Anti-inflammatory effects of Aureusidin in LPS-stimulated RAW264.7 macrophages via suppressing NF-κB and activating ROS- and MAPKs-dependent Nrf2/HO-1 signaling pathways.

Aureusidin, a naturally-occurring flavonoid, is found in various plants of Cyperaceae such as Heleocharis dulcis (Burm. f.) Trin., but its pharmacological effect and active mechanism are rarely reported. This study aimed to investigate the anti-inflammatory effect and action mechanism of Aureusidin in LPS-induced mouse macrophage RAW264.7 cells. The results suggested that lipopolysaccharide (LPS)-induced nitric oxide (NO), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) production were obviously inhibited by Aureusidin. Moreover, Aureusidin also significantly decreased the mRNA expression of various inflammatory factors in LPS-stimulated RAW264.7 cells. Furthermore, mechanistic studies showed that Aureusidin significantly inhibited nuclear transfer of nuclear factor-κB (NF-κB), while increasing the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) as well as expression of Nrf2 target genes such as heme oxygenase (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), but the addition of the HO-1 inhibitor Sn-protoporphyrin (Snpp) significantly abolished the anti-inflammatory effect of Aureusidin in LPS-stimulated RAW264.7 cells, confirming the view that HO-1 was involved in the anti-inflammatory effect. In addition, Aureusidin increased the levels of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) phosphorylation in RAW264.7 cells. Antioxidant N-acetylcysteine (NAC) or three MAPK inhibitors blocked the nuclear translocation of Nrf2 and HO-1 expression induced by Aureusidin, indicating that Aureusidin activated the Nrf2/HO-1 signaling pathway through ROS and MAPKs pathways. At the same time, co-treatment with the NAC blocked the phosphorylation of MAPKs. Results from molecular docking indicated that Aureusidin inhibited the NF-κB pathway by covalently binding to NF-κB. Thus, Aureusidin exerted the anti-inflammatory activity through blocking the NF-κB signaling pathways and activating the MAPKs and Nrf2/HO-1 signaling pathways. Based on the above results, Aureusidin may be an attractive therapeutic candidate for the inflammation-related diseases.

Ren J ，Su D ，Li L ，Cai H ，Zhang M ，Zhai J ，Li M ，Wu X ，Hu K ... -  《-》

A prenylated flavonoid, 10-oxomornigrol F, exhibits anti-inflammatory effects by activating the Nrf2/heme oxygenase-1 pathway in macrophage cells.

Prenylated flavonoids are a unique class of naturally occurring flavonoids that have various pharmacological activities. In the present study, we investigated the anti-inflammatory effect in murine macrophages of a prenylated flavonoid, 10-oxomornigrol F (OMF), which was isolated from the twigs of Morus alba (Moraceae). OMF inhibited the lipopolysaccharide (LPS)-induced production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 in RAW264.7 cells, as well as in mouse bone marrow-derived macrophages (BMMs). OMF also rescued LPS-induced septic mortality in ICR mice. LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α and IL-6 was also significantly suppressed by OMF treatment in RAW264.7 cells. Treatment of RAW264.7 cells with OMF induced heme oxygenase (HO)-1 mRNA and protein expression and increased the nuclear translocation of the nuclear factor-E2-related factor 2 (Nrf2) as well as the expression of Nrf2 target genes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1). Treatment of RAW264.7 cells with OMF increased the intracellular level of reactive oxygen species (ROS) and the phosphorylation levels of p38 mitogen-activated protein kinase (MAPK); co-treatment with the antioxidant N-acetyl-cysteine (NAC) blocked this OMF-induced p38 MAPK phosphorylation. Moreover, NAC, or SB203580 (a p38 MAPK inhibitor), blocked the OMF-induced nuclear translocation of Nrf2 and HO-1 expression, suggesting that OMF induces HO-1 expression by activating Nrf2 through the p38 MAPK pathway. Consistent with the notion that the Nrf2/HO-1 pathway has anti-inflammatory properties, inhibiting HO-1 significantly abrogated the anti-inflammatory effects of OMF in LPS-stimulated RAW264.7 cells. Taken together, these findings suggest that OMF exerts its anti-inflammatory effect by activating the Nrf2/HO-1 pathway, and may be a potential Nrf2 activator to prevent or treat inflammatory diseases.

Tran PL ，Tran PT ，Tran HNK ，Lee S ，Kim O ，Min BS ，Lee JH ... -  《-》

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 ... -  《-》

Anti-inflammatory activity of the water extract of Chloranthus serratus roots in LPS-stimulated RAW264.7 cells mediated by the Nrf2/HO-1, MAPK and NF-κB signaling pathways.

