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 mu 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-kappa B) and I-kappaB (I kappa B)alpha] 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)-alpha, interleukin (IL)-1 beta 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-kappa B in LPS-induced cells was significantly increased (P<0.01) and simultaneously I kappa B alpha decreased compared with the normal cell (P<0.05). The expressions of TNF-alpha, IL-beta, and IL-6 proteins in the LPS-induced RAW264.7 cells were significantly higher than in the normal cell (PP<0.01). LPS increased the percentage of cells in the G(0)/G(1)phase, which was recovered by emodin at different doses (12.5, 25, and 50 mu mol/L,PP<0.01). The medium-dose (25 mu ml/L) emodin decreased the expressions of NF-kappa B, P62 and p-mTOR (P<0.01) and increased I kappa B alpha expression, LC3B II/I ratio as well as LC3B fluorescence intensity (PP<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.

Automated summary

Emodin can inhibit inflammation of LPS-induced mice RAW264.7 macrophages by activating autophagy. Emodin reduces the expression of NF-kappa B, P62, and p-mTOR, while increasing I kappa B alpha expression, LC3B II/I ratio, and LC3B fluorescence intensity. The enhanced autophagic effects of emodin, such as increased LC3B II/I ratio and decreased P62 expression, are suppressed by the autophagy inhibitor 3-MA.