Anti-inflammatory activity of Khayandirobilide A from Khaya senegalensis via NF-κB, AP-1 and p38 MAPK/Nrf2/HO-1 signaling pathways in lipopolysaccharide-stimulated RAW 264.7 and BV-2 cells.

Immunocytes-involved inflammation is considered to modulate the damage in various diseases. Herein, novel therapeutics suppressing over-activation of immunocytes could prove an effective strategy to prevent inflammation-related diseases. The objective of this study is to evaluate the anti-inflammatory activity of Khayandirobilide A (KLA), a new andirobin-type limonoid with modified furan ring isolated from the Khaya senegalensis (Desr.) A. Juss., and to explore its potential underlying mechanisms in LPS-stimulated inflammatory models. The structure of KLA was elucidated on the basis of 1D- and 2D-NMR spectroscopic data as well as HR-ESI-MS. As for its anti-inflammatory effect, the production of pro-inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 and BV-2 cells were measured by Griess reagent, ELISA and qRT-PCR. The relevant proteins including nuclear factor κB (NF-κB), p-AKT, p-p38 and Nrf2/HO-1 were investigated by western blot. Nuclear localisations of NF-κB, activator protein-1 (AP-1) and Nrf2 were also examined by western blot and immunofluorescence. KLA could inhibit the production of LPS-induced NO with IC50 values of 5.04 ± 0.14 µM and 4.97 ± 0.5 µM in RAW 264.7 and BV-2 cells, respectively. KLA also attenuated interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels. Further mechanistic studies demonstrated the activation of NF-κB and AP-1 were reduced by KLA. Moreover, KLA elevated expression of heme oxygenase-1(HO-1) via inducing Keap1 autophagic degradation and promoting Nrf2 nuclear translocation. Despite KLA induced the phosphorylation of mitogen-activated protein kinases (MAPKs) family, inhibiting the phosphorylation of p38 by its specific inhibitor SB203580 attenuated the degradation of KLA-induced Keap1, and then reduced KLA-induced Nrf2 nuclear translocation and HO-1 expression. Furthermore, SB203580, Brusatol (a Nrf2 specific inhibitor) and ZnPP (a HO-1 specific inhibitor) could partly reverse the suppressive effects of KLA on LPS-induced NO production and mRNA levels of pro-inflammatory genes. These data displayed that KLA possessed anti-inflammatory activity, which was attributed to inhibit the release of LPS-stimulated inflammatory mediators via suppressing the activation of NF-κB, AP-1, and upregulating the induction of p38 MAPK/Nrf2-mediated HO-1.

Zhou MM ，Zhang WY ，Li RJ ，Guo C ，Wei SS ，Tian XM ，Luo J ，Kong LY ... -  《-》

7-Methoxy-(9H-β-Carbolin-1-il)-(E)-1-Propenoic Acid, a β-Carboline Alkaloid From Eurycoma longifolia, Exhibits Anti-Inflammatory Effects by Activating the Nrf2/Heme Oxygenase-1 Pathway.

Eurycoma longifolia is an herbal medicinal plant popularly used in Southeast Asian countries. In the present study, we show that 7-methoxy-(9H-β-carbolin-1-il)-(E)-1-propenoic acid (7-MCPA), a β-carboline alkaloid isolated from E. longifolia, exerted anti-inflammatory effects by activating the nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. 7-MCPA inhibited lipopolysaccharide (LPS)-induced production of nitric oxide (NO), prostaglandin E2 (PGE2 ), and interleukin-6 (IL-6) in RAW264.7 cells and rescued C57BL/6 mice from LPS-induced lethality in vivo. LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and IL-6 was also significantly suppressed by treatment of 7-MCPA in RAW264.7 cells. 7-MCPA induced nuclear translocation of Nrf2 and increased transcription of its target genes, such as HO-1. Treating RAW264.7 cells with 7-MCPA increased the intracellular level of reactive oxygen species (ROS) and the phosphorylation level of p38 mitogen-activated protein kinase (MAPK); however, co-treatment with the antioxidant N-acetyl-cysteine (NAC) blocked 7-MCPA-induced p38 MAPK phosphorylation. Moreover, NAC or SB203580 (p38 MAPK inhibitor) blocked 7-MCPA-induced nuclear translocation of Nrf2, suggesting that 7-MCPA activated Nrf2 via a ROS-dependent p38 pathway. 7-MCPA induced HO-1 protein and mRNA expression and knockdown of Nrf2 with siRNA or SB203580 blocked 7-MCPA-mediated induction of HO-1 expression. Inhibiting Nrf2 or HO-1 abrogated the anti-inflammatory effects of 7-MCPA in LPS-stimulated RAW264.7 cells. We also demonstrated that 7-MCPA suppressed LPS-induced nuclear factor κB (NF-κB) activation. These results provide the first evidence that 7-MCPA exerts its anti-inflammatory effect by modulating the Nrf2 and NF-κB pathways and may be a potential Nrf2 activator to prevent or treat inflammatory diseases.

