Immunomodulatory effects of sulfated polysaccharides of pine pollen on mouse macrophages.
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.
Key Laboratory of Animal Resistance Biology of Shandong Province, College of Life Science, Shandong Normal University, Jinan 250014, China. Electronic address: gengy@sdnu.edu.cn.
Yue
Y
Geng
Key Laboratory of Animal Resistance Biology of Shandong Province, College of Life Science, Shandong Normal University, Jinan 250014, China.
Li
L
Xing
Key Laboratory of Animal Resistance Biology of Shandong Province, College of Life Science, Shandong Normal University, Jinan 250014, China.
Mengmeng
M
Sun
Key Laboratory of Animal Resistance Biology of Shandong Province, College of Life Science, Shandong Normal University, Jinan 250014, China.
Fangchen
F
Su
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D007155:Immunologic Factors / Q000737:chemistry / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D010587:Phagocytosis / Q000187:drug effects*; D028223:Pinus / Q000737:chemistry*; D011058:Pollen / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry / Q000494:pharmacology*
D007155:Immunologic Factors; D011134:Polysaccharides
Macrophages; Sulfated polysaccharide; [Ca(2+)](i)