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Activator of NRF2


Int Urol Nephrol.2017 Nov 1. doi: 10.1007/s11255-017-1734-4.

Pterostilbene protects against uraemia serum-induced endothelial cell damage via activation of Keap1/Nrf2/HO-1 signaling.

Chen ZW1, Miu HF1, Wang HP2, Wu ZN2, Wang WJ1, Ling YJ2, Xu XH2, Sun HJ2, Jiang X3.

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Chronic kidney disease causes uremia-related endothelial cell dysfunction associated with high risk for cardiovascular diseases. The vascular endothelium is permanently exposed to uraemic toxins including indoxyl sulfate, which provokes endothelial damage in subjects with end-stage renal disease. Pterostilbene (PT) is identified to be homologous derivative of resveratrol and exerts antioxidant and anti-inflammatory actions. However, the effects of PT on uraemic serum-induced endothelial cell damage have not been elucidated. In this study, we investigated the effects and mechanisms of PT on uraemic serum (US)-mediated injury in human umbilical vein endothelial cells (HUVECs). Treatment of US obviously reduced cell viability, inhibited superoxide dismutase activity and catalase activity, suppressed phosphorylated endothelial nitric oxide synthase (eNOS) protein level and eNOS activity, whereas promoted lactate dehydrogenase leakage, increased malondialdehyde, hydrogen peroxide, superoxide anions levels and NAD(P)H activity accompanied with increased nitrative stress and inflammatory response in HUVECs, and these changes were reversed after PT treatment. Under US environment, PT downregulated Kelch-like ECH-associated protein 1 (Keap1) and upregulated nuclear factor erythroid-2-related factor 2 (Nrf2) and its downstream target heme oxygenase-1 (HO-1) protein levels. Of note, the level of HO-1 was decreased after the transfection of cells with Nrf2-siRNA, and HO-1 inhibitor Snpp abolished the protective effects of PT on HUVECs in response to US. Collectively, our study demonstrated that PT is effective in reducing US-evoked endothelial cell dysfunction via suppression of oxidative/nitrative stress and inflammatory response, which at least partly depended on Keap1/Nrf2/HO-1 signaling pathway.

KEYWORDS: Chronic kidney disease; Endothelial cell; Nrf2; Pterostilbene; Uremia

PMID: 29094331 DOI: 10.1007/s11255-017-1734-4

Redox Rep.2017 Nov;22(6):501-507. doi: 10.1080/13510002.2017.1329917. Epub 2017 May 22.

Pterostilbene protects against UVB-induced photo-damage through a phosphatidylinositol-3-kinase-dependent Nrf2/ARE pathway in human keratinocytes. 

Li H1, Jiang N1,2, Liang B1, Liu Q1,3, Zhang E1, Peng L1, Deng H1, Li R1, Li Z1, Zhu H1.

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OBJECTIVE: Ultraviolet B (UVB) irradiation is the initial etiological factor for various skin disorders, including erythema, sunburn, photoaging, and photocarcinogenesis. Pterostilbene (Pter) displayed remarkable antioxidant, anti-inflammatory, and anticarcinogenic activities. This study aimed to investigate the effective mechanism of Pter against UVB-induced photodamage in immortalized human keratinocytes.

METHODS: Human keratinocytes were pretreated with Pter (5 and 10 μM) for 24 h prior to UVB irradiation (300 mJ/cm2). Harvested cells were analyzed by MTT, DCFH-DA, comet, western blotting, luciferase promoter, small interference RNA transfection, and quantitative real-time polymerase chain reaction assay.

RESULTS: Pter significantly attenuated UVB-induced cell death and reactive oxygen species (ROS) generation, and effectively increased nuclear translocation of NF-E2-related factor-2 (Nrf2), expression of Nrf2-dependent antioxidant enzymes, and DNA repair activity. Moreover, the protective effects of Pter were abolished by small interference RNA-mediated Nrf2 silencing. Furthermore, Pter was also found to induce the phosphorylation of Nrf2 and the known phosphatidylinositol-3-kinase (PI3K) phosphorylated kinase, Akt. The specific inhibitor of PI3K, LY294002, successfully abrogated Pter-induced Nrf2 phosphorylation, activation of Nrf2-antioxidant response element pathway, ROS scavenging ability, and DNA repair activity.

CONCLUSION: The present study indicated that Pter effectively protected against UVB-induced photodamage by increasing endogenous defense mechanisms, scavenging UVB-induced ROS, and aiding in damaged DNA repair through a PI3K-dependent activation of Nrf2/ARE pathway.

