Attenuation of inflammatory responses by (+)-syringaresinol via MAP-Kinase-mediated suppression of NF-?B signaling in vitro and in vivo.
ABSTRACT: We examined the anti-inflammatory effects of (+)-syringaresinol (SGRS), a lignan isolated from Rubia philippinensis, in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells using enzyme-based immuno assay, Western blotting, and RT-PCR analyses. Additionally, in vivo effects of SGRS in the acute inflammatory state were examined by using the carrageenan-induced hind paw edema assay in experimental mice. As a result, treatment with SGRS (25, 50, and 100??M) inhibited protein expression of lipopolysaccharide-stimulated inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-?B) as well as production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-?), interleukin-1beta (IL-1?), and interleukin-6 (IL-6) induced by LPS. Moreover, SGRS also reduced LPS-induced mRNA expression levels of iNOS and COX-2, including NO, PGE2, TNF-?, IL-1?, and IL-6 cytokines in a dose-dependent fashion. Furthermore, carrageenan-induced paw edema assay validated the in vivo anti-edema effect of SGRS. Interestingly, SGRS (30?mg/kg) suppressed carrageenan-induced elevation of iNOS, COX-2, TNF-?, IL-1?, and IL-6 mRNA levels as well as COX-2 and NF-?B protein levels, suggesting SGRS may possess anti-inflammatory activities.
Project description:According to the GC-MS analysis, compositional variation was observed between samples of patchouli oil, of which an unknown compound identified as patchoulene epoxide (PAO) was found only in the long-stored oil, whose biological activity still remains unknown. Therefore, the present study aimed to evaluate the potential anti-inflammatory activity with three in vivo inflammatory models: xylene-induced ear edema, acetic acid-induced vascular permeability, and carrageenan-induced paw edema. Further investigation into its underlying mechanism on carrageenan-induced paw edema was conducted. Results demonstrated that PAO significantly inhibited the ear edema induced by xylene, lowered vascular permeability induced by acetic acid and decreased the paw edema induced by carrageenan. Moreover, PAO markedly decreased levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and nitric oxide (NO), but increased levels of interleukin-4 (IL-4) and interleukin-10 (IL-10). PAO was also shown to significantly downregulate the protein and mRNA expressions of cyclooxygenase-2 (COX-2) and inducible nitric-oxide synthase (iNOS). Western blot analysis revealed that PAO remarkably inhibited p50 and p65 translocation from the cytosol to the nucleus by suppressing IKKβ and IκBα phosphorylation. In conclusion, PAO exhibited potent anti-inflammatory activity probably by suppressing the activation of iNOS, COX-2 and NF-κB signaling pathways.
Project description:Diallyl disulfide (DADS) is the major organosulfur constituent in garlic, with a variety of pharmacological activities including antioxidant and anti-inflammatory. Here, we examined the potential antiedematous impact of DADS- versus carrageenan-mediated paw edema in mice. Carrageenan injection potentiated an inflammatory reaction as presented by the elevated serological C-reactive protein (CRP) levels and transcription of tumor necrosis factor-alpha (TNF-?, Tnf?), interleukin-1 beta (IL-1?, Il1b), interleukin-2 (IL-2, Il2), inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2 (COX-2, Ptgs2), prostaglandin E2 (PGE2), monocyte chemoattractant protein-1 (MCP-1, Ccl1), nuclear factor kappa B (NF-?B), and myeloperoxidase (MPO) activity, while interleukin-10 (IL-10) was declined in the injured paw tissue. Additionally, carrageenan elevated lipid peroxidation in terms of malondialdehyde (MDA) and decreased glutathione content (GSH). Remarkably, DADS was found to inhibit the inflammatory signaling, suppressed the developed oxidative damage, and protected the histopathological alterations in the inflamed paw tissue in response to carrageenan injection. Our findings suggest that DADS could be used as an alternative therapy used to alleviate the pathophysiological changes associated with the genesis of paw edema through its potent anti-inflammatory and antioxidant impacts.
