Effects Of Ploysavone On NO,IL-6,TNF-α Production And Its Mechanism In Mouse Macrophages

Macrophages which are the basis of innate immune response play an important role in hostdefense in phagocytosis, cytotoxicity, anti-presenting, cytokines secretion, regulating internalenvironment and antitumor effects. In previous study, we found that Polysavone increased the immunityin broilers and promote the proliferation of lymphocytes. To further investigate its effects on immunesystem and its mechanism in mouse macrophages, vivo observation, cell culture, cytokines productionand its mRNA expression, signal transduction molecule were determined.Exp. One Effects of Polysavone on phagocytic capacity, metabolism ability in macrophage invivo.To investigate the effects of Polysavone on phagocytic capacity and metabolism ability in vivo, 50male BABL/c mouse was allotted randomly to 4 groups with 10 for each group and were administratedwith Poysavone at 300mg/kg·d,500 mg/kg·d,1000 mg/kg·d, respectively. Saline (0.9％) substitutePoysavone in the control group. The results showed that Polysavone increased phagocytic capacitysignificantly (P＜0.05) in peritoneal macrophages. But Polysavone have no significant effect onmetabolism ability.Exp. Two Effects of Polysavone on phagocytic capacity, the production of NO, IL-6 and TNF-αinRAW264.7In order to demonstrate the effects of Polysavone on phagocytic capacity, the production of NO,IL-6 and TNF-αin macrophages, macrophages was treated with different concentration ofPolysavone(0.2, 0.4, 0.6, 0.8, 1.0mg/ml), the results show a increased phagocytic capacity in adose-dependent manner.The phagocytic capacity of macrophage response to Polysavone was increasedsignificantly from 0.4 to 1.0mg/ml (P＜0.05). Meanwhile, the production of TNF-αwas increased byPolysavone from 0.6 to 1.0 mg/ml (P＜0.05). In contrast, NO and IL-6 production induced by LPS wasdecreased by Polysavone in a dose-dependent manner and began to display significant effects at0.6mg/ml (P＜0.05) and 0.2mg/ml (P＜0.01), respectively. These results demonstrated that Polysavonehave different roles in regulating secretion and phagocytic capacity in macrophage.Exp. Three Effects of Polysavone on NO, IL-6 and TNF-αmRNA expression in macrophageTo investigate to the molecular mechanism reponsbile for regulation of NO, IL-6. TNF-αsecretionin macrophage by Polysavone, the sceond experiment, mRNA quantitative experiments were carried outon iNOS, IL-6,TNF-α. genes. First, optimal condition of RT-PCR was determined.Then RAW264.7 wastreated with different concentration of Polysavone (0.2, 0.4, 0.6, 0.8, 1.0mg/ml) and LPS (10μg/ml)and determination of iNOS,IL-6 and TNF-α. mRNA expression was followed. From the experiment,wefound that the of TNF-αmRNA expression was dose-dependently increased by Polysavone.On the contratry, iNOS,IL-6 mRNA expression induced by LPS was inhibited by Polysavone dose-dependently. These results demonstrate Polysavone mediated NO, IL-6, TNF-αseretion at the level of transcription.Exp. Four Secretion and mRNA expression of TNF-αinduced by Polysavone were medieated through p38 MAPK in macrophagep38 MAPK plays an important role in regulation of TNF-αand inflammation in innate immune system. To further investigate if p38MAPK was involved in activation of TNF-αproduction, we first determined the optimum stimulative concentration and time of Polysavone, then treated macrophage with different concentration (0.01, 0.1, 1, 5, 10, 20μg/ml)of p38MAPK inhibitor (SB203580)followed with treatment of Polysavone at optimum stimulative concentration and time. It is found that SB203580 inhibited TNF-αproduction and its mRNA expression induced by Polysavone in dose-dependent manner. These results suggested that TNF-αproduction induced by Polysavone was mediated through p38MAPK by regulating its gene expression.