METHODS: Genes and proteins of phase II detoxifying antioxidant enzymes were analyzed by QuantiGenePlex 2.0 Assay and Western blot analysis.
RESULTS: PB significantly induced genes and proteins of phase II and antioxidant enzymes, NAD(P)H quinone oxidoreductase 1, and catalase in aging mice (p < 0.05). The expression of these enzymes were stimulated via translocation of Nrf2 into the nucleus, indicating the involvement of ARE, a cis-acting motif located in the promoter region of nearly all phase II genes.
CONCLUSIONS: PB was testified for the first time to induce cytoprotective genes through the Nrf2/ARE signaling pathway, thus unraveling the antioxidant mechanism of PB during the aging process.
Methods: Antioxidant properties were assessed through various radical (DPPH, ABTS, and nitric oxide) scavenging assays and determination of total phenolic content and ferric reducing antioxidant power level. ARPE-19 cells were preincubated with samples before the addition of GO (to generate H2O2). Cell viability, change in intracellular reactive oxygen species (ROS), H2O2 levels in cell culture supernatant, and gene expression were assessed.
Results: F2 showed higher antioxidant levels than the extract when assessed for radical scavenging activities and ferric reducing antioxidant power. F2 protected the ARPE-19 cells against GO-H2O2-induced oxidative stress by reducing the production of H2O2 and intracellular reactive oxygen species. This was achieved by the activation of nuclear factor erythroid 2-related factor 2 (Nrf2/NFE2L2) and superoxide dismutase (SOD2), as well as downregulation of nitric oxide producer (NOS2) at the transcriptional level.
Conclusions: The results showed that myricetin derivatives from S. malaccense have the capacity to exert considerable exogenous antioxidant activities and stimulate endogenous antioxidant activities. Therefore, these derivatives have excellent potential to be developed as therapeutic agents for managing DR.
OBJECTIVE: To assess the ability of liquorice root samples to induce Nrf2 activation correlating to their potential chemopreventive property.
METHODS: The ability of nine methanolic extracts of liquorice root samples, collected from various geographical origins, to induce Nrf2 activation was determined by the luciferase reporter assay using the ARE-reporter cell line, AREc32. The antioxidant properties were determined by the 2,2-diphenyl-1-picryhydrazyl (DPPH) and the ferric-reducing antioxidant power (FRAP) assays.
RESULTS: All extracts exhibited free-radical-scavenging property (RC50 = 136.39-635.66 µg/mL). The reducing capacity of ferrous ion was 214.46-465.59 μM Fe(II)/g. Nrf2 activation indicated that all extracts induced expression of ARE-driven luciferase activity with a maximum induction of 2.3 fold relative to control. These activities varied for samples from one geographical location to another.
CONCLUSIONS: The present findings add to the existing knowledge of cancer chemoprevention by plant-derived extracts or purified phytochemicals, particularly the potential use of liquorice for this purpose. Copyright © 2016 John Wiley & Sons, Ltd.
METHODS: Rat CIRI models were established via middle cerebral artery occlusion (MCAO). Primary nerve cells were isolated and cultured in fetal rat cerebral cortex in vitro, and oxygen-glucose deprivation/reperfusion (OGD/R) models of primary nerve cells were induced. After intervention with DN with different concentrations in MCAO rats and OGD/R nerve cells, 2,3,5-triphenyltetrazolium chloride staining was used to quantify cerebral infarction size in CIRI rats. Modified neurological severity score was utilized to assess neurological performance. Histopathologic staining and live/dead cell-viability staining was used to observe apoptosis. Levels of glutathione (GSH), superoxide dismutase (SOD), reactive oxygen species (ROS) and malondialdehyde (MDA) in tissues and cells were detected using commercial kits. DN level in serum and cerebrospinal fluid of MCAO rats were measured by liquid chromatography tandem mass spectrometry. In addition, expression levels of proteins like Kelch like ECH associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nfr2) and heme oxygenase 1 (HO-1) in the Nrf2/HO-1 pathway, and apoptosis-related proteins like Cleaved caspase-3, BCL-2-associated X protein (Bax) and B-cell lymphoma-2 (Bcl-2) were determined by Western blot and immunofluorescence.
RESULTS: DN can significantly enhance neurological function recovery by reducing cerebral infarction size and weakening neurocytes apoptosis in MCAO rats. It was further found that DN could improve oxidative stress (OS) injury of nerve cells by bringing down MDA and ROS levels and increasing SOD and GSH levels. Notably, DN exerts its pharmacological influences through entering blood-brain barrier. Mechanically, DN can reduce Keap1 expression while activate Nrf2 and HO-1 expression in neurocytes.
CONCLUSIONS: The protective effect of DN on neurocytes have been demonstrated in both in vitro and in vivo circumstances. It deserves to be developed as a potential neuroprotective agent through regulating the Nrf2/HO-1 signaling pathway to ameliorate neurocytes impairment caused by OS.
MATERIALS AND METHODS: An in vitro monocyte recruitment model utilizing THP-1 and HUVECs was developed to evaluate TNF-α-induced monocyte adhesion and trans-endothelial migration. To study the role of Nrf2 for MA-mediated anti-inflammatory effects, Nrf2 inhibitor ML385 was used as the pharmacological inhibitor. The expression of Nrf2, monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1), cluster of differentiation 36 (CD36), and scavenger receptor type A (SR-A) in HUVECs and THP-1 macrophages were investigated using RT-qPCR and Western blotting. The NF-κB activity was determined using NF-κB (p65) Transcription Factor Assay Kit.
KEY FINDINGS: The results showed opposing effects of MA on Nrf2 expression in HUVECs and THP-1 macrophages. MA suppressed TNF-α-induced Nrf2 expression in HUVECs, but enhanced its expression in THP-1 macrophages. Combined effects of MA and ML385 suppressed MCP-1, VCAM-1, and SR-A expressions. Intriguingly, at the protein level, ML385 selectively inhibited SR-A but enhanced CD36 expression. Meanwhile, ML385 further enhanced MA-mediated inhibition of NF-κB activity in HUVECs. This effect, however, was not observed in THP-1 macrophages.
SIGNIFICANCE: MA attenuated foam cell formation by suppressing VCAM-1, MCP-1, and SR-A expression, as well as NF-κB activity, possibly through Nrf2 inhibition. The involvement of Nrf2 for MA-mediated anti-inflammatory effects however differs between HUVECs and macrophages. Future investigations are warranted for a detailed evaluation of the contributing roles of Nrf2 in foam cells formation.
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.