Phillygenin inhibits LPS-induced activation and inflammation of LX2 cells by TLR4/MyD88/NF-κB signaling pathway

J Ethnopharmacol. 2020 Feb 10:248:112361. doi: 10.1016/j.jep.2019.112361. Epub 2019 Nov 1.

Abstract

Ethnopharmacological relevance: The traditional Chinese medicine Forsythiae Fructus is the dried fruit of Forsythia suspensa (Thunb.) Vahl. It is commonly used to clear heat and detoxify, reduce swelling and disperse knot, and evacuate wind and heat.

Aim of the study: Inflammation is involved in liver fibrosis. Phillygenin (PHI) is a kind of lignans extracted and separated from Forsythiae Fructus, which has been reported to have a good anti-inflammatory effect. Therefore, we aimed to explore whether PHI has a therapeutic effect on liver fibrosis caused by inflammation.

Materials and methods: Firstly, the induction of the LX2 cells inflammatory model and fibrosis model by LPS with different concentrations were studied. Then, high, medium and low doses PHI was given for intervention therapy. The secretion of IL-6, IL-1β and TNF-α inflammatory factors were detected by ELISA kit, and the expression of collagen I and α-SMA was detected by Western blot and RT-qPCR. The possible mechanism of PHI on TLR4/MyD88/NF-κB signal pathway was studied by computer-aided drug design software and the results were further verified by Western blot and RT-qPCR experiments.

Results: The results showed that LPS could promote the expression of IL-6, IL-1β and TNF-α and the expression of collagen I and α-SMA, indicating that LPS could induce inflammation and fibrosis in LX2 cells. PHI could inhibit LX2 cell activation and fibrotic cytokine expression by inhibiting LPS-induced pro-inflammatory reaction. Molecular docking results showed that PHI could successfully dock with TLR4, MyD88, IKKβ, p65, IκBα, and TAK1 proteins. Subsequently, Western blot and qPCR results further proved that PHI could inhibit the proteins expression in TLR4/MyD88/NF-κB signal pathway which were consistent with the molecular docking results.

Conclusion: PHI can inhibit LPS-induced pro-inflammatory reaction and LX2 cell activation through TLR4/MyD88/NF-κB signaling pathway, thereby inhibiting liver fibrosis.

Keywords: Inflammation; LX2; Liver fibrosis; Phillygenin; TLR4/MyD88/NF-κB.

MeSH terms

  • Actins / metabolism
  • Anti-Inflammatory Agents / pharmacology*
  • Cell Line
  • Collagen Type I / metabolism
  • Hepatic Stellate Cells / drug effects*
  • Hepatic Stellate Cells / metabolism
  • Hepatic Stellate Cells / pathology
  • Hepatitis / metabolism
  • Hepatitis / pathology
  • Hepatitis / prevention & control*
  • Humans
  • Interleukin-1beta / metabolism
  • Interleukin-6 / metabolism
  • Lignans / pharmacology*
  • Lipopolysaccharides / toxicity*
  • Liver Cirrhosis / metabolism
  • Liver Cirrhosis / pathology
  • Liver Cirrhosis / prevention & control*
  • Myeloid Differentiation Factor 88 / metabolism*
  • NF-kappa B / metabolism*
  • Signal Transduction
  • Toll-Like Receptor 4 / metabolism*
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • ACTA2 protein, human
  • Actins
  • Anti-Inflammatory Agents
  • Collagen Type I
  • IL1B protein, human
  • IL6 protein, human
  • Interleukin-1beta
  • Interleukin-6
  • Lignans
  • Lipopolysaccharides
  • MYD88 protein, human
  • Myeloid Differentiation Factor 88
  • NF-kappa B
  • TLR4 protein, human
  • TNF protein, human
  • Toll-Like Receptor 4
  • Tumor Necrosis Factor-alpha
  • lipopolysaccharide, E coli O55-B5
  • phillygenin