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

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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 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.

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Authors and Affiliations

  1. Research Centre on Application of Classical Prescriptions, Basic Medical College, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China

    Yan-jie Tu, Lei Jiang, Zhong-hua Wu, Hong-ji Yu, Xiao-qian Li & Ai-dong Yang

  2. Department of Febrile Disease, Basic Medical College, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China

    Yan-jie Tu, Lei Jiang, Zhong-hua Wu, Hong-ji Yu, Xiao-qian Li & Ai-dong Yang

  3. Clinical Pharmacokinetic Laboratory, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China

    Bo Tan

Authors
  1. Yan-jie Tu
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  2. Bo Tan
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  3. Lei Jiang
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  4. Zhong-hua Wu
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  5. Hong-ji Yu
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  6. Xiao-qian Li
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  7. Ai-dong Yang
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Contributions

TU YJ and Tan B contributed equally to this work. Yang AD was responsible for designing the experiments. Tu YJ and Tan B were responsible for analyzing the experimental data and writing the paper. Jiang L, Wu ZH, Yu HJ, Li XQ were responsible for experimental operation e.g., cell culture, MTS assay, Western blot analysis, ELISA and Immunofluorescence analysis. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ai-dong Yang.

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The authors declare no conflict of interests in this work.

Additional information

Supported by Ministry of Science and Technology of China (No. 2018YFC1704102) and the National Natural Science Foundation of China (Nos. 81673855 and 81904072)

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Tu, Yj., Tan, B., Jiang, L. et al. Emodin Inhibits Lipopolysaccharide-Induced Inflammation by Activating Autophagy in RAW 264.7 Cells. Chin. J. Integr. Med. 27, 345–352 (2021). https://doi.org/10.1007/s11655-020-3477-9

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  • DOI: https://doi.org/10.1007/s11655-020-3477-9

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