cellular homeostasis and response to stimuli.
Purpose of the Study:
- To investigate the anti-inflammatory and autophagy-modulating effects of emodin in LPS-induced RAW 264.7 macrophages.
- To elucidate the underlying molecular mechanisms of emodin's action.
- To determine emodin's impact on cell cycle progression under inflammatory conditions.
Main Methods:
- RAW 264.7 macrophages were treated with varying concentrations of emodin followed by LPS exposure.
- Cell viability was assessed using MTS assay.
- Protein expression (NF-κB, IκBα, LC3B, P62, mTOR) and cytokine levels (TNF-α, IL-1β, IL-6) were analyzed via Western blot and ELISA.
- Cell cycle, autophagy markers (LC3B fluorescence), and the effect of autophagy inhibitor 3-MA were evaluated.
Main Results:
- LPS significantly increased NF-κB, P62, p-mTOR, and pro-inflammatory cytokines while decreasing IκBα and altering cell cycle (G0/G1 phase).
- Emodin treatment dose-dependently reversed these LPS-induced changes, decreasing NF-κB, P62, and p-mTOR, and increasing IκBα.
- Emodin enhanced autophagy, evidenced by increased LC3B II/I ratio and fluorescence, which was attenuated by the autophagy inhibitor 3-MA.
Conclusions:
- Emodin exhibits significant anti-inflammatory effects on LPS-induced RAW 264.7 macrophages.
- Emodin-mediated inflammation inhibition is potentially achieved through the activation of autophagy.
- Emodin may serve as a potential therapeutic agent for inflammatory conditions by modulating autophagy.
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