The Study On The Bidirectional Immunomodulatory Effects Of Ganoderma Applanatum Polysaccharides

Ganoderma applanatum polysaccharides is separated out from the Submerged FermentationFungal of Ganoderma applanatum (Pers.), GAP is helpful to clear heat, removestagnation,dissipate phlegm and relieve pain, however the effects and mechanism of GAP onimmune and excessive-immune were still unclearly. The experiment studied the bi-directionalimmunomodulatory activity of polysaccharides extracts from Submerged Fermentation Fungalof Ganoderma applanatum (Pers.) and two kinds of homogeneous polysaccharide asexperimental subjects. In my experiments, mouse macrophage RAW264.7cells were stimulatedand then analysed the effects of GAP on normal and LPS stimulated cells.The results about neural red method showed that the phagocytic activities of theRAW264.7cells were promoted after incubated with5,25,100μg/ml of GAP-0，GAP-1，GAP-2along for24hours respectively (p<0.01), and the highest phagocytic indexes were1.37(100μg/ml，GAP-0)，1.39(100μg/ml，GAP-1) and1.31(5μg/ml，GAP-2).The results aboutGriess reaction showed that these extracts also promoted NO release, and the extractconcentration at100μg/ml did the best, which the NO productions were significantly higher thanthat of control group (p<0.01). The results about Enzyme-Linked Immunosorbent Methodshowed that compared to the control group, the TNF-α, IL-1βand IL-6production were alsopromoted by GAP-0，GAP-1and GAP-2significantly, while GAP-0，GAP-1and GAP-2at theconcentration100μg/ml were the best. The productions of TNF-α at these respective extractconcentrations were164.09,198.91,157.44pg/ml respectively.The productions of IL-1β at theserespective extract concentrations were102.04,131.85,81.94pg/ml respectively.The productionsof IL-6at these respective extract concentrations were80.06,98.91,67.41pg/mlrespectively.Three kinds of GAP can promote macrophages to produce NO,TNF-α,IL-1βandIL-6. Anti TLR4(2.0μg/ml) antibody have a significant effect of antagonism the promotion;SB203580(20μM) is a specific inhibitor of p38MAPK and BAY11-7082(10μM) is a specificinhibitor of NF-κB also have significant inhibition to this promoting (P <0.01).LPS (1μg/ml) stimulated RAW264.7cells to produce a large amount of NO(35.91μM)after24hours.This action could be suppressed to some extent by the5,25,100μg/ml of theGAP-0,GAP-1,GAP-2(p<0.01).The maximum effective concentrations of GAP-0,GAP-1andGAP-2were25μg/ml, which could reduce the NO production to only21.58,18.70,11.37μMrespectively. Similarly, these extracts could suppress over TNF-α, IL-1βand IL-6productionstimulated by LPS. GAP-0,GAP-1and GAP-2could reduce the LPS stimulated TNF-α production from226.46pg/ml to153.66pg/ml (25μg/ml GAP-0),128.67pg/ml (25μg/ml GAP-1)and110.26pg/ml (100μg/ml GAP-2) respectively. GAP-0,GAP-1and GAP-2could reduce theLPS stimulated IL-1βproduction from102.49pg/ml to70.16pg/ml (25μg/ml GAP-0),68.16pg/ml (25μg/ml GAP-1) and48.52pg/ml (100μg/ml GAP-2) respectively. Similarly,GAP-0,GAP-1and GAP-2could reduce the LPS stimulated IL-6production from152.76pg/mlto81.28pg/ml (25μg/ml GAP-0),72.11pg/ml(25μg/ml GAP-1) and49.77pg/ml (100μg/mlGAP-2) respectively.These three kinds of polysaccharide can reduce macrophages produceexcessive NO,TNF-α,IL-1βand IL-6.Anti-TLR4(2.0μg/ml) antibody that have a significantlyantagonism on the downward effect (P <0.01); SB203580(20μM) is a specific inhibitor ofp38MAPK and BAY11-7082(10μM) is a specific inhibitor of NF-κB also have significantlyinhibited on the downward effection (P <0.01).These results showed that, GAP can activate normal macrophages, and weakly increasedthe production of NO, TNF-α, IL-1, and IL-6. That role may be due to GAP through TLR4toactivate macrophages, to improve the phagocytic activity of macrophages, to release of largeamounts of NO, Thereby to improve the dissolving ability of macrophage; P38MAPK signaltransduction pathway was activated, and then NF-κB a transcription factor als be activated,NF-κB starts TNF-α, IL-1β, IL-6gene to transcription,at last leading to the increase aboutsynthesis and secretion of TNF-α, IL-1β,IL-6,To improve body’s immune function.However,when cells were over-stimulated, GAP inhibited the high expression of NO, TNF-α, IL-1, andIL-6induced by LPS. The down-regulated effects associated with GAP could interfere with LPSbinding to cells. Therefore, the effect of GAP on macrophages is a two-way modulation, GAP isa bidirectional immunomodulator.