A Study Of Regulatory Effects Of Trametes Versicolor Polysaccharopeptide(PSP) On ROS Production And MAPK Signaling In Mouse Macrophages Stimulated With LPS

Trametes versicolor Polysaccharopeptide (PSP) that derives from the deep-layer cultivated Trametes versicolor mycelia Cov-1 strain is a polysaccharide-protein conjugated. It has been used to enhance the immune function, to antagonize the side-effect induced by chemotherapy, to resist tumor activity and to strengthen the immunity of sub-health population in clinical. Animal experiments also showed that PSP has anti-inflammatory activity but the mechanism of its bidirectional regulatory effects are still not fully understood.Mouse macrophage cell line Raw264.7 was used as a model and Flow cytometry, ELISA and Western blotting methods were applied to analyze the Reactive Oxygen Species (ROS) production, Interleukin-6 (IL-6) expression and signaling molecules regulation in MAPK pathway.Flow cytometric results showed that PSP (25??100?g/mL) alone could promote the production of ROS and increased ROS fluorescent intensity to 8.58%, 9.69% (p<0.05) higher than that of control group, respectively. However, in LPS stimulated cells, PSP (5, 25??100?g/mL) could reduce the production of ROS and decreased ROS fluorescent intensity to 7.16%, 6.97%, 9.04% (p<0.05) lower than that of LPS group.ELISA results showed that PSP (25??100?g/mL) could increase IL-6 levels in a dose-dependent manner when it acted on macrophages alone. The expression of IL-6 were 20.89±1.46pg/mL and 31.16±3.78pg/mL after the cells treated with PSP at 25??100?g/mL, respectively, which were 57.54%, 135.05% higher than the level in control group (13.26±0.20pg/mL)(p<0.05). However, when cells stimulated with LPS were co-treated with PSP (25??100?g/mL) which was prior to LPS administration, the IL-6 expression levels were 44.46% and 31.88% less (p<0.01) than that in LPS group (1825.34±18.32pg/mL).Western blotting results demonstrated that PSP (5, 25??100?g/mL) alone could promote the phosphorylation of p38, JNK and ERK in a dose dependent manner. The expression levels of p-p38 were 63.74%, 90.11%, 168.13% more than that in control group. The expression levels of p-JNK were 4.27%, 23.45%, 35.17% higher than that in control group. The expression levels of p-ERK were 49.51%, 44.66% and 54.37% higher than that in control group and reached statistical significance in the high-concentration PSP group. In LPS stimulated groups, PSP could down-regulate the phosphorylation of p-p38, p-JNK and p-ERK and decreased p-p38 levels to 26.17%(5?g/mL), 31.71%(25?g/mL), 59.76%(100?g/mL) (p<0.05) less than that in LPS group, decreased p-JNK levels to 19.17%(5?g/mL), 39.39%(25?g/mL), 43.75%(100?g/mL) (p<0.05) lower than that in LPS group. The production of p-ERK levels were also decreased to 4.9%(5?g/mL), 50.64%(25?g/mL) (p<0.05), 57.72%(100?g/mL) (p<0.05) less than that in LPS group.In summary, PSP could activate normal macrophages and increase the production of ROS, IL-6 and MAPK signaling molecules p-p38, p-JNK, p-ERK. However, when macrophages were stimulated with LPS, PSP could reduce the high production of those LPS induced pro-inflammatory mediatorsTherefore PSP plays a double role that can regulate immune response of macrophage at different status by affecting the production of ROS, IL-6 and regulating MAPK signaling molecules.