Chloranthus serratus is a traditional Chinese medicine for treating arthritis and bruises. To investigate the dose-effect relationship and molecular mechanisms of the water extract of C. serratus roots (WECR) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The cell viability was detected by CCK-8 method. One-step method, DCFH-DA fluorescence probe method and immunofluorescence method were used to detect nitric oxide (NO), reactive oxygen species (ROS) and p65 nuclear transcription, respectively. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) were detected by enzyme linked immunosorbent assay. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA were detected by quantitative real-time PCR. Western blotting was taken to determine the contents of the relevant proteins in the nuclear transcription factor E2 related factor 2/heme oxygenase-1 (Nrf2/HO-1), mitogen-activated protein kinases (MAPK) and nuclear factor-kappa B (NF-κB) pathways. The concentrations of 3, 30 and 300 μg/mL were optimized as low, medium and high concentrations of the WECR, respectively, and 1 μg/mL was selected as the optimal concentration of LPS to activate macrophages. The dose of the positive drug dexamethasone was 0.13 mg/mL. The WECR could not only inhibit LPS-induced cell differentiation and the overexpression of NO, IL-6, TNF-α, PGE2 and ROS but also promote the expression of Nrf2 and HO-1, and down-regulate the phosphorylation levels of ERK, JNK, p38 and p65. After the WECR treatment, the expression levels of iNOS and COX-2 mRNA and nuclear translocation of p65 were all inhibited. The WECR exerts its anti-inflammatory activity by inhibiting the MAPK and NF-κB pathways, activating the Nrf2/HO-1 pathway and down-regulating inflammatory factor levels in a dose-dependent manner.

Sun S ，Zhang J ，Li H ，Du Y ，Li S ，Li A ，Suo X ，Wang Y ，Sun Q ... -  《-》

Anti-inflammatory activity of Anchusa italica Retz. in LPS-stimulated RAW264.7 cells mediated by the Nrf2/HO-1, MAPK and NF-κB signaling pathways.

Anchusa italica Retz. (Boraginaceae) is an important medicinal plant for the treatment of meningitis and pneumonia in traditional Uygur medicines. To clarify the anti-inflammatory activity of A. italica, to reveal its molecular mechanisms, and to discover the anti-inflammatory active ingredients. Dried and crushed aerial parts of A. italica were extracted with 75% ethanol to yield crude extract (AICE) and AICE was fractionated to obtain petroleum ether extract (AIPE), dichloromethane extract (AIDE), ethyl acetate extract (AIEE), n-butanol extract (AIBE) and residues (AIW). By measuring the effects of AIPE, AIDE, AIEE, AIBE and AIW on cell viability and nitric oxide (NO) in Lipopolysaccharide (LPS) stimulated RAW264.7 cell lines, AIDE with the lowest cytotoxicity and NO contents was finally selected for further chemical and anti-inflammatory investigations. LC-MS/MS experiment was applied to analyze the chemical composition of AIDE. MTT and Griess methods were used to detect the cell viability and to quantify the nitrite levels in culture supernatants, respectively. Prostaglandin E2 (PGE2), interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) production was examined by ELISA assays. Real-time quantitative PCR was used to detect the expression of hemeoxygenase-1 (HO-1), Nrf2-mediated quinone oxidoreductase 1 (NQO-1), glutathione S-transferase A 1 (GSTA1) and glutathione S-transferase M 1 (GSTM1) mRNA. Western blot analysis was employed to examine the protein expression and enzymatic activities. In preliminary anti-inflammatory screening, AIDE showed the lowest cytotoxicity and the most significant inhibitory effect on the production of NO (the inhibitory is 89%) induced by LPS among the tested five extracts. Thirty-three compounds including twenty-five triterpenoids were identified by LC-MS/MS analysis. AIDE could inhibit LPS-induced the over-expression of NO, IL-6, PGE2, IL-1β and TNF-α and down-regulate the levels of extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), P38-MAPK (P38) and nuclear transcription factors κB-P65 (P65) phosphorylation. It promoted the mRNA expression level of HO-1, NQO-1, GSTA1 and GSTM1 and the protein expression level of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1. After the treatment of AIDE, P65 nuclear translocation was inhibited and Nrf2 nuclear translocation was increased. In addition, the protein expression of pyrolytic relevant protein nod-like receptor family pyrin domain-containing 3 (NLRP3) and IL-1β were decreased after the AIDE treatment. Anchusa italica Retz. exerted its anti-inflammatory activity by inhibiting the mitogen-activated protein kinase (MAPK), nuclear transcription factors κB (NF-κB) and pyrolytic relevant proteins, down-regulating inflammatory factor levels, and activating the Nrf2/HO-1 pathway. Triterpenoids might be its major active anti-inflammatory ingredients.

Duan X ，Li J ，Cui J ，Dong Y ，Xin X ，Aisa HA ... -  《-》