Triggers for the Nrf2/ARE Signaling Pathway and Its Nutritional Regulation: Potential Therapeutic Applications of Ulcerative Colitis.

Ulcerative colitis (UC), which affects millions of people worldwide, is characterized by extensive colonic injury involving mucosal and submucosal layers of the colon. Nuclear factor E2-related factor 2 (Nrf2) plays a critical role in cellular protection against oxidant-induced stress. Antioxidant response element (ARE) is the binding site recognized by Nrf2 and leads to the expression of phase II detoxifying enzymes and antioxidant proteins. The Nrf2/ARE system is a key factor for preventing and resolving tissue injury and inflammation in disease conditions such as UC. Researchers have proposed that both Keap1-dependent and Keap1-independent cascades contribute positive effects on activation of the Nrf2/ARE pathway. In this review, we summarize the present knowledge on mechanisms controlling the activation process. We will further review nutritional compounds that can modulate activation of the Nrf2/ARE pathway and may be used as potential therapeutic application of UC. These comprehensive data will help us to better understand the Nrf2/ARE signaling pathway and promote its effective application in response to common diseases induced by oxidative stress and inflammation.

Chemical Space Charting of Different Parts of Inula nervosa Wall.: Upregulation of Expression of Nrf2 and Correlated Antioxidants Enzymes.

The edible and medicinal part of Inula nervosa Wall. (Xiaoheiyao) is confined to its root without sufficient phytochemical and biological investigation. In this study, the secondary metabolites of root, stem, leaf, and flower of I. nervosa Wall. were visualized using Global Natural Products Social Molecular Networking (GNPS), MolNetEnhancer, XCMS(xcmsonline.scripps.edu) analysis, and `ili mapping based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) data to reveal their chemical differences. Among the 11 kinds of chemical repertoires annotated by MolNetEnhancer and 16 hits against the GNPS library, 10-isobutyryloxy-8,9-epoxythymol isobutyrate (1) was revealed as the most dominant and responsible marker between the roots and the other parts. Moreover, a battery of unique MS features as well as differential markers were discovered from different parts of the plant. The chemical differences contribute to the bioactivity differences, which presented in the 2,2-diphenyl-1-picryl-hydrazyl (DPPH)assay and H2O2-insulted HepG2 cells and were in significant correlations with the contents of 1. real-time reverse transcription polymerase chain reaction (RT-PCR)results demonstrated that I. nervosa Wall. extracts upregulated the mRNA expression of nuclear factor E2-related factor 2(Nrf2), heme oxygenase 1(HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), manganese superoxide dismutase (MnSOD), and glutamate-cysteine ligase catalytic subunit (GCLC) actors involved in antioxidative response in H2O2-challenged HepG2 cells. These findings support the roots of I. nervosa Wall. as active parts of Xiaoheiyao, and also indicate the potential antioxidant activities of other parts.

Farrerol Directly Targets GSK-3ß to Activate Nrf2-ARE Pathway and Protect EA.hy926 Cells against Oxidative Stress-Induced Injuries.

Oxidative stress-mediated endothelial injury is considered to be involved in the pathogenesis of various cardiovascular diseases. Farrerol, a typical natural flavanone from the medicinal plant Rhododendron dauricum L., has been reported to show protective effects against oxidative stress-induced endothelial injuries in our previous study. However, its action molecular mechanisms and targets are still unclear. In the present study, we determined whether farrerol can interact with glycogen synthase kinase 3ß- (GSK-3ß-) nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling, which is critical in defense against oxidative stress. Our results demonstrated that farrerol could specifically target Nrf2 negative regulator GSK-3ß and inhibit its kinase activity. Mechanistic studies proved that farrerol could induce an inhibitory phosphorylation of GSK-3ß at Ser9 without affecting the expression level of total GSK-3ß protein and promote the nuclear translocation of Nrf2 as well as the mRNA and protein expression of its downstream target genes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in EA.hy926 cells. Further studies performed with GSK-3ß siRNA and specific inhibitor lithium chloride (LiCl) confirmed that GSK-3ß inhibition was involved in farrerol-mediated endothelial protection and Nrf2 signaling activation. Moreover, molecular docking and molecular dynamics studies revealed that farrerol could bind to the ATP pocket of GSK-3ß, which is consistent with the ATP-competitive kinetic behavior. Collectively, our results firstly demonstrate that farrerol could attenuate endothelial oxidative stress by specifically targeting GSK-3ß and further activating the Nrf2-ARE signaling pathway.

