## 3 Results
### Inhibitory Effect of Compound K on LPS-Induced NO Production and iNOS and COX-2 Expression Levels  
To assess the inhibitory effects of compound K on LPS-induced NO production, RAW 264.7 cells were treated with various concentration of compound K and then incubated with or without LPS (1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) for 24 h. As shown in Figure 2A, compound K treatment in LPS-treated RAW 264.7 cells significantly inhibited the production of NO in a dose-dependent manner. From the results of RT-PCR and Western blot analysis, we found that compound K effectively suppressed LPS-induced mRNA and protein expression of iNOS, which is one of key enzymes generating NO from the amino acid L-arginine (Figure 2B,D). In addition, compound K suppressed LPS-induced COX-2 mRNA and protein levels (Figure 2B,D). Expression of COX-2 and iNOS in mRNA was reduced by 90% and 70%, respectively, and in protein levels was reduced by 68% and 54%, respectively, by 40 \(\mathrm{\SIUnitSymbolMicro m}\) of compound K (Figure 2C,E). These data indicate that compound K effectively inhibited LPS-induced inflammatory responses by suppressing the expression of iNOS and COX-2 in RAW264.7 macrophages. Inhibitory Effect of Compound K on LPS-Induced IL-1\(\beta\) and IL-6 Release and Their Expressions  
To investigate whether compound K could inhibit LPS-induced pro-inflammatory cytokines production, cells were pretreated with compound K for 1 h and then stimulated with LPS (1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) for 24 h. From the results of RT-PCR and EIA kit analyses, we found that LPS treatment in RAW 264.7 macrophages enhanced mRNA and release levels of pro-inflammatory cytokines IL-1\(\beta\) and IL-6 compared with those of untreated cells (Figure 3A). Our RT-PCR analysis showed that mRNA expression levels of IL-1\(\beta\) and IL-6, which are crucial inflammatory cytokines, were reduced with treatment with compound K (Figure 3A). Compound K (40 \(\mathrm{\SIUnitSymbolMicro m}\)) reduced the mRNA expression levels of IL-1\(\beta\) and IL-6 by 34% and 21%, respectively, compared with the control (Figure 3B). In an enzyme immunoassay, release levels of these cytokines IL-6 and IL-1\(\beta\) secreted from LPS-treated macrophages were significantly decreased by compound K treatment in a dose-dependent way (Figure 3C). In particular, compound K, at a concentration of 40 \(\mathrm{\SIUnitSymbolMicro m}\), effectively inhibited LPS-induced IL-1\(\beta\) release compared with those of the LPS-only control (Figure 3C). These results indicate that compound K inhibited LPS-induced pro-inflammatory cytokines production in RAW 264.7 macrophages. Figure 1: The chemical structure of compound K and its effect on cell viability. (**A**) A chemical contracture of compound K. (**B**) Raw 264.7 cells (1 \(\times\) 10\({}^{4}\)/well) were treated with Compound K (0–60 \(\mathrm{\SIUnitSymbolMicro m}\)) for 24 h. Cell viability was determined using the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazol-5-carboxanilide (XTT) assay. Values are means \(\pm\) S.D. of three independent experiments. (a \(p<\) 0.05, b \(p<\) 0.01). Figure 3: Effect of compound K on mRNA expression of IL-1\(\beta\) and IL-6 their secreted protein levels in LPS-stimulated Raw 264.7 cells. Cells were treated with compound K (20, 30, or 40 \(\upmu\)M), LPS was then added, and cells were incubated for a further 4 or 24 h. (**A**) The mRNA expression of IL-1\(\beta\) and IL-6 was determined by using RT-PCR and visualized on the gel. (**B**) The results were quantified using the ImageJ software. (**C**) Cytokine in the culture media were determined using enzyme immunoassay kit. Values are means \(\pm\) S.D. of three independent experiments (\({}^{a}\)\(p<\) 0.05, \({}^{b}\)\(p<\) 0.01, \({}^{c}\)\(p<\) 0.001). Figure 2: Inhibitory effect of compound K on lipopolysaccharide-induced NO production and iNOS and COX-2 expression levels in Raw 264.7. Raw 264.7 activated by 1 ug/mL LPS was treated with compound K (20, 30 or 40 \(\upmu\)M) for 4.