Project description:Houttuynia cordata Thunb. has been used as a traditional medicine to treat a variety of ailments in Asian countries such as China, Japan, South Korea, and Thailand. In Thailand, H. cordata fermentation products (HCFPs) are commercially produced and popularly consumed throughout the country without experimental validation. Anti-inflammatory activity of H. cordata fresh leaves or aerial parts has previously been reported, however, the anti-inflammatory activity of the commercially available HCFPs produced by the industrialized process has not yet been investigated. The aim of this study was to evaluate in vitro and in vivo anti-inflammatory potential of the selected industrialized HCFP. LPS-induced RAW264.7 and carrageenan-induced paw edema models were used to evaluate the anti-inflammatory activity of HCFP. The phenolic acid components of HCFP aqueous and methanolic extracts were investigated using HPLC analysis. In RAW264.7 cells, the HCFP aqueous and methanolic extracts reduced NO production and suppressed LPS-stimulated expression of PGE2, iNOS, IL-1?, TNF-? and IL-6 levels in a concentration-dependent manner, however, less effect on COX-2 level was observed. In Wistar rats, 3.08 and 6.16 mL/kg HCFP reduced paw edema after 2 h carrageenan stimulation, suggesting the second phase anti-edematous effect similar to diclofenac (150 mg/kg). Whereas, 6.16 mL/kg HCFP also reduced paw edema after 1 h carrageenan stimulation, suggesting the first phase anti-edematous effect. Quantitative HPLC revealed the active phenolic compounds including syringic, vanillic, p-hydroxybenzoic and ferulic acids, which possess anti-inflammatory activity. Our results demonstrated for the first time the anti-inflammatory activity of the industrialized HCFP both in vitro and in vivo, thus validating its promising anti-inflammation potential.
Project description:: Prolonged inflammatory responses can lead to the development of several chronic diseases, such as autoimmune disorders and the development of natural therapeutic agents is required. A murine model was used to assess the anti-inflammatory effects of the megastigmane glucoside, icariside B2 (ICSB), and the assessment was carried out in vitro, and in vivo. The in vitro anti-inflammatory effects of ICSB were tested using LPS-stimulated BV2 cells, and the protein expression levels of inflammatory genes and cytokines were assessed. Mice were subcutaneously injected with 1% carrageenan (CA) to induce acute phase inflammation in the paw. Inflammation was assessed by measuring paw volumes hourly; subsequently, the mice were euthanized and the right hind paw skin was expunged and processed for reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. ICSB inhibits LPS-stimulated nitric oxide (NO) and prostaglandin E2 (PGE2) generation by reducing the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). ICSB also inhibits the COX-2 enzyme with an IC50 value of 7.80±0.26 µM. Molecular docking analysis revealed that ICSB had a strong binding affinity with both murine and human COX-2 proteins with binding energies of -8 kcal/mol and -7.4 kcal/mol, respectively. ICSB also reduces the manifestation of pro-inflammatory cytokines, such as TNF-?, IL-6, and IL-1?, at their transcriptional and translational level. ICSB hinders inhibitory protein ?B? (I?B?) phosphorylation, thereby terminating the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-?B) nuclear translocation. ICSB also represses the mitogen-activated protein kinases (MAPKs) signaling pathways. ICSB (50 mg/kg) showed an anti-edema effect in CA-induced mice and suppressed the CA-induced increases in iNOS and COX-2 protein levels. ICSB attenuated inflammatory responses by downregulating NF-?B expression through interference with extracellular signal-regulated kinase (ERK) and p38 phosphorylation, and by modulating the expression levels of iNOS, COX-2, TNF-?, IL-1?, and IL-6.
Project description:The present study aimed to test the anti-inflammatory and xanthine oxidase inhibitory activities of two synthesized molecules and compare them to routinely prescribed nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac and the serum urate-lowering drug, allopurinol. The anti-inflammatory effects of the designed compounds (A and B) were evaluated in carrageenan (CAR)-induced paw edema in mice. The levels of nitric oxide and myeloperoxidase activity were measured in paw skin using biochemical methods. Additionally, prostaglandin E2 (PGE2), C-reactive protein (CRP), cyclooxygenase-2 (Cox-2), tumor necrosis factor-? (TNF?), interleukin (IL)-1?, IL-2 and IL-10, and monocyte chemoattractant protein-1 (MCP1) were assessed by enzyme-linked immunosorbent assay (ELISA). The expression of inflammation-related genes was confirmed by real-time qPCR. The expression of inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-?B) were estimated using immunohistochemistry, and xanthine oxidase inhibitory activity was evaluated using an in vitro assay. The results revealed that compounds A and B decreased inflammation, as was observed by a reduction in the elevation of all the tested markers. In addition, the tested compounds markedly decreased paw swelling, mobilization of inflammatory cells, iNOS-, and NF-?B-immunoreactive cells in a mouse model of paw edema. Interestingly, both compounds were potent xanthine oxidase inhibitors as well as Cox inhibitors with higher activity in favor of compound B providing potential dual acting series of anti-hyperuricemic and anti-inflammatory therapeutic agents.
