Potential Mechanism Of Polysaccharide From Agaricus Blazei Murrill In Regulating The Cell-mediated Immunity Of RAW 264.7 Through MAPK Signal Transduction Pathway

Agaricus blazei Murrill (ABM) a common folk remedy anticancer. The active ingredients, polysaccharides have been isolated and shown to have indirect tumor-suppressing activity via an immunological activation. The effect of polysaccharide from Agaricus blazei Murrill (ABMP) on RAW 264.7 by western blot and real-time reverse transcription polymerase chain reaction (RT-PCR) technology. The main results are as follows:1. The effect of ABMP at those concentrations of 4000,2000,1000,500,250,125,62.5,31.25, 15.62,7.8125,3.906,1.953 ?g/mL on the cell growth by MTT test. After 24 h the cell vitality by its determined. The test results show that the ABMP for RAW 264.7 macrophage inhibitory rate increased with the concentration increased, the optimum concentration of cell growth for 1000 ?g/mL. At the same time, it was found that treatment group which compared with control group, the greater JNK, ERK, p38 inhibitors concentration are 6.25 ?mol,20 ?mol and 6.25 ?mol.2. From RT-PCR results, gene expression quantity of JNK, ERK, p38 were extremely decreased in LPS-induced RAW 264.7 cells, with different concentrations of ABMP (500 ?g/mL,1000 ?g/mL,2000 ?g/mL) act on RAW 264.71 h, then used ABMP containing different concentration and LPS which the concentration was 1 ?g/mL to culture cells, after 6 h cultivating, cells were collected for RT-PCR detection to found relative expression of related genes. The results showed that different concentrations of ABMP can significantly suppress the JNK, ERK, p38 relative expression of mRNA, with RAW 264.7 induced by LPS. At the same time, the result found that the largest inhibition rate of each gene, when ABMP concentrations was 1000 ?g/mL. These results can show that ABMP can inhibit the mRNA expression of JNK, ERK, and p38 that RAW 264.7 macrophages in mice treated with LPS.3. From Western blot results, in RAW 264.7 cells treated with LPS, protein expression of JNK, ERK, p38 were decreased, phosphorylated of JNK, ERK, p38 were also as well. To serve as an internal calibration beta actin, protein, according to the results of western blot ABMP concentrations within 500-2000 ?g/mL, of LPS induced mice macrophage RAW 264.7 MAPK signaling pathways protein has a certain inhibitory effect, and the effect on the protein phosphorylation is evident. Among them, when ABMP concentrations for 1000 ?g/mL, the JNK protein, p-JNK, ERK1/2, p38 inhibition effect is the most significantly. When the concentration of polysaccharide of 500 ?g/mL, of p-ERK protein, p-p38 inhibition effect is the most significantly. Conclusion:Base on all the observations, the conclusion is MAPK signal transduction pathway was the potential mechanism of ABMP in regulating cell-mediated immunity of RAW 264.7 cells. ABMP promote macrophage RAW 264.7 cell proliferation, and through the MAPK signal transduction pathway adjust the immune function of macrophages RAW 264.7, JNK, ERK, p38 is the role of molecular targets.