Keap1/Nrf2/ARE signaling unfolds therapeutic targets for redox imbalanced-mediated diseases and diabetic nephropathy.

Hyperglycemia/oxidative stress has been implicated in the initiation and progression of diabetic complications while the components of Keap1/Nrf2/ARE signaling are being exploited as therapeutic targets for the treatment/management of these pathologies. Antioxidant agents like drugs, nutraceuticals and pure compounds that target the proteins of this pathway and their downstream genes hold the therapeutic strength to put the progression of this disease at bay. Here, we elucidate how the modulation of Keap1/Nrf2/ARE had been exploited for the treatment/management of end-stage diabetic kidney complication (diabetic nephropathy) by looking into (1) Nrf2 nuclear translocation and phosphorylation by some protein kinases at specific amino acid sequences and (2) Keap1 downregulation/Keap1-Nrf2 protein-protein inhibition (PPI) as potential therapeutic mechanisms exploited by Nrf2 activators for the modulation of diabetic nephropathy biomarkers (Collagen IV, Laminin, TGF-ß1 and Fibronectin) that ultimately lead to the amelioration of this disease progression. Furthermore, we brought to limelight the relationship between diabetic nephropathy and Keap1/Nrf2/ARE and finally elucidate how the modulation of this signaling pathway could be further explored to create novel therapeutic milestones.

KLF2 Protects against Osteoarthritis by Repressing Oxidative Response through Activation of Nrf2/ARE Signaling In Vitro and In Vivo.

Osteoarthritis (OA) is a multifactorial and inflammatory disease characterized by cartilage destruction that can cause disability among aging patients. There is currently no effective treatment that can arrest or reverse OA progression. Kruppel-like factor 2 (KLF2), a member of the zinc finger family, has emerged as a transcription factor involved in a wide variety of inflammatory diseases. Here, we identified that KLF2 expression is downregulated in IL-1ß-treated human chondrocytes and OA cartilage. Genetic and pharmacological overexpression of KLF2 suppressed IL-1ß-induced apoptosis and matrix degradation through the suppression of reactive oxygen species (ROS) production. In addition, KLF2 overexpression resulted in increased expression of heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) through the enhanced nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Further, Nrf2 inhibition abrogated the chondroprotective effects of KLF2. Safranin O/fast green and TUNEL staining demonstrated that adenovirus-mediated overexpression of KLF2 in joint cartilage protects rats against experimental OA by inhibiting cartilage degradation and chondrocyte apoptosis. Immunohistochemical staining revealed that KLF2 overexpression significantly decreases MMP13 expression caused by OA progression in vivo. This in vitro and in vivo study is the first to investigate the antioxidative effect and mechanisms of KLF2 in OA pathogenesis. Our results collectively provide new insights into OA pathogenesis regulated by KLF2 and a rationale for the development of effective OA intervention strategies.

Anti-neuroinflammatory effects of Ephedra sinica Stapf extract-capped gold nanoparticles in microglia.

