The binding of kaempferol-3-O-rutinoside to vascular endothelial growth factor potentiates anti-inflammatory efficiencies in lipopolysaccharide-treated mouse macrophage RAW264.7 cells

Phytomedicine. 2021 Jan:80:153400. doi: 10.1016/j.phymed.2020.153400. Epub 2020 Oct 26.

Abstract

Background: Vascular Endothelial Growth Factors (VEGFs) are a group of growth factor in regulating development and maintenance of blood capillary. The VEGF family members include VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C and VEGF-D. VEGF receptor activation leads to multiple complex signaling pathways, particularly in inducing angiogenesis. Besides, VEGF is produced by macrophages and T cells, which is playing roles in inflammation. In macrophages, VEGF receptor-3 (VEGFR-3) and its ligand VEGF-C are known to attenuate the release of pro-inflammatory cytokines.

Methods: Immunoprecipitation and molecular docking assays showed the binding interaction of kaempferol-3-O-rutinoside and VEGF-C. Western blotting and qRT-PCR methods were applied to explore the potentiating effect of kaempferol-3-O-rutinoside in VEGF-C-mediated expressions of proteins and genes in endothelial cells and LPS-induced macrophages. Enzyme-linked immunosorbent assay (ELISA) was employed to reveal the release of proinflammatory cytokines in LPS-induced macrophages. Immunofluorescence assay was performed to determine the effect of kaempferol-3-O-rutinoside in regulating nuclear translocation of NF-κB p65 subunit in the VEGF-C-treated cultures. In addition, Transwell® motility assay was applied to detect the ability of cell migration after drug treatment in LPS-induced macrophages.

Results: We identified kaempferol-3-O-rutinoside, a flavonoid commonly found in vegetable and fruit, was able to act on cultured macrophages in inhibiting inflammatory response, and the inhibition was mediated by its specific binding to VEGF-C. The kaempferol-3-O-rutinoside-bound VEGF-C showed high potency to trigger the receptor activation. In LPS-treated cultured macrophages, applied kaempferol-3-O-rutinoside potentiated inhibitory effects of exogenous applied VEGF-C on the secretions of pro-inflammatory cytokines, i.e. IL-6 and TNF-α, as well as expressions of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). This inhibition was in parallel to transcription and translocation of NF-κB. Moreover, the binding of kaempferol-3-O-rutinoside with VEGF-C suppressed the LPS-induced migration of macrophage.

Conclusion: Taken together, our results suggested the pharmacological roles of kaempferol-3-O-rutinoside in VEGF-C-mediated anti-inflammation.

Keywords: Herbal medicine; Inflammation; Kaempferol-3-O-rutinoside; Phytochemical; VEGF-C; VEGFR-3.

MeSH terms

  • Animals
  • Anti-Inflammatory Agents, Non-Steroidal / chemistry
  • Anti-Inflammatory Agents, Non-Steroidal / pharmacology*
  • Cyclooxygenase 2 / metabolism
  • Cytokines / metabolism
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Inflammation / drug therapy
  • Inflammation / metabolism
  • Kaempferols / chemistry
  • Kaempferols / metabolism*
  • Kaempferols / pharmacology*
  • Lipopolysaccharides / toxicity
  • Macrophages / drug effects
  • Mice
  • Molecular Docking Simulation
  • NF-kappa B / genetics
  • NF-kappa B / metabolism
  • Nitric Oxide Synthase Type II / metabolism
  • RAW 264.7 Cells
  • Vascular Endothelial Growth Factor C / metabolism*
  • Vascular Endothelial Growth Factor C / pharmacology*

Substances

  • Anti-Inflammatory Agents, Non-Steroidal
  • Cytokines
  • Kaempferols
  • Lipopolysaccharides
  • NF-kappa B
  • Vascular Endothelial Growth Factor C
  • kaempferol-3-O-rutinoside
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse
  • Ptgs2 protein, mouse
  • Cyclooxygenase 2