LPS-induced Intracellular Reactive Oxygen Species Research Articles

Abarema cochliacarpos reduces LPS-induced inflammatory response in murine peritoneal macrophages regulating ROS-MAPK signal pathway

Ethnopharmacological relevanceAbarema cochliacarpos (Gomes) Barneby and Grimes (Fabaceae), known by the vulgar name of Babatenã, has been traditionally used in Northeast Brazil, as an anti-inflammatory remedy. Previous studies have demonstrated its anti-inflammatory and antiulcer effects in skin lesion, alcohol gastric ulcer and acute and chronic colitis. AimsThe present study was designed to evaluate the antioxidant and anti-inflammatory effects of the butanolic fraction from A. cochliacarpos (BFAC) and its major flavonoid, (+)-catechin, in LPS-stimulated murine peritoneal macrophages. Moreover, we studied the role of mitogen-activated protein kinase (MAPK)s and NF-kB signaling pathways possibly involved in the beneficial effects. Materials and methodsThe quantification of the extract was carried out by ultra-performance liquid chromatography analysis. Cell viability was determined using SRB assay. Nitric oxide (NO) production was analyzed by Griess method and intracellular reactive oxygen species (ROS) by fluorescence analysis. In addition, cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) expression, MAPK activation and IkappaBalpha (IKBα) degradation, were determined by Western blot. ResultsAfter BFAC characterization, (+)-catechin was revealed as its major constituent. Both BFAC and (+)-catechin, exerted significant anti-oxidant and anti-inflammatory effects inhibiting LPS-induced intracellular ROS and NO production in peritoneal macrophages. Additionally, the extract but also its major component reduced pro-inflammatory proteins expression probably through c-Jun N-terminal kinase and p38 MAPK signaling pathways. ConclusionThese data suggest that the beneficial effects of BFAC might be mediated, at least in part, by the presence of (+)-catechin. Conclusively our findings confirm the potential of A. cochliacarpos as a new therapeutic strategy for the management of inflammatory and oxidative stress-related diseases.

Inhibitory effects and molecular mechanisms of garlic organosulfur compounds on the production of inflammatory mediators

Garlic is used for both culinary and medicinal purposes by many cultures. The garlic organosulfur compounds (GOSCs) are thought to be bioactive components. This study aims to clarify the antiinflammatory effects and molecular mechanisms of GOSCs in both cell and animal models. RAW264.7 cells were treated with six kinds of GOSCs to screen their influence on cyclooxygenase-2 and inducible nitric oxide synthase expression by Western blotting. Prostaglandin E2 and nitrite were measured by ELISA and Griess reaction, respectively. Cytokines in culture medium were assayed by the multiplex technology. Proteins were detected by Western blotting. Mouse paw edema was induced by LPS. The results revealed that diallyl trisulfide (DATS) was a strongest inhibitor for cyclooxygenase and inducible nitric oxide synthase among GOSCs, and reduced the levels of LPS-induced IL-6, IL-10, IL-12(p70), KC, MCP-1, and TNF-α. Cellular signaling analysis revealed that DATS downregulated AKT1/TGF-β-activated kinase-mediated mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. Furthermore, DATS activated Nrf2-mediated expression of HO-1 and NQO1 and reduced LPS-induced intracellular reactive oxygen species, which may contribute to suppress inflammatory mediator production. Finally, in vivo data demonstrated that DATS attenuated LPS-induced mouse paw edema. DATS as a potential inhibitor revealed antiinflammatory effect in both cell and animal models by downregulating AKT1/TGF-β-activated kinase-mediated NFκB and MAPK signaling pathways.

Antioxidant and Anti-Inflammatory Potential of Hesperetin Metabolites Obtained from Hesperetin-Administered Rat Serum: An Ex Vivo Approach

In recent years much attention has been focused on the pharmaceutical relevance of bioflavonoids, especially hesperidin and its aglycon hesperetin in terms of their antioxidant and anti-inflammatory actions. However, the bioactivity of their metabolites, the real molecules in vivo hesperetin glucuronides/sulfates produced after ingestion, has been poorly understood. Thus, the study using an ex vivo approach is aimed to compare the antioxidant and anti-inflammatory activities of hesperidin/hesperetin or hesperetin metabolites derived from hesperetin-administered rat serum. We found that hesperetin metabolites (2.5-20 μM) showed higher antioxidant activity against various oxidative systems, including superoxide anion scavenging, reducing power, and metal chelating effects, than that of hesperidin or hesperetin. The data also showed that pretreatment of hesperetin metabolites (1-10 μM) within the range of physiological concentrations, compared to hesperetin, significantly inhibited nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production, as evidenced by the inhibition of their precursors, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein levels without appreciable cytotoxicity on LPS-activated RAW264.7 macrophages or A7r5 smooth muscle cells. Concomitantly, hesperetin metabolites dose-dependently inhibited LPS-induced intracellular reactive oxygen species (ROS). Furthermore, hesperetin metabolites significantly downregulate LPS-induced nuclear factor-κB (NF-κB) activation followed by the suppression of inhibitor-κB (I-κB) degradation and phosphorylation of c-Jun N-terminal kinase1/2 (JNK1/2) and p38 MAPKs after challenge with LPS. Hesperetin metabolites ex vivo showed potent antioxidant and anti-inflammatory activity in comparison with hesperidin/hesperetin.