Background: Combination therapy remains a promising strategy for treating neurodegenerative diseases, although green synthesis of gold nanoparticles for treating chronic neuroinflammation and studying their efficacy in treating neuroinflammation-mediated neurodegenerative diseases is not well assessed. Results: Here, Ephedra sinica Stapf (ES) extract was used as the reducing, capping, and stabilizing agent for gold nanoparticle synthesis. We developed ES extract-capped gold nanoparticles (ES-GNs) and investigated their anti-neuroinflammatory properties in microglia. ES-GNs displayed maximum absorption at 538 nm in ultraviolet-visible spectroscopy. Dynamic light scattering assessment revealed that ES-GN diameter was 57.6±3.07 nm, with zeta potential value of -24.6±0.84 mV. High resolution-transmission electron microscopy confirmed the spherical shape and average diameter (35.04±4.02 nm) of ES-GNs. Crystalline structure of ES-GNs in optimal conditions was determined by X-ray powder diffraction, and elemental gold presence was confirmed by energy-dispersive X-ray spectroscopy. Fourier transform-infrared spectroscopy confirmed gold nanoparticle synthesis using ES. Anti-neuroinflammatory properties of ES-GNs on production of pro-inflammatory mediators (nitric oxide, prostaglandin E2, and reactive oxygen species) and cytokines (tumor necrosis factor-α, IL-1ß, and IL-6) in lipopolysaccharide (LPS)-stimulated microglia were investigated by ELISA and flow cytometry. ES-GNs significantly attenuated LPS-induced production of pro-inflammatory mediators and cytokines, which was related to suppressed transcription and translation of inducible nitric oxide synthase and cyclooxygenase-2, determined by RT-PCR and western blotting. ES-GNs downregulated upstream signaling pathways (IκB kinase-α/ß, nuclear factor-κB, Janus-activated kinase /signal transducers and activators of transcription, mitogen-activated protein kinase , and phospholipase D) of pro-inflammatory mediators and cytokines in LPS-stimulated microglia. Anti-neuroinflammatory properties of ES-GNs were mediated by ES-GNs-induced AMP-activated protein kinase)-mediated nuclear erythroid 2-related factor 2 /antioxidant response element signaling. Conclusion: Collectively, these findings provide a new insight on the role of ES-GNs in treating chronic neuroinflammation-induced neurodegenerative diseases.

Neuroprotective effect of Dictyopteris divaricata extract-capped gold nanoparticles against oxygen and glucose deprivation/reoxygenation.

Combination therapy remains a promising approach to ameliorate cerebral ischemia injury. Nevertheless, the primary mechanism of the neuroprotective properties of Dictyopteris divaricata extract-capped gold nanoparticles (DD-GNPs) is not completely understood. DD-GNPs displayed maximum absorption at 525 nm and a diameter of 62.6 ± 1.2 nm, with a zeta potential value of -26.1 ± 0.6 mV. High resolution-transmission electron microscopy confirmed the spherical shape and average diameter (28.01 ± 2.03 nm). Crystalline structure and gold nanoparticle synthesis of DD-GNPs were determined by X-ray powder diffraction, and the presence of elemental gold was confirmed by energy-dispersive X-ray spectroscopy and Fourier transform-infrared spectroscopy. We examined the neuroprotective properties of DD-GNPs and explored their potential mechanisms in human SH-SY5Y neuroblastoma cells treated with oxygen and glucose deprivation/reoxygenation (OGD/R). We found that DD-GNPs inhibited OGD/R-induced release of lactate dehydrogenase (LDH), loss of cell viability, and production of reactive oxygen species. This neuroprotection was accompanied by regulation of apoptosis-related proteins, as indicated by decreased levels of cleaved-caspase-3, cleaved-PARP, cleaved-caspase-9, p53, p21, and Bax, as well as an increased level of Bcl-2. Notably, the neuroprotective effects of DD-GNPs were partially abolished by HO-1, NQO1, Nrf2, and AMPK knockdown. Our results established that DD-GNPs effectively attenuated OGD/R-stimulated neuronal injury, as evidenced by reduced neuronal injury. Even though the accumulating evidence has indicated the low toxicity and minimal side effects of GNPs, experimental clinical trials of DD-GNPs are still limited because of the lack of knowledge regarding the effects of DD-GNPs as neuroprotective agents against neurodegenerative diseases.

Lotus seed skin proanthocyanidin extract exhibits potent antioxidant property via activation of the Nrf2-ARE pathway.

