Effects Of Apple Polyphenols On Gene Expression And Production Of NO And Cytokines In Murine Macrophage

Apple polyphenols is a multicomponent polyphenol extract from apple,which has functions of free radical scavenging,bacteriostatic action,anticancer,anti-mutagenize,anti-aging and so on.In this study,effects of different concentrations of apple polyphenols on the vitality and growth of murine macrophages was detected by MTT assay and morphological observationin in this study.Effects of apple polyphenols on gene expression profile in murine macrophages was analyze by using gene chip;6 differentially expressed gene were identified by real-time fluorescence quantitative PCR;Impacts of apple polyphenols on production of NO,TNF-α,IL-1α and IFN-γ in murine macrophages was measured by NO micromethod assay kit and ELISA kit,the results are as follows:MTT assay and morphological observation showed that the concentration of apple polyphenols was more than 20μg/mL,the growth of macrophages was significantly inhibited in comparison with the control grou(p<0.05),10μg/mL of apple polyphenols were significantly promoted on the growth of murine macrophages(p<0.05),5μg/mL of apple polyphenols was no statistically significant difference(p>0.05);Polyphenols concentration was more than 25μg/mL,the volume of murine macrophages became small round,necrosis and crushed,apple polyphenols concentration was at the range of 5-20μg/mL,the majority of the murine macrophages were round or oval and a very few were spindle shaped.As a result,10μg/mL of apple polyphenols is the maximum safe concentration.Data of apple polyphenols on murine macrophages were analyzed by gene chip technique,the results showed that there were 3149 differentially expressed genes based on standard deviation of two times,1540 genes were up-regulated expression and 1609 genes down-regulated expression.Gene ontology results showed that these differentially expressed genes were mainly related to protein and nucleotide binding,DNA repairation、cytoplasm,cytoskeleton and the nucleus,etc.The related pathways included steroid biosynthesis,ribosome synthesis in eukaryotes,biosynthesis of antibiotic,etc.And then 6 differential genes of the TNF signaling pathway were verified by qPCR.As a result,Map3k8,Mlkl and Tnf were significantly down-regulated(p<0.05),Pik3 ca,Cflar,Atf2 were not significantly down-regulated(p>0.05).Results of NO and cytokines tests showed that 2.5μg/mL,5μg/mL and 10μg/mL of apple polyphenols on the production of NO was significantly decreased in comparison with the control group(p<0.05),0.625μg/mL and 1.25μg/mL of apple polyphenols on the production of NO was no significant difference(p>0.05);10μg/mL of apple polyphenols could significantly down-regulate the expression of inflammatory factors TNF-α,IL-1α and IFN-γ(p<0.05).This study showed that 1540 genes were up-regulated expression and 1609 genes down-regulated expression,these differentially expressed genes are mainly related to protein and nucleotide binding,DNA repairation,etc,which provided the basis for the further study of the biological effects of apple polyphenols;Apple polyphenols may mediate anti-tumor mechanism or the occurrence of inflammatory reaction on macrophages by affecting the expression of related genes by TNF signaling pathway;Apple polyphenols could exert anti-inflammatory or immunomodulatory effect through decreasing the level of TNF-α,IL-1α,IFN-γ as well as generation of NO.