Crocin is a water-soluble carotenoid pigment that is primarily used in various cuisines as a seasoning and coloring agent, as well as in traditional medicines for the treatment of edema, fever, and hepatic disorder. In this study, we demonstrated that crocin markedly induces the expression of heme oxygenase-1 (HO-1) which leads to an anti-inflammatory response. Crocin inhibited inducible nitric oxide synthase (iNOS) expression and nitric oxide production via downregulation of nuclear factor kappa B activity in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. These effects were abrogated by blocking of HO-1 expression or activity. Crocin also induced Ca2+ mobilization from intracellular pools and phosphorylation of Ca2+/calmodulin-dependent protein kinase 4 (CAMK4). CAMK4 knockdown and kinase-dead mutant inhibited crocin-mediated HO-1 expression, Nrf2 activation, and phosphorylation of Akt, indicating that HO-1 expression is mediated by CAMK4 and that Akt is a downstream mediator of CAMK4 in crocin signaling. Moreover, crocin-mediated suppression of iNOS expression was blocked by CAMK4 inhibition. Overall, these results suggest that crocin suppresses LPS-stimulated expression of iNOS by inducing HO-1 expression via Ca2+/calmodulin-CAMK4-PI3K/Akt-Nrf2 signaling cascades. Our findings provide a novel molecular mechanism for the inhibitory effects of crocin against endotoxin-mediated inflammation.

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BackgroundThe stem bark of Kalopanax pictus (KP) has been used in traditional medicine to treat rheumatoidal arthritis, neurotic pain and diabetes mellitus in China and Korea. In this study, the mechanism responsible for anti-inflammatory effects of KP was investigated.MethodsWe examined the effects of KP on NO production, nitric oxide synthase (iNOS) and HO-1 expression, NF-κB, Nrf2 and MAPK activation in mouse peritoneal macrophages.ResultsThe aqueous extract of KP inhibited LPS-induced NO secretion as well as inducible iNOS expression, without affecting cell viability. KP suppressed LPS-induced NF-κB activation, phosphorylation and degradation of IκB-α, phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Furthermore, KP induced HO-1 expression and Nrf2 nuclear translocation.ConclusionThese results suggest that KP has the inhibitory effects on LPS-induced NO production in macrophages through NF-κB suppression and HO-1 induction.

Achyranthoside E dimethyl ester (AEDE) is an oleanolic acid glycoside from Achyranthes japonica. In this study, we investigated the effects of AEDE on nitric oxide (NO) production and underlying molecular mechanisms in lipopolysaccharide (LPS)-stimulated macrophages. AEDE inhibited LPS-induced NO secretion as well as inducible NO synthase (iNOS) expression, without affecting cell viability. Further study demonstrated that AEDE induced heme oxygenase-1 (HO-1) gene expression. In addition, the inhibitory effects of AEDE on iNOS expression were abrogated by small interfering RNA-mediated knock-down of HO-1. Moreover, AEDE induced nuclear translocation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor that regulates HO-1 expression. AEDE-induced expression of HO-1 was inhibited by inhibitors of phosphatidylinositol 3-kinase (PI-3K) and extracellular signal regulated kinase (ERK1/2). AEDE phosphorylated Akt and ERK1/2 as well. Therefore, these results suggest that AEDE suppresses the production of pro-inflammatory mediator such as NO by inducing HO-1 expression via PI-3K/Akt/ERK-Nrf2 signaling. These findings provide the scientific rationale for anti-inflammatory therapeutic use of AEDE.

Achyranthoside C dimethyl ester (ACDE) is an oleanolic acid glycoside from Achyranthes japonica which has been used in traditional medicine for the treatment of edema and arthritis. In this study, we investigated the anti-inflammatory effects of ACDE in RAW264.7 macrophages. ACDE significantly induced heme oxygenase-1 (HO-1) gene expression in RAW264.7 cells, while ACDE improved LPS-induced toxicity of cells. And ACDE induced nuclear translocation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor that regulates HO-1 expression. Further study demonstrated that ACDE-induced expression of HO-1 was inhibited by inhibitors of phosphatidylinositol 3-kinase (PI-3K) (LY294002), c-Jun kinase (JNK) (SP600125), extracellular signal regulated kinase (ERK) (PD98059) and p38 kinase (SB203580). Moreover, ACDE phosphorylated Akt, JNK, ERK, and p38 MAPK. In addition, ACDE inhibited LPS-induced NO secretion as well as inducible NO synthase (iNOS) expression in a dose-dependent manner. The inhibitory effects of ACDE on iNOS expression were abrogated by small interfering RNA (siRNA)-mediated knock-down of HO-1. Therefore, these results suggest that ACDE suppresses the production of pro-inflammatory mediator such as NO by inducing HO-1 expression via PI-3K/Akt/MAPK-Nrf2 signaling pathway. These findings could help us to understand the active principle included in the roots of A. japonica and the molecular mechanisms underlying anti-inflammatory action of ACDE.Key words : Achyranthoside C dimethyl ester, heme oxygenase-1, inducible nitric oxide synthase, NF-E2-related factor 2, nitric oxide