Lotus seed is well known as traditional food and medicine, but its skin is usually discarded. Recent studies have shown that lotus seed skin contains a high concentration of proanthocyanidins that have multi-functions, such as antioxidation, anti-inflammation, and anti-cancer effects. In the present study, we aimed to isolate and purify the proanthocyanidins from lotus seed skin by acetone extraction and rotary evaporation, identify their chemical structures by HPLC-MS-MS and NMR, and further investigate the antioxidant properties of the extract purified by macroporous resin (PMR) from lotus seed skin both in vitro and in vivo. The results showed that PMR mainly contained oligomeric proanthocyanidins, especially dimeric procyanidin B1 (PB1), procyanidin B2 and procyanidin B4. Although it had limited ability to directly scavenge radicals in vitro, PMR could significantly enhance the expressions of antioxidant proteins via activation of nuclear factor-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway in HepG2 cells. Molecular data revealed that PB1, a major component in PMR, stabilized Nrf2 by inhibiting the ubiquitination of Nrf2, which led to subsequent activation of the Nrf2-ARE pathway, including the enhancements of Nrf2 nuclear translocation, Nrf2-ARE binding and ARE transcriptional activity. Moreover, the in vivo results in high fat diet-induced mice further verified the powerful antioxidant property of PMR. These results revealed that lotus seed skin is a promising resource for functional food development.

Suppression of NRF2/ARE by convallatoxin sensitises A549 cells to 5-FU-mediated apoptosis.

NF-E2-related factor 2 (NRF2) regulates transcription of phase II cytoprotective enzymes to protect normal cells against oxidative stress. However, a high level of NRF2 offers a growth advantage, chemoresistance, and radioresistance in cancer. In the present study, we have identified convallatoxin as a novel inhibitor of NRF2/ARE. Suppression of NRF2 by convallatoxin was not transcriptionally mediated, but regulated at the level of proteolysis. Convallatoxin activated GSK-3ß and suppression of NRF2 by convallatoxin required the Neh6 domain. Convallatoxin sensitised A549 cells to 5-fluorouracil-mediated cell death by promoting apoptosis. Together, our results provide evidence that convallatoxin might be useful as a chemotherapeutic adjuvant due to its ability to suppress NRF2/ARE.

Effects of bioactive constituents in the Traditional Chinese Medicinal formula Si-Wu-Tang on Nrf2 signaling and neoplastic cellular transformation.

BACKGROUND: The nuclear factor erythroid 2-related factor 2 (Nrf2) is a potential molecular target for cancer chemoprevention. Si-Wu-Tang (SWT), a popular traditional Chinese medicine for women's health, was reported with a novel activity of cancer prevention. PURPOSE: The present study was aimed to identify the bioactive constituents in SWT responsible for the Nrf2 activating and cancer preventive activity and explore the pharmacological mechanisms. METHODS: Nine compounds detectable from various batches of SWT were ranked using in silico molecular docking based on their ability to interfere the forming of Nrf2-Keap1 complex. The predicted Nrf2 activating effect was validated using the antioxidant response element (ARE) luciferase reporter assay and quantitative RT-PCR analysis for select Nrf2 regulated genes Hmox1, Nqo1 and Slc7a11. The antimutagenic activity of the compounds were determined by the Ames test. The chemopreventive activity of these compounds were assessed on EGF-induced neoplastic transformation of JB6 P+ cells, an established non-cancerous murine epidermal model for studying tumor promotion and identifying cancer preventive agents. These compounds were further characterized using luciferase reporter assay on EGF-induced activation of AP-1, a known transcription factor mediating carcinogenesis. RESULTS: Three of the nine compounds predicted as Nrf2 activators by molecular docking, gallic acid (GA), Z-liguistilide (LIG), and senkyunolide A (SA), were confirmed with highest potency of increasing the Nrf2/ARE promoter activity and upregulating the expression of Hmox1, Nqo1 and Slc7a11. In addition, GA, LIG and SA exhibited an antimutagenic activity against the direct mutagen 2-nitrofluorene while no mutagenic effects were observed at the same time in Ames test. At nontoxic concentrations, GA, LIG, and SA inhibited EGF-induced neoplastic transformation of JB6 P+ cells. Combined treatment of GA, LIG and SA, in the same ratio as detected in SWT, showed enhanced effect against JB6 transformation compared with that of the single compound alone. GA, LIG and SA, alone or in combination, suppressed EGF-induced activation of AP-1. CONCLUSION: We identified three bioactive constituents in SWT responsible for the Nrf2 activating and cancer preventive activity. This study provides evidence supporting novel molecular basis of SWT in cancer prevention.

l-Arginine induces antioxidant response to prevent oxidative stress via stimulation of glutathione synthesis and activation of Nrf2 pathway.

