Immunomodulatory effects of sulfated polysaccharides of pine pollen on mouse macrophages

Int J Biol Macromol. 2016 Oct;91:846-55. doi: 10.1016/j.ijbiomac.2016.06.021. Epub 2016 Jun 8.

Abstract

This study was undertaken to explore the effects of sulfated polysaccharide (SPPM60-D) from masson pine pollen on [Ca(2+)]i and immune function of RAW264.7 macrophages. SPPM60-D was obtained by subjecting Masson pine pollen to boiling water and alcohol precipitation, 60% ethanol grading precipitation, Sephacryl S-400HR purification, and chlorosulfonic acid-pyridine method sulfation. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the effect of SPPM60-D on relative activity and proliferation of RAW264.7 cells, and a fluorescence spectrophotometer was used to determine [Ca(2+)]i. Phagocytosis of neutral red was used to determine phagocytosis capacity. Adherence, scratch healing, and transwell assays were used to assess migration and adhesion abilities of macrophages. An enzyme-linked immuno sorbent assay (ELISA) assay was used to assess the secretion of cytokines and inflammatory mediators. A dexamethasone (DEX) inhibition method was used to measure the recovery of RAW264.7 immune activity by SPPM60-D. SPPM60-D significantly increased relative activity, proliferation, and [Ca(2+)]i levels of mouse RAW264.7 cells. It also significantly enhanced the immune function of macrophages from normal and immune-suppressed mouse. The results showed that SPPM60-D mainly bound to TLR4 on macrophages. This activated the TLR4-PI3K-PLC-IP3R signaling pathway, leading to the opening of calcium release-activated calcium channels (CRAC), increasing [Ca(2+)]i and activating the macrophages, and thus improving immunity.

Keywords: Macrophages; Sulfated polysaccharide; [Ca(2+)](i).

MeSH terms

  • Animals
  • Calcium Signaling / drug effects
  • Cell Line
  • Cell Proliferation / drug effects
  • Immunologic Factors / chemistry
  • Immunologic Factors / pharmacology*
  • Macrophages, Peritoneal / immunology
  • Macrophages, Peritoneal / metabolism*
  • Mice
  • Phagocytosis / drug effects*
  • Pinus / chemistry*
  • Pollen / chemistry*
  • Polysaccharides / chemistry
  • Polysaccharides / pharmacology*

Substances

  • Immunologic Factors
  • Polysaccharides