Glutathione peroxidase-1 modulates lipopolysaccharide-induced adhesion molecule expression in endothelial cells by altering CD14 expression.

CD14 contributes to LPS signaling in leukocytes through formation of toll-like receptor 4/CD14 receptor complexes; however, a specific role for endogenous cell-surface CD14 in endothelial cells is unclear. We have found that suppression of glutathione peroxidase-1 (GPx-1) in human microvascular endothelial cells increases CD14 gene expression compared to untreated or siControl (siCtrl)-treated conditions. Following LPS treatment, GPx-1 deficiency augmented LPS-induced intracellular reactive oxygen species accumulation, CD14 expression, and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein expression compared to LPS-treated control cells. GPx-1 deficiency also transiently augmented LPS-induced vascular cell adhesion molecule-1 (VCAM-1) expression. Adenoviral overexpression of GPx-1 significantly diminished LPS-mediated responses in adhesion molecule expression. Consistent with these findings, LPS responses were also greater in endothelial cells derived from GPx-1-knockout mice, whereas adhesion molecule expression was decreased in cells from GPx-1-overexpressing transgenic mice. Knockdown of CD14 attenuated LPS-mediated up-regulation of ICAM-1 and VCAM-1 mRNA and protein, and it mitigated the effects of GPx-1 deficiency on LPS-induced adhesion molecule expression. Taken together, these data suggest that GPx-1 modulates the endothelial cell response to LPS, in part, by altering CD14-mediated effects.

Anti-inflammatory Effects of the Roots of Alpinia pricei Hayata and Its Phenolic Compounds

Alpinia pricei Hayata is cultivated throughout Asia and is an endemic plant in Taiwan. The leaf and root of this plant are used for traditional wrapping of food and as a cooking substitute for fresh ginger. The aim of this work was to study the in vitro anti-inflammatory effects of ethanol extracts from A. pricei Hayata (EEAP) and its phenolic compounds. High-performance liquid chromatography (HPLC) profiling indicated that EEAP contained caffeic acid, chlorogenic acid, ferulic acid, p-hydroxybenzoic acid, rutin, apigenin, curcumin and pinocembrin. EEAP and its phenolic compounds, apigenin, curcumin, and pinocembrin, inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in RAW 264.7 cells. Furthermore, EEAP, apigenin, curcumin, and pinocembrin decreased LPS-mediated induction of protein and mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW 264.7 cells. In addition, EEAP and its major active compound pinocembrin inhibited LPS-induced nuclear translocation of nuclear factor-kappaB (NF-kappaB) and NF-kappaB-mediated reporter gene expression. EEAP and pinocembrin also significantly inhibited LPS-induced intracellular reactive oxygen species (ROS) production in RAW 264.7 cells. When these results are taken together, they indicate that EEAP and pinocembrin suppressed LPS-induced NO and PGE(2) production by inhibition of NF-kappaB nuclear translocation and ROS generation.

Nobiletin from citrus fruit peel inhibits the DNA-binding activity of NF-κB and ROS production in LPS-activated RAW 264.7 cells

Post-treatment with nobiletin (5,6,7,8,3′,4′-hexamethoxy flavone), which was purified from the fruit peel of Citrus sunki Hort. ex Tanaka, at concentration 6–50 μM significantly suppressed NF-κB transcriptional activation, NO and PGE 2 production, and iNOS and COX-2 protein expression in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Nobiletin inhibited neither LPS-induced phosphorylation/degradation of inhibitory κB-α nor LPS-induced nuclear translocation of NF-κB. However, it interrupted the DNA-binding activity of activated NF-κB. As reactive oxygen species (ROS) are also known to regulate the activation of NF-κB, we tested the effect of nobiletin on LPS-induced ROS generation. Nobiletin significantly inhibited LPS-induced intracellular ROS production in RAW 264.7 cells. These results suggest that nobiletin may exert an anti-inflammatory effect through the interruption of NF-κB DNA-binding activity and the suppression of ROS generation.

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by phenolic (3 E)-4-(2-hydroxyphenyl)but-3-en-2-one in RAW 264.7 macrophages

The large amount of nitric oxide (NO) produced by inducible NO synthase (iNOS) contributes to cellular injury in inflammatory disease. In the present study, a novel synthetic compound (3 E)-4-(2-hydroxyphenyl)but-3-en-2-one (HPB) was found to inhibit lipopolysaccharide (LPS)-induced NO generation, but not through the inhibition of iNOS activity, in RAW 264.7 macrophages. Administration of HPB into mice also inhibited the LPS-induced increase in serum nitrite/nitrate levels. To evaluate the underlying mechanisms of HPB inhibition of NO generation, the expression of the iNOS gene in RAW 264.7 macrophages was examined. HPB abolished the LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in a similar concentration-dependent manner. LPS-induced nuclear factor-κB (NF-κB) DNA binding and NF-κB-dependent reporter gene activity were both significantly inhibited by HPB. This effect was mediated through the inhibition of inhibitory factor-κBα (IκBα) phosphorylation and degradation, and of p65 nuclear translocation. HPB had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinases (MAPK), and c-Jun NH 2-terminal kinase (JNK). However, HPB suppressed the LPS-induced intracellular reactive oxygen species (ROS) production. These results indicate that HPB down-regulates iNOS gene expression probably through the inhibition of LPS-induced intracellular ROS production, which has been implicated in the activation of NF-κB.