Arginine is a conditionally essential amino acid. To elucidate the influence of l-arginine on the activation of endogenous antioxidant defence, male Wistar rats were orally administered daily with l-arginine at different levels of 25, 50, 100 mg/100 g body weight. After 7 and 14 days feeding, the antioxidative capacities and glutathione (GSH) contents in the plasma and in the liver were uniformly enhanced with the increasing consumption of l-arginine, whereas the oxidative stress was effectively suppressed by l-arginine treatment. After 14 days feeding, the mRNA levels and protein expressions of Keap1 and Cul3 were gradually reduced by increasing l-arginine intake, resulting that the nuclear factor Nrf2 was activated. Upon activation of Nrf2, the expressions of antioxidant responsive element (ARE)-dependent genes and proteins (GCLC, GCLM, GS, GR, GST, GPx, CAT, SOD, NQO1, HO-1) were up-regulated by l-arginine feeding, indicating an upward trend in antioxidant capacity uniformly with the increasing consumption of l-arginine. The present study demonstrates that the supplementation of l-arginine stimulates GSH synthesis and activates Nrf2 pathway, leading to the up-regulation of ARE-driven antioxidant expressions via Nrf2-Keap1 pathway. Results suggest the availability of l-arginine is a critical factor to suppress oxidative stress and induce an endogenous antioxidant response.

Oyster (Ostrea plicatula Gmelin) polysaccharides intervention ameliorates cyclophosphamide-Induced genotoxicity and hepatotoxicity in mice via the Nrf2-ARE pathway.

In this study, we explored the protective effects of oyster (Ostrea plicatula Gmelin) polysaccharides (OPS) against genotoxicity and liver injury induced by cyclophosphamide (CP) in BALB/c mice. OPS was administered to mice at doses of 100 and 200mg/kg for 7 consecutive days, then 50mg/kg CP was injected via abdomen. Then mice were sacrificed and samples were collected. Bone marrow micronuclei (MN) and polychromatic erythrocytes (PCE): normochromatic erythrocytes (NCE) ratio were calculated to evaluate CP induced genetic toxicity. Activites of transaminase and antioxidants in serum as well as liver histopathology were examined to assess the severity of liver damage. We further investigate the molecular mechanism by Western blot analysis. When CP induced group pretreated with OPS, the generation of MN was obviously reduced accompanying by the restoration of PCE: NCE ratio. We also found that pretreatment of mice with OPS markedly reduced the release of serum alanine transaminase (ALT) and aspartate transferase (AST). Histological examination and grading evaluation also showed that OPS could significantly attenuated CP-induced liver damage. At the same time, OPS supplementation attenuated CP-induced oxidative stress as evident by the alternation of malondialdehyde (MDA) and superoxide dismutase (SOD) activity. Furthermore, CP induced mice showed the downregulation of Nrf2 (nuclear factor E2 - related factor 2) - ARE (antioxidant response element) and the downstream genes i.e. NAD(P)H: quinine oxidoreductase-1 (NQO - 1) and Hemoxygenase-1 (HO - 1), which were obviously reversed by OPS pretreatment. In conclusion, OPS protects against the genotoxicity and hepatotoxicity induced by CP in vivo. The beneficial effect may depend on activation of Nrf2 - ARE pathway and subsequent suppression of oxidative stress and genetic toxicity.

Self-protection against triptolide-induced toxicity in human hepatic cells via Nrf2-ARE-NQO1 pathway.

