Emodin Inhibits Lipopolysaccharide-Induced Inflammation by Activating Autophagy in RAW 264.7 Cells

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.

Keywords: autophagy; emodin; inflammation; lipopolysaccharide.