AIM OF THE STUDY: The molecular mechanisms of the anti-inflammatory properties of M. accedens are not yet understood. Therefore, we examined those mechanisms using a methanol extract of M. accedens (Ma-ME) and determined the target molecule in macrophages.
MATERIALS AND METHODS: We evaluated the anti-inflammatory effects of Ma-ME in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and in an HCl/EtOH-triggered gastritis model in mice. To investigate the anti-inflammatory activity, we performed a nitric oxide (NO) production assay and ELISA assay for prostaglandin E2 (PGE2). RT-PCR, luciferase gene reporter assays, western blotting analyses, and a cellular thermal shift assay (CETSA) were conducted to identify the mechanism and target molecule of Ma-ME. The phytochemical composition of Ma-ME was analyzed by HPLC and LC-MS/MS.
RESULTS: Ma-ME suppressed the production of NO and PGE2 and the mRNA expression of proinflammatory genes (iNOS, IL-1β, and COX-2) in LPS-stimulated RAW264.7 cells without cytotoxicity. Ma-ME inhibited NF-κB activation by suppressing signaling molecules such as IκBα, Akt, Src, and Syk. Moreover, the CETSA assay revealed that Ma-ME binds to Syk, the most upstream molecule in the NF-κB signal pathway. Oral administration of Ma-ME not only alleviated inflammatory lesions, but also reduced the gene expression of IL-1β and p-Syk in mice with HCl/EtOH-induced gastritis. HPLC and LC-MS/MS analyses confirmed that Ma-ME contains various anti-inflammatory flavonoids, including quercetin, daidzein, and nevadensin.
CONCLUSIONS: Ma-ME exhibited anti-inflammatory activities in vitro and in vivo by targeting Syk in the NF-κB signaling pathway. Therefore, we propose that Ma-ME could be used to treat inflammatory diseases such as gastritis.
AIM OF THE STUDY: To evaluate the anti-inflammatory activity as well as the preliminary mechanism of S. ferruginea parasitizing on Tecoma stans.
MATERIALS AND METHODS: The anti-inflammatory capability of freeze-dried stem aqueous extract was assessed via inhibition of inflammatory cytokines interleukin- (IL-) 1β, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) production in lipopolysaccharide (LPS) and interferon-γ (IFN-γ) stimulated RAW 264.7 macrophages. The underlying anti-inflammatory mechanism was deciphered through reverse transcriptase and real time quantitative polymerase chain reactions (RT-PCR and qPCR) for inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1β, and TNF-α mRNA expression.
RESULTS: The results exhibited that aqueous extract of freeze-dried S. ferruginea stem sample concentration-dependently inhibited IL-1β protein production along with the down regulation of iNOS and IL-1β mRNA expression. Moreover, it significantly suppressed the protein release of IL-6 and IL-10 in a concentration-dependent manner. However, it slightly reduced TNF-α at higher sample concentration (250 μg/mL) without affecting the mRNA expression levels of COX-2 and TNF-α.
CONCLUSIONS: This study suggests that S. ferruginea parasitizing on Tecoma stans exerted anti-inflammatory capability attributed to inhibition of iNOS and IL-1β mRNA expression, NO creation, IL-1β, IL-6, IL-10, and TNF-α protein production, indicating this plant might be a useful plant-derived candidate against inflammation.
OBJECTIVE: The antioxidant, cytotoxic, and protective effects of a series of synthesized 2- trifluoromethylquinazolines (2, 4, and 5) and quinazolinones (6-8) in lipopolysaccharide (LPS)- murine microglia (BV2) and hydrogen peroxide (H2O2)-mouse neuroblastoma-2a (N2a) cells were investigated.
METHOD: The antioxidant activity of synthesized compounds was evaluated with ABTS and DPPH assays. The cytotoxic activities were determined by MTS assay in BV2 and N2a cells. The production of nitric oxide (NO) in LPS-induced BV2 microglia cells was quantified.
RESULTS: The highest ABTS and DPPH scavenging activities were observed for compound 8 with 87.7% of ABTS scavenge percentage and 54.2% DPPH inhibition. All compounds were noncytotoxic in BV2 and N2a cells at 5 and 50 μg/mL. The compounds which showed the highest protective effects in LPS-induced BV2 and H2O2-induced N2a cells were 5 and 7. All tested compounds, except 4, also reduced NO production at concentrations of 50 μg/mL. The quinazolinone series 6-8 exhibited the highest percentage of NO reduction, ranging from 38 to 60%. Compounds 5 and 8 possess balanced antioxidant and protective properties against LPS- and H2O2-induced cell death, thus showing great potential to be developed into anti-inflammatory and neuroprotective agents.