KEYWORDS: Nrf2; Pterostilbene; antioxidants; photoprotection; ultraviolet

PMID: 28532341 DOI: 10.1080/13510002.2017.1329917

Oncotarget.2017 Jun 27;8(26):41988-42000. doi: 10.18632/oncotarget.16716.

Pterostilbene inhibits inflammation and ROS production in chondrocytes by activating Nrf2 pathway.

Xue EX1, Lin JP2, Zhang Y1, Sheng SR1, Liu HX1, Zhou YL1, Xu H1.

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Pterostilbene has been reported as a potential drug to inhibit oxidative stress and inflammation. However, the effect of pterostilbene on chondrocytes and osteoarthritis remains to be elucidated. We sought to investigate whether pterostilbene could protect chondrocytes from inflammation and ROS production through factor erythroid 2-related factor 2 (Nrf2) activation. The pterostilbene toxicity on chondrocytes collected from cartilages of Sprague-Dawley rats was assessed by CCK-8 test. Immunofluorescence and Western blotting explored the nuclear translocation of Nrf2. Nrf2 expression was silenced by siRNA to evaluate the involvement of Nrf2 in the effect of pterostilbene on chondrocytes. Finally, osteoarthritis model was established by the transection of anterior cruciate ligament and partial medial meniscectomy in rats, and then these rats received pterostilbene 30 mg/kg, daily, p.o. for 8 weeks. Histology and immunohistochemistry were used to assess histopathological change and Nrf2 expression in cartilage. Nuclear translocation of Nrf2 was stimulated by pterostilbene without cellular toxicity. Pterostilbene inhibited the level of COX-2, iNOS, PGE2, and NO, as well as the mitochondrial and total intracellular ROS production induced by IL-1β in chondrocytes, partially reversed by the Nrf2 silencing. Pterostilbene prevented cartilage degeneration and promoted the nuclear translocation of Nrf2 in cartilage. These results suggest that pterostilbene could inhibit the IL-1β-induced inflammation and ROS production in chondrocytes by stimulating the nuclear translocation of Nrf2.

KEYWORDS: chondrocyte; inflammation; nuclear factor erythroid 2-related factor 2; pterostilbene; reactive oxygen species

PMID: 28410217 PMCID: PMC5522043DOI: 10.18632/oncotarget.16716

J Nutr Biochem.2017 Jun;44:11-21. doi: 10.1016/j.jnutbio.2017.02.015. Epub 2017 Mar 6.

Role of pterostilbene in attenuating immune mediated devastation of pancreatic beta cells via Nrf2 signaling cascade.

Sireesh D1, Ganesh MR2, Dhamodharan U1, Sakthivadivel M3, Sivasubramanian S3, Gunasekaran P3, Ramkumar KM4.

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Nrf2 (nuclear factor erythroid 2-related factor-2) is a transcription factor that regulates oxidative/xenobiotic stress response and also suppress inflammation. Nrf2 signaling is associated with an increased susceptibility to various kinds of stress. Nrf2 has been shown as a promising therapeutic target in various human diseases including diabetes. Our earlier studies showed Pterostilbene (PTS) as a potent Nrf2 activator, and it protects the pancreatic β-cells against oxidative stress. In this study, we investigated PTS confer protection against cytokine-induced β-cell apoptosis and its role on insulin secretion in streptozotocin (STZ)-induced diabetic mice. The Nrf2 activation potential of PTS was assessed by dissociation of the Nrf2-Keap1 complex and by expression of ARE-driven downstream target genes in MIN6 cells. Further, the nuclear Nrf2 translocation and blockage of apoptotic signaling as demonstrated by the reduction of BAX/Bcl-2 ratio, Annexin-V positive cells and caspase-3 activity conferred the cyto-protection of PTS against cytokine-induced cellular damage. In addition, PTS treatment markedly improved glucose homeostasis and abated inflammatory response evidenced by the reduction of proinflammatory cytokines in diabetic mice. The inhibition of β-cell apoptosis by PTS as assessed by BAX/Bcl-2 ratio and caspase-3 activity in the pancreas was associated with the activation of Nrf2 and the expression of its downstream target genes. PTS also inhibited the activation of iNOS and decreased nitric oxide (NO) formation in the pancreas of diabetic animals. The results obtained from both in vitro and in vivo experiments showed that PTS improves β-cell function and survival against cytokine stress and also prevents STZ-induced diabetes.

KEYWORDS: Cytokine cocktail; Diabetes; MIN6; Nrf2; Streptozotocin

PMID: 28343084 DOI: 10.1016/j.jnutbio.2017.02.015

Biochim Biophys Acta.2017 Apr;1863(4):827-837. doi: 10.1016/j.bbadis.2017.01.005. Epub 2017 Jan 9.

