## 4 Discussion  
We have demonstrated that compound K effectively inhibited the inflammatory response in LPS-treated RAW 264.7 macrophages and zebrafish. Our previous study compared the anti-inflammatory effects of ginseng Rb1 and compound K to investigate how the biotransformation of ginseng affects their biological activity, and we showed that compound K, rather than Rb1, significantly inhibited the inflammatory response. However, the mechanisms by which compound K is effective against inflammation are unknown. Therefore, in this study we confirmed the anti-inflammatory effect of compound K in LPS-stimulated Raw 264.7 macrophages and zebrafish models and investigated its molecular signaling pathways. Compound K reduced NO production dose-dependently both in vivo and in vitro. Excessive levels of NO, induced by LPS in macrophages, are associated with inflammatory diseases and are involved in other negative cellular physiologies such as mutagenesis, DNA structural damage, and formation of N-nitrooamines [29; 30; 31]. Our data provide evidence that compound K reduces LPS-induced NO production dose-dependently and down-regulates iNOS, which is closely related to the synthesis of NO. COX-2, another inflammatory marker, is also associated with the production of pro-inflammatory molecules, such as prostaglandin E2, and promotes pro-inflammatory signaling cascades. [PERSON] et al. (2001) reported that COX-2 was stimulated in angiogenesis-related diseases, such as rheumatoid arthritis and psoriasis [32]. In the present study, we reconfirm that compound K effectively suppressed LPS-induced mRNA and protein expression of COX-2 in macrophages. Inflammatory cytokines play important roles in the extent of inflammation and recruit other immune cells implicated in the pathogenesis of inflammatory conditions, such as rheumatoid arthritis and septic shock [33]. IL-1B is an important inflammatory cytokine produced by LPS-activated macrophages. During the inflammatory response, it increased secretion of IL-1B leads to cell or tissue damage. IL-6, a pro-inflammatory cytokine, acts as an endogenous pyrogen in addition to its multiple effects on the immune system and particularly on hematopoiesis [34; 35; 36]. Reduced secretion of IL-1B and IL-6, pro-inflammatory cytokines, from macrophages may retard inflammatory responses to LPS stimulation. In the present study, IL-1B and IL-6 mRNA expression levels were decreased markedly in LPS-treated cells after treatment with compound K. In addition, compound K significantly attenuated the release of pro-inflammatory cytokines in a dose-dependent manner. Therefore, these results indicate that compound K may have an anti-inflammatory effect. The regulation of inflammatory cytokines and inflammatory responses are regulated transcriptionally by NF-kB transcription factors. Macrophages activated by LPS accelerate IxB\(\alpha\) phosphorylation, which leads to its subsequent degradation [37]. After the degradation of IxB\(\alpha\), the activated NF-kB is translocated into the nucleus and binds to NF-kB-binding sites in the promoter regions of inflammatory mediators to induce their transcription, promoting the expression of inflammatory genes, including iNOS, COX-2, and the inflammatory cytokines, IL-6 and IL-1B [38]. In the present study, to evaluate the effect of compound K on NF-kB translocation, we determined the expression levels of phosphorylated IxB\(\alpha\) and nuclear and cytosolic p65 by Western blot analysis using anti-NF-kB p65 and IxB\(\alpha\) antibodies. Our results showed that compound K suppressed the expression levels of phosphorylated IxB\(\alpha\) and inhibited p65 translocation into the nucleus in LPS-treated cells, suggesting that compound K has anti-inflammatory effects via the suppression of IxB\(\alpha\) phosphorylation and NF-kB translocation in LPS-stimulated Raw 264.7 cells. MAPK signaling pathways regulate signal transduction and inflammatory mediators via activation of NF-kB [8].