The Regulation Effects Of Polyporus Polysaccharide On Polarized Macrophages

ObjectiveTo study the regulation effects of Polyporus polysaccharide (PPS) on polarized macrophages.Method:1. RAW264.7cells were polarized to M1subtype macrophage by treating with100ng/ml IFN-γ.The xpression of CD16/32, CD14, CD40and CD86on me mbrane surface of IFN-γ-induced RAW264.7cells were determined by flow c ytometry, and the expression of TNF-α, IL-1β and IL-6mRNA were detected by real-time quantitative PCR (RT-PCR).2. IFN-γ-induced RAW264.7cells were treated with PPS for24h. The exp ression of CD40, CD86, CD16/32and CD14were detected by flow cytometry. The expression of TNF-α, IL-1β, IL-6, ARG1and iNOS mRNA were detected by usi ng RT-PCR, and the expression of TNF-α, IL-1β,IL-6, IL-17, IL-10and TGF-β protein were detected by ELISA. The release of nitric oxide(NO) was as sayed by Grass.3. CD206and CD23was used as biomarkers to confirm IL-4induced-M2m acrophages by treating RAW264.7with20ng/ml of IL-4. IL-4-induced RAW264.7cells were treated with PPS of50μg/mL,100μg/mL or200μg/mL for24h. Then the expression of CD206, CD16/32and CD40were analyzed by flow cyto metry, and the mRNA expression of IL-10, TNF-α, IL-10and iNOS were detect ed by RT-PCR.4. RAW264.7cells were treated with PPS of50μg/mL,100μg/mL or200μg/mL for24h. The expression of CD40, CD16/32and CD86were detected by flow cytometry. The expression of IL-10, IL-1β, TNF-α and IL-6protein wer e analyzed by ELISA, and the expression of IL-10, IL-1β, TNF-α and IL-6m RNA were detected by RT-PCR.Results:1. After treated with INF-γ, the positive rate of CD16/32and CD86o f RAW264.7cells were high, and the mRNA expression of TNF-α, IL-10, Argl, IL-6and iNOS were increased.2. After treated with PPS of50μg/mL,100μg/ml,200μg/ml for24h, Th e rate of CD40,CD16/32and CD86were increased of IFN-γ-induced Raw264.7cells., pps can significantly enhance The mRNA levels of IL-10, IL-1β, TN F-α and IL-6increased, and protein of these cytokines also increased. The release of NO increased.3. After treated with IL-4, the positive rate of CD206of RAW264.7cel ls were high. After treated with PPS, the rate of CD16/32and CD86in IL-4-induced RAW264.7cells were high, the expression of CD206decreased, and the mRNA level of IL-1β, TNF-α increased.4. After PPS treatment, compared with blank control group, the expressio n of CD40, CD16/32and CD86increased, and the mRNA expression of TNF-α, IL-10, IL-1β and IL-6increased significantly.Conclusion:RAW264.7cells can be polarlized to M1subtype macrophages by using1OOng/mL of IFN-γ. RAW264.7cells can be polarlized to M2subtype macroph age by using20ng/mL of IL-4. PPS can not only enhances the release of inf lammatory cytokines such as IL-β, TNF-α and IL-6, but also enhanses the release of anti-inflammatory cytokines such as IL-10and TGF-Pin IFN-γ-induced RAW264.7cells. PPS enhances IL-1β, TNF-α in IL-4-induced RAW264.7cells. These results indicate PPS has the bi-directional regulation.