Immunomodulatory activities of phlorizin metabolites in lipopolysaccharide-stimulated RAW264.7 cells

The immunomodulatory effects of two new phlorizin metabolites, phloretin 4-O-β-d-glucuronide (1), 6-methoxyl-phloretin-2-O-β-d-glucuronide (2), together with phloretin-2-O-β-d-glucuronide (3) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells were determined. 1-3 (1-5 μg/ml) significantly inhibited the production of NO (p < 0.01). At the concentration of 5 μg/ml, 2 and 3 further inhibited iNOS mRNA expression (p < 0.01 or 0.05), and 1-3 inhibited iNOS protein expression (p < 0.01). Conversely, they all promoted the proinflammatory cytokine TNF-α mRNA expression (p < 0.01). For IL-10 mRNA, 1 and 3, which are main metabolism forms in rat plasma, obviously promoted its expression (p < 0.01), while metabolite 2, which was only detected in rat urine, showed inhibitory activity, but 1-3 alone without LPS stimulation had no effect on the expression of both TNF-α and IL-10 mRNA expression. 1 further inhibited VEGF, CCL2 and CXCL1 mRNA expression at the concentration of 5-25 μg/ml (p < 0.01). These results indicated phloretin's metabolites with different structural forms showed different immunomodulatory activities.