Pterostilbene attenuates high glucose-induced oxidative injury in hippocampal neuronal cells by activating nuclear factor erythroid 2-related factor 2.

Yang Y1, Fan C2, Wang B3, Ma Z2, Wang D4, Gong B4, Di S2, Jiang S5, Li Y6, Li T6, Yang Z6, Luo E7.

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In the present study, neuroblastoma (SH-SY5Y) cells were used to investigate the mechanisms mediating the potential protective effects of pterostilbene (PTE) against mitochondrial metabolic impairment and oxidative stress induced by hyperglycemia for mimicking the diabetic encephalopathy. High glucose medium (100mM) decreased cellular viability after 24h incubation which was evidenced by: (i) reduced mitochondrial complex I and III activities; (ii) reduced mitochondrial cytochrome C; (iii) increased reactive oxygen species (ROS) generation; (iv) decreased mitochondrial membrane potential (ΔΨm); and (v) increased lactate dehydrogenase (LDH) levels. PTE (2.5, 5, and 10μM for 24h) was nontoxic and induced the nuclear transition of Nrf2. Pretreatment of PTE (2.5, 5, and 10μM for 2h) displayed a dose-dependently neuroprotective effect, as indicated by significantly prevented high glucose-induced loss of cellular viability, generation of ROS, reduced mitochondrial complex I and III activities, reduced mitochondrial cytochrome C, decreased ΔΨm, and increased LDH levels. Moreover, the levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and glutathione S-transferase (GST) were elevated after PTE treatment. In addition, the elevation of nuclear Nrf2 by PTE treatment (10μM for 2h) was abolished by Nrf2 siRNA. Importantly, Nrf2 siRNA induced the opposite changes in mitochondrial complex I and III activities, mitochondrial cytochrome C, reactive species generation, ΔΨm, and LDH. Overall, the present findings were the first to show that pterostilbene attenuated high glucose-induced central nervous system injury in vitro through the activation of Nrf2 signaling, displaying protective effects against mitochondrial dysfunction-derived oxidative stress.

KEYWORDS: High glucose; Neuroprotection; Nuclear factor erythroid 2-related factor 2 signaling; Oxidative stress; Pterostilbene

PMID: 28089584 DOI: 10.1016/j.bbadis.2017.01.005

J Pharm Pharmacol.2017 Jan;69(1):73-81. doi: 10.1111/jphp.12657. Epub 2016 Nov 23.

The resveratrol derivatives trans-3,5-dimethoxy-4-fluoro-4′-hydroxystilbene and trans-2,4′,5-trihydroxystilbene decrease oxidative stress and prolong lifespan in Caenorhabditis elegans. 

Fischer N1, Büchter C1, Koch K1, Albert S2, Csuk R2, Wätjen W1.

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OBJECTIVES: Resveratrol (trans-3,4′,5-trihydroxystilbene (1)) was previously shown to extend the lifespan of different model organisms. However, its pharmacological efficiency is controversially discussed. Therefore, the bioactivity of four newly synthesized stilbenes (trans-3,5-dimethoxy-4-fluoro-4′-hydroxystilbene (3), trans-4′-hydroxy-3,4,5-trifluorostilbene (4), trans-2,5-dimethoxy-4′-hydroxystilbene (5), trans-2,4′,5-trihydroxystilbene (6)) was compared to (1) and pterostilbene (trans-3,5-dimethoxy-4′-hydroxystilbene (2)) in the established model organism Caenorhabditis elegans.

METHODS: Trolox equivalent antioxidant capacity (TEAC), 2′,7′-dichlorofluorescein (DCF), thermotolerance assays, C. elegans lifespan analyses.

KEY FINDINGS: All compounds exert a strong in-vitro radical scavenging activity (6 > 1 > 5 > 2 = 3 = 4), but in vivo, only (3) and (6) reduce reactive oxygen species (ROS) accumulation. Furthermore, (3) and (6) increased the mobility of aged nematodes and prolonged their mean lifespans, while these compounds decreased the thermal stress resistance. Using daf-16 (FoxO), skn-1 (Nrf2) and sir-2.1 (sirtuin) loss-of-function mutant strains, the in vivo antioxidant effects of compounds (3) and (6) were abolished, showing the necessity of these evolutionary highly conserved factors. However, short-time treatment with stilbenes (3) and (6) did not modulate the cellular localization of the transcription factors DAF-16 and SKN-1.

CONCLUSION: In contrast to resveratrol, the synthetic stilbene derivatives (3) and (6) increase the lifespan of C. elegans, rendering them promising candidates for pharmacological anti-ageing purposes.