CONCLUSION: Compounds 5 and 7 were able to protect the BV2 and N2a cells against LPS and H2O2 toxicity, respectively, at a low concentration (5 μg/mL). Compounds 6-8 showed potent reduction of NO production in BV2 cells.
RESULTS: The BAEC of Groups 1 and 2 demonstrated moderate to severe endothelial lysis, suggestive of acute cellular injury. In general, severity of the ultrastructural changes increased with the time of incubation but no significant difference (p > 0.05) in the severity of the cellular changes between Groups 1 and 2 was observed in the first 18 h. The severity of lesions became significant (p
METHODS: Endotoxemic shock was induced in sheep by administration of an escalating dose of lipopolysaccharide, after which they subsequently received either no fluid bolus resuscitation or a 0.9% saline bolus. Lung tissue, bronchoalveolar fluid (BAL) and plasma were analysed by real-time PCR, ELISA, flow cytometry and immunohistochemical staining to assess inflammatory cells, cytokines, hyaluronan and matrix metalloproteinases.
RESULTS: Endotoxemia was associated with decreased serum albumin and total protein levels, with activated neutrophils, while the glycocalyx glycosaminoglycan hyaluronan was significantly increased in BAL. Quantitative real-time PCR studies showed higher expression of IL-6 and IL-8 with saline resuscitation but no difference in matrix metalloproteinase expression. BAL and tissue homogenate levels of IL-6, IL-8 and IL-1β were elevated.
CONCLUSIONS: This data shows that the inflammatory response is enhanced when a host with endotoxemia is resuscitated with saline, with a comparatively higher release of inflammatory cytokines and endothelial/glycocalyx damage, but no change in matrix metalloproteinase levels.
MATERIALS AND METHODS: In this study, DET (0.625. 1.25 and 2.5 mg/kg, i.p.) was administered in rats for 21 days and those animals were challenged with single injection of LPS (250 μg/kg, i.p.) for 7 days. Cognitive and behavioral assessment was carried out for 7 days followed by molecular assessment on brain hippocampus. Statistical significance was analyzed with one-way analysis of variance followed by Dunnett's test to compare the treatment groups with the control group.
KEY FINDINGS: DET ameliorated LPS-induced neuroinflammation by suppressing major pro-inflammatory mediators such as iNOS and COX-2. Furthermore, DET enhanced the anti-inflammatory cytokines and concomitantly suppressed the pro-inflammatory cytokines and chemokine production. DET treatment also reversed LPS-induced behavioral and memory deficits and attenuated LPS-induced elevation of the expression of AD markers. DET improved synaptic-functionality via enhancing the activity of pre- and post-synaptic markers, like PSD-95 and SYP. DET also prevented LPS-induced apoptotic neurodegeneration via inhibition of PARP-1, caspase-3 and cleaved caspase-3.
SIGNIFICANCE: Overall, our studies suggest DET can prevent neuroinflammation-associated memory impairment and neurodegeneration and it could be developed as a therapeutic agent for the treatment of neuroinflammation-mediated and neurodegenerative disorders, such as AD.
METHODS: The release of prostaglandin E2 (PGE2) and pro-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β in a culture supernatant was determined by ELISA. Determination of cyclooxygenase-2 (COX-2) protein and the activation of MAPKs molecules (JNK, ERK and p38 MAPK), NF-κB and Akt in LPS-induced U937 human macrophages were investigated by immunoblot technique. The relative gene expression levels of COX-2 and pro-inflammatory cytokines were measured by using qRT-PCR. The major metabolites of P. amarus were qualitatively and quantitatively analyzed in the extract by using validated reversed-phase high performance liquid chromatography (HPLC) methods.
