Demethyleneberberine alleviated the inflammatory response by targeting MD-2 to inhibit the TLR4 signaling

Front Immunol. 2023 Apr 24:14:1130404. doi: 10.3389/fimmu.2023.1130404. eCollection 2023.

Abstract

Introduction: The colitis induced by trinitrobenzenesulfonic acid (TNBS) is a chronic and systemic inflammatory disease that leads to intestinal barrier dysfunction and autoimmunedisorders. However, the existing treatments of colitis are associated with poor outcomes, and the current strategies remain deep and long-time remission and the prevention of complications. Recently, demethyleneberberine (DMB) has been reported to be a potential candidate for the treatment of inflammatory response that relied on multiple pharmacological activities, including anti-oxidation and antiinflammation. However, the target and potential mechanism of DMB in inflammatory response have not been fully elucidated.

Methods: This study employed a TNBS-induced colitis model and acute sepsis mice to screen and identify the potential targets and molecular mechanisms of DMB in vitro and in vivo. The purity and structure of DMB were quantitatively analyzed by high-performance liquid chromatography (HPLC), mass spectrometry (MS), Hydrogen nuclear magnetic resonance spectroscopy (1H-NMR), and infrared spectroscopy (IR), respectively. The rats were induced by a rubber hose inserted approximately 8 cm through their anus to be injected with TNBS. Acute sepsis was induced by injection with LPS via the tail vein for 60 h. These animals with inflammation were orally administrated with DMB, berberine (BBR), or curcumin (Curc), respectively. The eukaryotic and prokaryotic expression system of myeloid differentiation protein-2 (MD-2) and its mutants were used to evaluate the target of DMB in inflammatory response.

Resluts: DMB had two free phenolic hydroxyl groups, and the purity exceeded 99% in HPLC. DMB alleviated colitis and suppressed the activation of TLR4 signaling in TNBS-induced colitis rats and LPS-induced RAW264.7 cells. DMB significantly blocked TLR4 signaling in both an MyD88-dependent and an MyD88-independent manner by embedding into the hydrophobic pocket of the MD-2 protein with non-covalent bonding to phenylalanine at position 76 in a pi-pi T-shaped interaction. DMB rescued mice from sepsis shock induced by LPS through targeting the TLR4-MD-2 complex.

Conclusion: Taken together, DMB is a promising inhibitor of the MD-2 protein to suppress the hyperactivated TLR4 signaling in inflammatory response.

Keywords: TLR4 signaling; TNBS-induced colitis; demethyleneberberine; inflammatory response; myeloid differentiation protein-2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Colitis* / chemically induced
  • Colitis* / drug therapy
  • Colitis* / metabolism
  • Lipopolysaccharides / toxicity
  • Lymphocyte Antigen 96
  • Mice
  • Myeloid Differentiation Factor 88 / metabolism
  • NF-kappa B / metabolism
  • Rats
  • Toll-Like Receptor 4* / metabolism

Substances

  • Toll-Like Receptor 4
  • NF-kappa B
  • Myeloid Differentiation Factor 88
  • demethyleneberberine
  • Lipopolysaccharides
  • Lymphocyte Antigen 96
  • Tlr4 protein, mouse
  • Tlr4 protein, rat

Grants and funding

This study was funded by the following sources: The Basic Research Project of China Pharmaceutical University (No. 2632018PY06) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education institutions (PAPD) and the National Natural Science Foundation of China (No. 81573484) to YBZ.