Platycodin D and D3 isolated from the root of Platycodon grandiflorum modulate the production of nitric oxide and secretion of TNF-α in activated RAW 264.7 cells

Abstract

Platycodon D (PD) and D3 (PD3) isolated from Platycodon grandiflorum has been previously reported to show anti-inflammatory activities in rats. In this study, the production of proinflammatory cytokines, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) was examined in a macrophage like cell line, RAW 264.7 cells, in the presence of PD and PD3, oligosaccharide derivatives of oleanolic acid. RAW 264.7 cells activated with lipopolysaccharide (LPS; 1 μg/ml) and recombinant interferon-γ (rIFN-γ; 50 U/ml) were treated with various doses of PD and PD3 for 24 h. Supernatants were analyzed for the production of NO and TNF-α using Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. NO was inhibited in a dose-dependent manner by PD and PD3 (IC 50 of platycodin D≈15 uM, IC 50 PD3≈55 uM). The expression of inducible NOS (iNOS) was inhibited by these compounds, as measured by Western blot analysis, as well as the expression of iNOS mRNA, as measured by Northern blot analysis. RAW 264.7 cells were treated at various times after LPS and activation with PD. Treatment with PD up to 8 h after activation showed significant inhibition of NO, indicating that early signal transduction of NOS synthesis may be inhibited by PD. In contrast to NO, secretion of TNF-α as well as expression of TNF-α mRNA was increased by PD and PD3. TNF-α secretion from RAW 264.7 cells was measured at various times after LPS and rIFN-γ activation. Secretion of TNF-α was also increased up to 8 h postactivation, suggesting that PD may stimulate TNF-α synthesis or inhibit degradation of TNF-α mRNA. Oleanolic acid was without effect on both the production of NO and secretion of TNF-α. These data suggest a dichotomous regulation of these important proinflammatory mediators by PD and PD3.