8-Prenylkaempferol suppresses inducible nitric oxide synthase expression through interfering with JNK-mediated AP-1 pathway in murine macrophages

8-Prenylkaempferol is a prenylflavonoid isolated from the roots of Sophora flavescens, a Chinese herb with anti-inflammatory properties. However whether 8-prenylkaempferol itself displayed an anti-inflammatory activity remained unclear. In this study, we evaluated the effect of 8-prenylkaempferol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages. 8-Prenylkaempferol inhibited significantly LPS-induced NO production through suppressing inducible NO synthase (iNOS) expression at both protein and mRNA levels but failed to affect sodium nitroprusside-triggered NO production, iNOS enzyme activity, and cell viability. Further investigation of the mechanisms revealed that 8-prenylkaempferol inhibited LPS-induced c-Jun phosphorylation (a major component of activator protein-1, AP-1), but did not attenuate IkB-alpha degradation nor NF-kappaB nuclear translocation. Cellular signaling analysis using mitogen-activating protein kinase (MAPK) inhibitors including 2'-amino-3'-methoxyflavone (PD98059, MEK1/2 inhibitor), 4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580, p38 kinase inhibitor) and anthra[1-9-cd]pyrazol-6(2H)-one (SP600125, c-Jun N-terminal kinase inhibitor) demonstrated that extracellular signal-regulated kinase1/2 (ERK1/2), p38 and JNK all participated in LPS-stimulated iNOS expression and NO production, but 8-prenylkaempferol interfered selectively with JNK phosphorylation. On the other hand, LPS-induced c-Jun phosphorylation was attenuated in the presence of SP600125. We suggested that interfering with JNK-mediated c-Jun phosphorylation and thus blocking AP-1 activation might contribute to the suppression effects of 8-prenylkaempferol on iNOS. These findings provided the first molecular basis that 8-prenylkaempferol is an effective agent for attenuating pro-inflammatory NO induction.     

Arctigenin, a phenylpropanoid dibenzylbutyrolactone lignan, inhibits MAP kinases and AP-1 activation via potent MKK inhibition: the role in TNF-alpha inhibition

Arctigenin, naturally occurring in Bardanae fructus, Saussurea medusa, Arctium lappa L., Torreya nucifera and Ipomea cairica, is a phenylpropanoid dibenzylbutyrolactone lignan with antioxidant and anti-inflammatory activities. Previously, we showed that arctigenin potently inhibited the induction of nitric oxide synthase (iNOS) by lipopolysaccharide (LPS), which involved suppression of NF-kappaB activation. In the present study, we examined the effects of arctigenin on mitogen-activated protein (MAP) kinase activation in Raw264.7 cells and MAP kinase kinase (MKK) activity. The effect of arctigenin on activator protein-1 (AP-1) activation was also studied in association with tumor necrosis factor-alpha (TNF-alpha) expression. Immunoblot analysis showed that arctigenin inhibited phosphorylation of MAP kinases ERK1/2, p38 kinase and JNK and their activities in Raw264.7 cells treated with LPS. Arctigenin potently inhibited the activity of MKK1 in vitro with the IC(50) value of 1 nM. Gel shift and reporter gene analyses revealed that arctigenin inhibited LPS-inducible AP-1 binding to the AP-1 consensus oligonucleotide and AP-1-mediated reporter gene expression. In view of the potential role of AP-1 in the induction of TNF-alpha, we next examined the inhibitory effects of arctigenin on the expression of TNF-alpha. Arctigenin blocked TNF-alpha production and decreased the level of TNF-alpha mRNA in the cells exposed to LPS. These results showed that arctigenin inhibited activation of MAP kinases including ERK1/2, p38 kinase and JNK through the inhibition of MKK activities, leading to AP-1 inactivation, which might, at least in part, contribute to the inhibition of TNF-alpha production.     

Antioxidant activity of Vaccinium stamineum: exhibition of anticancer capability in human lung and leukemia cells

Fruit of deerberry [Vaccinium stamineum L.] were evaluated for their antioxidant capacity and anticancer properties in JB6 P (+) mouse epidermal cells, human lung and leukemia cells. Deerberries contain potent free radical scavenging activities. Pretreatment of JB6 P (+) mouse epidermal cells with deerberry fruit extracts produced an inhibition on the activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) induced by either 12- O-tetradecanoylphorbol 13-acetate (TPA) or ultraviolet-B (UVB). Deerberry fruit extracts also blocked TPA- or UVB-induced phosphorylation of ERKs and MEK 1/2, two upstream regulators of AP-1 and inhibited proliferation of human leukemia HL-60 cancer cells and human lung epithelial cancer A549 cells and induced apoptosis of HL-60 cells. These results suggest that the inhibition of TPA- or UVB-induced AP-1 and NF-kappaB activity, inhibition of HL-60 cells and cancer A549 cells proliferation and induction of apoptotic in human leukemia HL-60 cancer cells may be mediated through the ERKs and MEK 1/2 signal pathway.