Nguyen HD ，Choo YY ，Nguyen TD ，Nguyen HN ，Chau VM ，Lee JH ... -  《-》

Xanthii fructus inhibits inflammatory responses in LPS-stimulated RAW 264.7 macrophages through suppressing NF-κB and JNK/p38 MAPK.

Xanthii fructus (XF) has long been used to treat a variety of inflammatory conditions in Korean traditional medicine, but the underlying mechanisms that could explain the anti-inflammatory actions of XF remain largely unknown. This study aimed to elucidate the anti-inflammatory effects of X. fructus (XF) and to examine its underlying molecular mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The effect of XF on LPS-induced mRNA and protein expressions of inflammatory mediators and cytokines were determined. Moreover, the activation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and the expression of heme oxygenase-1 (HO-1) were explored to elucidate the anti-inflammatory mechanisms. XF significantly inhibited LPS-induced production of inflammatory mediators, interleukin-6 (IL-6), nitric oxide (NO), and prostaglandin E2 (PGE2), without any cytotoxicity. However, it did not affect tissue necrosis factor (TNF)-α or IL-1β production in LPS-stimulated RAW 264.7 cells. Expression levels of inducible nitric oxide synthase (iNOS) mRNA and protein were inhibited dose-dependently by XF in LPS-stimulated RAW 264.7 cells, but there were no changes in cyclooxygenase-2 (COX-2) mRNA and protein. XF significantly attenuated LPS-induced phosphorylation and degradation of inhibitory kappa Bα (IκBα) and consequently reduced the nuclear translocation of p65 NF-κB. Pretreatment with XF also strongly inhibited the LPS-induced phosphorylation of p38 kinase and JNK, whereas the phosphorylation of ERK1/2 was not affected. In addition, XF led to an increase in HO-1 expression. Taken together, our findings support that XF inhibits LPS-induced inflammatory responses by blocking NF-κB activation, inhibiting JNK/p38 MAPK phosphorylation, and enhancing HO-1 expression in macrophages, suggesting that it could be an attractive therapeutic candidate for various inflammatory diseases.

Yeom M ，Kim JH ，Min JH ，Hwang MK ，Jung HS ，Sohn Y ... -  《-》

Herbal formula SC-E1 suppresses lipopolysaccharide-stimulated inflammatory responses through activation of Nrf2/HO-1 signaling pathway in RAW 264.7 macrophages.

Park JY ，Kwon YW ，Lee SC ，Park SD ，Lee JH ... -  《BMC Complementary and Alternative Medicine》

A new steroidal saponin, furotrilliumoside from Trillium tschonoskii inhibits lipopolysaccharide-induced inflammation in Raw264.7 cells by targeting PI3K/Akt, MARK and Nrf2/HO-1 pathways.

A new steroidal saponin, furotrilliumoside (FT) was isolated from the roots and rhizomes of Trillium tschonoskii Maxim. Its structure was elucidated on the basis of 1D- and 2D-NMR spectroscopic data as well as HR-ESI-MS analysis. FT showed superior activity of inhibiting NO production of RAW264.7 cells induced by lipopolysaccharide (LPS) in the preliminary biological screening. In order to develop novel therapeutic drug for acute and chronic inflammatory disorders, the anti-inflammatory activity and underlying mechanism of FT were investigated in LPS-induced RAW264.7 cells. The results showed that FT could reduce LPS-induced expression of inducible nitric oxide synthase (iNOS) and then resulted in the decrement of NO production. More meaningful, FT could down-regulate the expression of cyclooxygenase-2 (COX-2) and decrease the expressions of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β), in both gene and protein levels. In mechanism study, FT blocked the LPS-induced upregulation of phosphorylated phosphoinositide-3-kinase and Akt (PI3K/Akt). Furthermore, FT inhibited the translocation of nuclear factor-kappa B (NF-κB) through the prevention of inhibitory factor kappa B alpha (IκBα) phosphorylation and degradation and also suppressed the mitogen-activated protein kinases (MAPK) signaling pathway in LPS-stimulated RAW264.7 macrophages. In addition, FT upregulated heme oxygenase-1 (HO-1) expression via nuclear translocation of nuclear factor E2-related factor 2 (Nrf2). Taken together, FT might act as a natural agent to treat some inflammatory diseases by targeting PI3K/Akt, MARK and Nrf2/HO-1 pathways.

Yan T ，Yu X ，Sun X ，Meng D ，Jia JM ... -  《-》