OBJECTIVE: To find the signaling pathway of triptolide (TP)-induced liver injury and to reveal whether NF-E2-related factor 2 (Nrf2) plays an important role in cellular self-protection. METHODS: The L-02 and HepG2 cells were cultured and treated with various concentrations of TP. The cell viability was observed, and the cell medium was collected for detecting the aspartate aminotransferase (ALT), alanine aminotransferase (AST), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and L-glutathione production (GSH) levels. Nrf2 and its downstream target NAD(P)H: quinine oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) expression, the nuclear translocation of Nrf2, and the binding ability of Nrf2 and antioxidant response element (ARE) were also identified. Meanwhile, shRNA was used to silence Nrf2 in L-02 cells to find out whether Nrf2 plays a protective role. RESULTS: The viability of the L-02 and HepG2 cells treated with TP decreased in a doseand time-dependent manner, and TP (20-80 µg/mL) markedly induced the release of ALT, AST and LDH (P<0.05 or P<0.01), reduced the levels of SOD and GSH (P<0.01), and increased the intracellular reactive oxygen species. Meanwhile, TP augmented the Nrf2 expression in L-02 and HepG2 cells (P<0.05 or P<0.01), induced Nrf2 nuclear translocation, increased the Nrf2 ARE binding activity, and increased HO-1 and NQO1 expressions. Nrf2 knockdown revealed a more severe toxic effect of TP (P<0.05 or P<0.01). CONCLUSIONS: Human hepatic cells treated with TP induced oxidative stress, and led to cytotoxicity. Self-protection against TP-induced toxicity in human hepatic cells might be via Nrf2-ARE-NQO1 transcriptional pathway.

Thymus vulgaris alleviates UVB irradiation induced skin damage via inhibition of MAPK/AP-1 and activation of Nrf2-ARE antioxidant system.

Solar ultraviolet (UV) radiation-induced reactive oxidative species is mainly responsible for the development of photoageing. Rosmarinic acid was one of the main bioactive components detected in Thymus vulgaris (TV) we extracted. In this study, UVB-induced skin damages have been shown to be ameliorated by treatment with TV in hairless mice (HR-1) skin, demonstrated by decreased matrix metalloproteinases (MMPs) and increased collagen production. However, the underlying molecular mechanism on which TV acted was unclear. We examined the photoprotective effects of TV against UVB and elucidated the molecular mechanism in normal human dermal fibroblasts. Thymus vulgaris remarkably prevented the UVB-induced reactive oxygen species and lactate dehydrogenase. Dose-dependent increase in glutathione, NAD(P)H: quinone oxidoreductase1 and heme oxygenase-1, by TV was confirmed by increased nuclear accumulation of Nrf2. Furthermore, 5-Methoxyindole-2-carboxylic acid was introduced as a specific inhibitor of dihydrolipoyl dehydrogenase (DLD). We demonstrated that Nrf2 expression was regulated by DLD, which was a tricarboxylic acid cycle-associated protein that decreased after UVB exposure. Besides, TV significantly diminished UVB induced phosphorylation of mitogen activated protein kinases pathway, containing extracellular signal-regulated kinase, Jun N-terminal kinase and p38, which consequently reduced phosphorylated c-fos and c-jun. Our results suggest that TV is a potential botanical agent for use against UV radiation-induced oxidative stress mediated skin damages.

Therapeutic advantage of pro-electrophilic drugs to activate the Nrf2/ARE pathway in Alzheimer's disease models.