Project description:Inflammation is part of self-limiting non-specific immune response, which occurs during bodily injury. In some disorders the inflammatory process becomes continuous, leading to the development of chronic inflammatory diseases including cardiovascular diseases, diabetes, cancer etc. Several Indian tribes used the bark of Odina wodier (OWB) for treating inflammatory disorders. Thus, we have evaluated the immunotherapeutic potential of OWB methanol extract and its major constituent chlorogenic acid (CA), using three popular in vivo antiinflammatory models: Carrageenan- and Dextran-induced paw edema, Cotton pellet granuloma, and Acetic acid-induced vascular permeability. To elucidate the possible anti-inflammatory mechanism of action we determine the level of major inflammatory mediators (NO, iNOS, COX-2-dependent prostaglandin E2 or PGE2), and pro-inflammatory cytokines (TNF-?, IL-1?, IL-6, and IL-12). Further, we determine the toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88), c-Jun N-terminal kinases (JNK), nuclear factor kappa-B cells (NF-?B), and NF-kB inhibitor alpha (IK-B?) by protein and mRNA expression, and Western blot analysis in drug treated LPS-induced murine macrophage model. Moreover, we determined the acute and sub-acute toxicity of OWB extract in BALB/c mice. Our study demonstrated a significant anti-inflammatory activity of OWB extract and CA along with the inhibition of TNF-?, IL-1?, IL-6 and IL-12 expressions. Further, the expression of TLR4, NF-?Bp65, MyD88, iNOS and COX-2 molecules were reduced in drug-treated groups, but not in the LPS-stimulated untreated or control groups, Thus, our results collectively indicated that the OWB extract and CA can efficiently inhibit inflammation through the down regulation of TLR4/MyD88/NF-kB signaling pathway.
Project description:We previously reported that 3-(benzo[d]-1,3-dioxol-5-yl)-4-phenylfuran-2,5-dione (BPD) showed strong inhibitory effects on PGE(2) production. However, the exact mechanism for the anti-inflammatory effect of BPD is not completely understood. In this study, we investigated the molecular mechanism involved in the effects of BPD on inflammatory mediators in LPS-stimulated macrophages and animal models of inflammation.The expressions of COX-2, inducible NOS (iNOS), TNF-?, IL-6 and IL-1?, in LPS-stimulated RAW 264.7 cells and murine peritoneal macrophages, were determined by Western blot and/or qRT-PCR, respectively. NF-?B activation was investigated by EMSA, reporter gene assay and Western blotting. Anti-inflammatory effects of BPD were evaluated in vivo in carrageenan-induced paw oedema in rats and LPS-induced septic shock in mice.BPD not only inhibited COX-2 activity but also reduced the expression of COX-2. In addition, BPD inhibited the expression of iNOS, TNF-?, IL-6 and IL-1? at the transcriptional level. BPD attenuated LPS-induced DNA-binding activity and the transcription activity of NF-?B; this was associated with a decrease in the phosphorylation level of inhibitory ?B-? (I?B-?) and reduced nuclear translocation of NF-?B. Furthermore, BPD suppressed the formation of TGF-?-activated kinase-1 (TAK1)/TAK-binding protein1 (TAB1), which was accompanied by a parallel reduction of phosphorylation of TAK1 and I?B kinase (IKK). Pretreatment with BPD inhibited carrageenan-induced paw oedema and LPS-induced septic death.Taken together, our data indicate that BPD is involved in the dual inhibition of COX-2 activity and TAK1-NF-?B pathway, providing a molecular basis for the anti-inflammatory properties of BPD.
