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Anti-inflammatory Potential of Petiveria alliacea on Activated RAW264.7 Murine Macrophages.

ABSTRACT: Defense and protection to multiple harmful stimuli are the inflammation, when is self-amplified and uncontrolled is the basis of the pathogenesis of a wide variety of inflammatory illness. The aim of this study was to evaluate if Petiveria alliacea could attenuate inflammation in a murine model of RAW264 macrophages the involved model and its involved mechanism.The ethanol extract from P. alliacea was precipitated with water and supernatant was used for this study (PW). The anti-inflammatory effects of PW were investigated through evaluating of the production of several cytokines, chemokines, and expression of nuclear factor-kappa B (NF-κB) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Also was determined the ability to decrease the oxidative stress in RAW264.7 cells with carboxy-2',7'-dichloro-dihydro-fluorescein diacetate.PW significantly suppress the secretion of prostaglandin E2, leukotriene C4, interleukin (IL)-1 β, IL-6, IL-10, interferon gamma nitric oxide (NO), inducible NO synthase, IL-1 β, IL-4, in RAW264.7 cells in a dose-dependent manner. In addition, PW also markedly inhibited the transcriptional activity of NF-κB. PW produced significant anti-inflammatory activity through inhibiting the production of inflammatory mediators through the NF-κB inactivation in the LPS-stimulated RAW24.7 cells.PW exerts significant antioxidant and anti-inflammatory activities, and this effect can be attributed in part, to the presence of dibenzyl disulfide, dibenzyl trisulfide pinitol, coumarin, myricetin, glutamyl-S-benzyl cysteine, and petiveriins A and B.Treatment with ethanol extract from Petiveria alliacea which was previously precipitated with water and supernatant (PE) was tested in LPS-stimulated RAW264.7 cells. PE suppressed the level of oxidative stress and the induction of proinflammatory mediators, as PGE2, LTC4, IL-1 ß, IL-6, IL-10, IFN- NO, iNOS, IL-1 ß, IL-4, in RAW264.7 macrophages through NF-B inactivation. These findings suggest that P. alliacea affords promising therapeutic in inflammatory diseases. Abbreviation used: COX-2: Ciclooxigenasa 2; DCFHDA: Carboxy-2',7'-dichloro-dihydro-fluorescein diacetate; DMEM: Dulbecco's modified eagle's medium; FBS: Fetal bovine serum; HSP70: Heat shock protein; IFN-γ: Interferon gamma; IL-1 β: Interleukin 1 β, IL-6: Interleukin 6; IL-10: Interleukin 10; IL-4: Interleukin 4; iNOS: Nitric oxide synthase; KCl: Potassium chloride; LPS: Lipopolysaccharides; LTC4: leukotriene C 4; MgCl2: Magnesium chloride; MTT: 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B-cells or transcriptional activity of nuclear factor-kB; NO: Nitric oxide; PBS: Phosphate-buffered saline; PGE2: Prostaglandin E2, PMSF: Phenylmethylsulfonyl fluoride; PTC: Chloroform extract from Petiveria alliacea; PE: Ethanol extract from Petiveria alliacea; PTH: Hexane extract from Petiveria alliacea; PW: Supernatant of PTE precipitated with water; RAW264.7: Cell line murine macrophages; ROS: Reactive oxygen species; TNF-α: Tumor necrosis factor.

SUBMITTER: Gutierrez RMP

PROVIDER: S-EPMC5538151 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

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Project description:Context Torin2 has various pharmacological properties. However, its anti-inflammatory activity has not been reported. Objective This study focused on the potential anti-inflammatory properties of Torin2 in lipopolysaccharide (LPS)-evoked RAW264.7 murine macrophages. The study aimed to shed light on the molecular mechanisms that ameliorate these effects. Methods Torin2 was applied to 100 ng/mL lipopolysaccharide-induced RAW 264.7 macrophages in vitro. Nitric oxide (NO) levels were detected using the Griess reagent kit. Prostaglandin E2 (PGE2), pro-inflammatory cytokines interleukin (IL)-1β, interleukin (IL)-6, and tumor necrosis factor in the supernatant fraction were determined using enzyme-linked immunosorbent assay (ELISA). Gene expression of pro-inflammatory cytokines, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were tested using real-time quantitative polymerase chain reaction (qPCR). Cyclooxygenase-2 and inducible nitric oxide synthase proteins, phosphorylation of mitogen-activated protein kinase (MAPK) subgroups, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, I-kappa-B-alpha (IκBα), and nuclear factor-kappa-B (NF-κB), and activation in extracts were detected via western blotting. Nuclear factor-kappa-B/p65 nuclear translocation was tested using an immunofluorescence assay. Results The results demonstrated that pre-treatment with Torin2 profoundly attenuated the lipopolysaccharide-stimulated levels of nitric oxide and prostaglandin E2, pro-inflammatory cytokines, messenger ribonucleic acid (mRNA), and protein expression of cyclooxygenase-2 and inducible nitric oxide synthase. Collectively, Torin2 pre-treatment notably weakened lipopolysaccharide-induced damage by reducing the phosphorylation of nuclear factor-kappa-B, p38, c-Jun N-terminal kinase, extracellular signal-regulated kinase proteins, and nuclear factor-kappa-B/p65 nuclear translocation. Conclusion Numerous pieces of evidence indicated that Torin2 reversed inflammatory activation by regulating nuclear factor-kappa-B and mitogen-activated protein kinase signaling pathways and provided a tentative potential candidate for preventing and treating inflammatory diseases. Torin2; RAW264.7 murine macrophages; Lipopolysaccharide; Anti-inflammation; Mechanisms.

Project description:ContextAjuga 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.ObjectiveTo assess the anti-inflammatory activity of ajudecumin A isolated from Ajuga ovalifolia var. calantha, and its possible mechanisms.Materials and methodsLipopolysaccharide (LPS, 0.5 μg/mL)-stimulated RAW264.7 macrophages were used to assess the anti-inflammatory activity of ajudecumin A (1-40 μM) in vitro. Nitric oxide levels were evaluated by Griess reagent. The mRNA levels of iNOS, COX-2, TNF-α, IL-1β and IL-6 were determined using qRT-PCR. Phosphorylation of ERK, JNK, p38 MAPK and IκBα 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.ResultsAjudecumin A (10-40 μM) decreased LPS-induced nitric oxide production with an IC50 value of 16.19 μM. Ajudecumin A (20 and 40 μM) 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-α (53.75-58.94%, p < 0.01-0.001), IL-1β (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 IκBα, as well as IκBα 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 conclusionsAjudecumin A exhibited a potent anti-inflammatory activity in vitro and in vivo through inhibition on NF-κB and ERK/p38 MAPK pathways, suggesting that ajudecumin A may be potentially developed as a lead compound in anti-inflammatory drug discovery.

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Anti-inflammatory Potential of Petiveria alliacea on Activated RAW264.7 Murine Macrophages.

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