Alzheimer's disease (AD) is characterized by synaptic and neuronal loss, which occurs at least partially through oxidative stress induced by oligomeric amyloid-ß (Aß)-peptide. Carnosic acid (CA), a chemical found in rosemary and sage, is a pro-electrophilic compound that is converted to its active form by oxidative stress. The active form stimulates the Keap1/Nrf2 transcriptional pathway and thus production of phase 2 antioxidant enzymes. We used both in vitro and in vivo models. For in vitro studies, we evaluated protective effects of CA on primary neurons exposed to oligomeric Aß. For in vivo studies, we used two transgenic mouse models of AD, human amyloid precursor protein (hAPP)-J20 mice and triple transgenic (3xTg AD) mice. We treated these mice trans-nasally with CA twice weekly for 3 months. Subsequently, we performed neurobehavioral tests and quantitative immunohistochemistry to assess effects on AD-related phenotypes, including learning and memory, and synaptic damage. In vitro, CA reduced dendritic spine loss in rat neurons exposed to oligomeric Aß. In vivo, CA treatment of hAPP-J20 mice improved learning and memory in the Morris water maze test. Histologically, CA increased dendritic and synaptic markers, and decreased astrogliosis, Aß plaque number, and phospho-tau staining in the hippocampus. We conclude that CA exhibits therapeutic benefits in rodent AD models and since the FDA has placed CA on the 'generally regarded as safe' (GRAS) list, thus obviating the need for safety studies, human clinical trials will be greatly expedited.

Piper betle Induced Cytoprotective Genes and Proteins via the Nrf2/ARE Pathway in Aging Mice.

BACKGROUND/AIMS: The objective of this study was to elucidate the underlying antioxidant mechanism of aqueous extract of Piper betle (PB) in aging rats. The nuclear factor erythroid 2-related factor 2 (Nrf2)/ARE pathway involving phase II detoxifying and antioxidant enzymes plays an important role in the antioxidant system by reducing electrophiles and reactive oxygen species through induction of phase II enzymes and proteins. 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.

Magnolia officinalis (Hou Po) bark extract stimulates the Nrf2-pathway in hepatocytes and protects against oxidative stress.

ETHNOPHARMACOLOGICAL RELEVANCE: The highly aromatic bark of Magnolia officinalis Rehder and EH Wilson, (magnolia bark) has been widely used in traditional Chinese medicine where it is known as Hou Po. Historically the bark of the tree has been used for treating variety of disorders the most common use of magnolia bark in traditional prescription has been to treat stress and anxiety disorders. Till date it is not clear regarding the fundamental cellular pathway it modulates. NRF2 signaling has emerged as the central pathway that protects cells from variety of stressors this led us to hypothesize that basis for magnolia bark's effects could be via activating NRF2 pathway. MATERIALS AND METHODS: We utilized variety of biochemical procedures like luciferase reporter assay, enzyme induction, gene expression to determine NRF2 inducing activity by magnolia bark extract and its significance. Further we identified the phytochemicals inducing this activity using bio-directed fractionation procedure. RESULTS: In this study, we demonstrate that magnolia bark extract activates Nrf2-dependent gene expression and protects against hydrogen peroxide mediated oxidative stress in hepatocytes. We further identified through HPLC fractionation and mass spectroscopy that magnolol, 4-methoxy honokiol and honokiol are the active phytochemicals inducing the Nrf2-mediated activity. This could be the molecular basis for its numerous beneficial activity.

Paeoniflorin protects Schwann cells against high glucose induced oxidative injury by activating Nrf2/ARE pathway and inhibiting apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin (PF) is the principal bioactive component of Paeonia lactiflora Pall., which an included in Tang Luo Ning recipe, a traditional Chinese herbal medicine based on Huangqi Guizhi Wuwu decoction. PF is also widely used in Traditional Chinese Medicine for the treatment of blood-arthralgia disease including diabetic peripheral neuropathy (DPN), but its underlying molecular mechanism of neuroprotective effects is not yet well understood. Diabetic hyperglycemia induced oxidative stress in Schwann cells, an important component of the peripheral nervous system, has been proposed as a unifying mechanism for DPN. The objective of this study is to determine the effects of PF on Schwann cells oxidative stress and apoptosis induced by high glucose. MATERIALS AND METHODS: RSC96 cells, a Schwann cell line, were treated with high glucose (150mM) and PF (1, 10 and 100µM). Subsequently, MTT assay was performed. The level of apoptosis was examined by flow cytometry and the oxidative stress was reflected by reactive oxygen species (ROS), malondialdehyde (MDA), glutathione S-transferases (GST) and glutathione peroxidase (GPX) levels. The mRNA expressions of Nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) were detected by qRT-PCR. The levels of Kelch-like ECH-associating protein 1 (Keap1), Nrf2, HO-1, γ-glutamylcysteine synthetase (γGCS), B-cell CLL/lymphoma 2 (Bcl-2), Bax and Caspase 3 were detected by High content analysis and/or Western blot. RESULTS: The role of PF markedly suppressed high glucose induced Schwann cells oxidative stress by decreasing ROS and MDA levels and increasing GST and GPX activity. Western blot analysis showed that PF induced nuclear translocation of Nrf2. High content analysis showed that PF promoted Nrf2 dissociation from Keap1 and upregulating the Nrf2/ antioxidant response element (ARE) pathway. Furthermore, PF reduced Schwann cells apoptosis by increasing Bcl-2 and inhibiting Bax and Caspase-3 expressions. CONCLUSIONS: PF in the management of Schwann cells oxidative stress induced by high glucose may be associated with activation of Nrf2/ARE pathway and Bcl-2-related apoptotic pathway.