Project description:The edible blue pigments produced by gardenia fruits have been used as value-added colorants for foods in East Asia for 20 years. However, the biological activity of the blue pigments derived from genipin has not been reported.The anti-inflammatory effect of blue pigments was studied in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophage in vitro. The secretions of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) were inhibited in concentration-dependent manner by blue pigments. Real-time reverse-transcription polymerase chain reaction (Real-time RT-PCR) analyses demonstrated that the mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-6, and tumor necrosis factor alpha (TNF-?) was inhibited, moreover, ELISA results showed that the productions of IL-6 and TNF-? were inhibited. Cell-based ELISA revealed the COX-2 protein expression was inhibited. The proteome profiler array showed that 12 cytokines and chemokines involved in the inflammatory process were down-regulated by blue pigments. Blue pigments inhibited the nuclear transcription factor kappa-B (NF-?B) activation induced by LPS, and this was associated with decreasing the DNA-binding activity of p65 and p50. Furthermore, blue pigments suppressed the degradation of inhibitor of ?B (I?B) ?, Inhibitor of NF-?B Kinase (IKK) ?, IKK-?, and phosphorylation of I?B-?. The anti-inflammatory effect of blue pigments in vivo was studied in carrageenan-induced paw edema and LPS-injecting ICR mice. Finally, blue pigments significantly inhibited paw swelling and reduced plasma TNF-? and IL-6 production in vivo.These results suggest that the anti-inflammatory properties of blue pigments might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1?, and TNF-? expression through the down-regulation of NF-?B activation, which will provide strong scientific evidence for the edible blue pigments to be developed as a new health-enhancing nutritional food for the prevention and treatment of inflammatory diseases.
Project description:Erigeron annuus is a naturalized plant belonging to Compositae (asteraceae) family, which is called the annual fleabane, and commonly found at meadows and roadside. This study investigated the anti-inflammatory effects of the extract of E. annuus roots (EER), as assessed by the paw edema formation and histological analysis in rat, and the productions of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines in Raw264.7 murine macrophages. Carrageenan treatment promoted infiltration of inflammatory cells and caused swelling in the hind paw. Oral administrations of EER (0.3?g/kg and 1?g/kg) attenuated acute inflammation similar to the result using dexamethasone (1?mg/kg). Treatment of macrophages with lipopolysaccharide (LPS) simulated inflammatory condition: LPS significantly increased the productions of NO, PGE2, and proinflammatory cytokines. EER suppressed activation of macrophages, preventing the induction of iNOS and COX-2 protein expressions. LPS treatment induced phosphorylation of I- ? B ? and increased the level of nuclear NF- ? B protein, both of which were suppressed by concomitant treatment of EER. In conclusion, EER ameliorated acute inflammation in rats, and the induction of NO, PGE2, and proinflammatory cytokines in Raw264.7 cells. EER's effects may be associated with its inhibition of NF- ? B activation, suggesting its effect on inflammatory diseases.
Project description:CONTEXT:Ajuga ovalifolia Bur. et Franch. var. calantha (Diels) C. Y. Wu et C. Chen (Labiatae), a traditional Chinese medicine, has been used to treat several inflammatory diseases. OBJECTIVE:To assess the anti-inflammatory activity of ajudecumin A isolated from Ajuga ovalifolia var. calantha, and its possible mechanisms. MATERIALS AND METHODS:Lipopolysaccharide (LPS, 0.5??g/mL)-stimulated RAW264.7 macrophages were used to assess the anti-inflammatory activity of ajudecumin A (1-40??M) in vitro. Nitric oxide levels were evaluated by Griess reagent. The mRNA levels of iNOS, COX-2, TNF-?, IL-1? and IL-6 were determined using qRT-PCR. Phosphorylation of ERK, JNK, p38 MAPK and I?B? were detected by western Blot. To further assess the anti-inflammatory of ajudecumin A in vivo, mice were oral treated with ajudecumin A (10?mg/kg) or dexamethasone (0.25?mg/kg, positive control) for 5?days before administration of carrageenan or xylene. Paw and ear edema were then measured, respectively. RESULTS:Ajudecumin A (10-40??M) decreased LPS-induced nitric oxide production with an IC50 value of 16.19??M. Ajudecumin A (20 and 40??M) also attenuated cell spreading and formation of pseudopodia-like structures, and decreased the mRNA levels of iNOS (55.23-67.04%, p?<?0.001), COX-2 (57.58-70.25%, p?<?0.001), TNF-? (53.75-58.94%, p?<?0.01-0.001), IL-1? (79.41-87.85%, p?<?0.001) and IL-6 (54.26-80.52%, p?<?0.01-0.001) in LPS-activated RAW264.7 cells. Furthermore, ajudecumin A suppressed LPS-induced phosphorylation of ERK, p38 MAPK, and I?B?, as well as I?B? degradation (p?<?0.05-0.001). Finally, ajudecumin A (10?mg/kg) attenuated carrageenan- and xylene-induced inflammation in mice by about 28 and 24%, respectively. DISCUSSION AND CONCLUSIONS:Ajudecumin A exhibited a potent anti-inflammatory activity in vitro and in vivo through inhibition on NF-?B and ERK/p38 MAPK pathways, suggesting that ajudecumin A may be potentially developed as a lead compound in anti-inflammatory drug discovery.