RESULTS: P. amarus extract significantly inhibited the production of pro-inflammatory mediators (TNF-α, IL-1β, PGE2) and COX-2 protein expression in LPS-induced U937 human macrophages. P. amarus-pretreatment also significantly downregulated the increased mRNA transcription of pro-inflammatory markers (TNF-α, IL-1β, and COX-2) in respective LPS-induced U937 macrophages. It downregulated the phosphorylation of NF-κB (p65), IκBα, and IKKα/β and restored the degradation of IκBα, and attenuated the expression of Akt, JNK, ERK, and p38 MAPKs phosphorylation in a dose-dependent manner. P. amarus extract also downregulated the expression of upstream signaling molecules, TLR4 and MyD88, which play major role in activation of NF-κB, MAPK and PI3K-Akt signaling pathways. The quantitative amounts of lignans, phyllanthin, hypophyllahtin and niranthin, and polyphenols, gallic acid, geraniin, corilagin, and ellagic acid in the extract were determined by HPLC analysis.
CONCLUSION: The study revealed that P. amarus targeted the NF-κB, MAPK and PI3K-Akt signaling pathways to exert its anti- inflammatory effects by downregulating the prospective inflammatory signaling mediators.
METHODS: This study evaluated the functional constituents, antioxidant and anti-inflammatory activities of Malaysian Ganoderma lucidum aqueous extract (GLE) and Egyptian Chlorella vulgaris ethanolic extract (CVE). Also, the synergistic, addictive or antagonistic activities of the combination between the two extracts (GLE-CVE) were studied. Expression of inducible nitric oxide synthase, cyclooxygenase-2, and nuclear factor-kappa B, as well as levels of nitric oxide, tumor necrosis factor (TNF)-α, lipid peroxidation, reduced glutathione and antioxidant enzymes were determined using in vitro model of lipopolysaccharide-stimulated white blood cells.
OBJECTIVE: In this study, we aim to investigate the involvement of heme oxygenase-1 (HO-1) in the anti-inflammatory effects of ZnC in lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages.
MATERIALS AND METHODS: We used immunoblotting analysis to evaluate the involvement of HO-1 in the anti-inflammatory effects of ZnC and the signaling pathway involved was measured using Dual luciferase reporter assay.
RESULTS: Results from immunoblotting analysis demonstrated that pretreatment of cells with ZnC enhanced the expression of HO-1 in RAW 264.7 cells. Pretreatment of cells with HO-1 inhibitor (tin protoporphyrin IX dichloride) significantly attenuated the inhibitory effects of ZnC on nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression and NF-κB activation in LPS-induced RAW 264.7 cells, suggesting that HO-1 play an important role in the suppression of inflammatory responses induced by ZnC. Furthermore, results from co-immunoprecipitation of Nrf2 and Keap1 and dual luciferase reporter assay showed that pretreatment of ZnC was able to activate the Nrf2 signaling pathway. Treatment of cells with p38 inhibitor (SB203580), c-Jun N-terminal kinase inhibitor (SP600125), and MEK 1/2 inhibitor (U0126) did not significantly suppress the induction of HO-1 by ZnC. Moreover, our present findings suggest that the effects of ZnC on NO production, HO-1 expression, and Nrf2 activation were attributed to its Zn subcomponent, but not l-carnosine.
CONCLUSION: Pretreatment with ZnC was able to activate Nrf2/HO-1 signaling pathway, thus suppressing the expression of inflammatory mediators, such as NO and iNOS in LPS-induced RAW 264.7 cells.
AIM OF THE STUDY: Endothelial barrier dysfunction is a pathological hallmark of many diseases and can be caused by lipopolysaccharides (LPS) stimulation. Therefore, this study aims to investigate the possible barrier protective effects of tHGA upon LPS-stimulated inflammatory responses in human umbilical vein endothelial cells (HUVECs).
MATERIALS AND METHODS: HUVECs were pretreated with tHGA prior to LPS stimulation, where inflammatory parameters including permeability, monocyte adhesion and migration, and release of pro-inflammatory mediators were examined. Additionally, the effect of tHGA on F-actin rearrangement and adhesion protein expression of LPS-stimulated HUVECs was evaluated.
RESULTS: It was found that pretreatment with tHGA inhibited monocyte adhesion and transendothelial migration, reduced endothelial hyperpermeability and secretion of prostaglandin E2 (PGE2). Additionally, tHGA inhibited cytoskeletal rearrangement and adhesion protein expression on LPS-stimulated HUVECs.
CONCLUSION: As the regulation of endothelial barrier dysfunction can be one of the therapeutic strategies to improve the outcome of inflammation, tHGA may be able to preserve vascular barrier integrity of endothelial cells following LPS-stimulated dysfunction, thereby endorsing its potential usefulness in vascular inflammatory diseases.