Phytoestrogens modulate hepcidin expression by Nrf2: Implications for dietary control of iron absorption.

Hepcidin is a liver-derived antimicrobial peptide that regulates iron absorption and is also an integral part of the acute phase response. In a previous report, we found evidence that this peptide could also be induced by toxic heavy metals and xenobiotics, thus broadening its teleological role as a defensin. However it remained unclear how its sensing of disparate biotic and abiotic stressors might be integrated at the transcriptional level. We hypothesized that its function in cytoprotection may be regulated by NFE2-related factor 2 (Nrf2), the master transcriptional controller of cellular stress defenses. In this report, we show that hepcidin regulation is inextricably linked to the acute stress response through Nrf2 signaling. Nrf2 regulates hepcidin expression from a prototypical antioxidant response element in its promoter, and by synergizing with other basic leucine-zipper transcription factors. We also show that polyphenolic small molecules or phytoestrogens commonly found in fruits and vegetables including the red wine constituent resveratrol can induce hepcidin expression in vitro and post-prandially, with concomitant reductions in circulating iron levels and transferrin saturation by one such polyphenol quercetin. Furthermore, these molecules derepress hepcidin promoter activity when its transcription by Nrf2 is repressed by Keap1. Taken together, the data show that hepcidin is a prototypical antioxidant response or cytoprotective gene within the Nrf2 transcriptional circuitry. The ability of phytoestrogens to modulate hepcidin expression in vivo suggests a novel mechanism by which diet may impact iron homeostasis.

Dietary Lycium barbarum polysaccharide induces Nrf2/ARE pathway and ameliorates insulin resistance induced by high-fat via activation of PI3K/AKT signaling.

Lycium barbarum polysaccharide (LBP), an antioxidant from wolfberry, displays the antioxidative and anti-inflammatory effects on experimental models of insulin resistance in vivo. However, the effective mechanism of LBP on high-fat diet-induced insulin resistance is still unknown. The objective of the study was to investigate the mechanism involved in LBP-mediated phosphatidylinositol 3-kinase (PI3K)/AKT/Nrf2 axis against high-fat-induced insulin resistance. HepG2 cells were incubated with LBP for 12 hrs in the presence of palmitate. C57BL/6J mice were fed a high-fat diet supplemented with LBP for 24 weeks. We analyzed the expression of nuclear factor-E2-related factor 2 (Nrf2), Jun N-terminal kinases (JNK), and glycogen synthase kinase 3ß (GSK3ß) involved in insulin signaling pathway in vivo and in vitro. First, LBP significantly induced phosphorylation of Nrf2 through PI3K/AKT signaling. Second, LBP obviously increased detoxification and antioxidant enzymes expression and reduced reactive oxygen species (ROS) levels via PI3K/AKT/Nrf2 axis. Third, LBP also regulated phosphorylation levels of GSK3ß and JNK through PI3K/AKT signaling. Finally, LBP significantly reversed glycolytic and gluconeogenic genes expression via the activation of Nrf2-mediated cytoprotective effects. In summary, LBP is novel antioxidant against insulin resistance induced by high-fat diet via activation of PI3K/AKT/Nrf2 pathway.