© 2016 Royal Pharmaceutical Society.

KEYWORDS: Nrf2; ageing; insulin-signalling; oxidative stress; secondary plant compounds

PMID: 27882602 DOI: 10.1111/jphp.12657

Eur J Pharmacol.2016 Oct 15;789:229-243. doi: 10.1016/j.ejphar.2016.07.046. Epub 2016 Jul 27.

Promising therapeutic potential of pterostilbene and its mechanistic insight based on preclinical evidence.

Kosuru R1, Rai U1, Prakash S1, Singh A2, Singh S3.

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Pterostilbene (PS) is a well-recognized antioxidant that primarily exists in blueberries, grapevines and heartwood of red sandalwood. Interest in this compound has been renewed in recent years, and studies have found that PS possesses an array of pharmacological properties, including chemopreventive, antiinflammatory, antidiabetic, antidyslipidemic, antiatherosclerotic and neuroprotective effects. However, the greater in vivo bioavailability of PS, as compared to resveratrol, is an added advantage for its efficacy. This review provides a summary regarding the sources, pharmacokinetic aspects and pharmacodynamics of PS, with a focus on the molecular mechanisms underlying its protective effects against cancer, brain injuries and heart disease. Studies regarding the safety profile of PS have also been included. Based on the presently available evidence, we conclude that PS represents an active phytonutrient and a potential drug with pleiotropic health applications.

KEYWORDS: AMPK; Cardiovascular disease; HO-1; NF-κB; Nrf2; Pterostilbene

PMID: 27475678 DOI: 10.1016/j.ejphar.2016.07.046

Bioorg Med Chem.2016 Aug 15;24(16):3378-86. doi: 10.1016/j.bmc.2016.05.011. Epub 2016 May 11.

Pterostilbene-mediated Nrf2 activation: Mechanistic insights on Keap1:Nrf2 interface.

Bhakkiyalakshmi E1, Dineshkumar K2, Karthik S1, Sireesh D3, Hopper W2, Paulmurugan R4, Ramkumar KM5.

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The discovery of Keap1-Nrf2 protein-protein interaction (PPI) inhibitors has become a promising strategy to develop novel lead molecules against variety of stress. Hence, Keap1-Nrf2 system plays an important role in oxidative/electrophilic stress associated disorders. Our earlier studies identified pterostilbene (PTS), a natural analogue of resveratrol, as a potent Nrf2 activator and Keap1-Nrf2 PPI inhibitor as assessed by luciferase complementation assay. In this study, we further identified the potential of PTS in Nrf2 activation and ARE-driven downstream target genes expression by nuclear translocation experiments and ARE-luciferase reporter assay, respectively. Further, the luciferase complementation assay identified that PTS inhibits Keap1-Nrf2 PPI in both dose and time-dependent manner. Computational studies using molecular docking and dynamic simulation revealed that PTS directly interacts with the basic amino acids of kelch domain of Keap1 and perturb Keap1-Nrf2 interaction pattern. This manuscript not only shows the binding determinants of Keap1-Nrf2 proteins but also provides mechanistic insights on Nrf2 activation potential of PTS.

KEYWORDS: ARE; Keap1; Molecular docking; Molecular dynamic simulation; Nrf2; Pterostilbene

PMID: 27312421 DOI: 10.1016/j.bmc.2016.05.011

Brain Res.2016 Jul 15;1643:70-9. doi: 10.1016/j.brainres.2016.04.048. Epub 2016 Apr 21.

Neuroprotective effects of pterostilbene against oxidative stress injury: Involvement of nuclear factor erythroid 2-related factor 2 pathway. 

Wang B1, Liu H1, Yue L1, Li X1, Zhao L1, Yang X1, Wang X1, Yang Y2, Qu Y3.

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Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) regulates multiple anti-oxidative enzymes and has neuroprotective effects. Pterostilbene (PTE) is a natural anti-oxidant found in blueberries. Its non-metabolized form exhibits high distribution in the brain after dietary administration. In this study, we aimed to explore the potential of PTE in protecting murine hippocampal neuronal HT22 cells against glutamate-induced oxidative stress injury and possible underlying mechanisms. PTE was nontoxic and induced the nuclear translocation of Nrf2 when HT22 cell cultures were incubated with different concentrations of PTE. Further, PTE displayed a dose-dependent neuroprotective effect, as indicated by increased cell viability and a reduction in lactate dehydrogenase (LDH) release after glutamate treatment. Nrf2 siRNA treatment inhibited PTE-induced neuroprotective effects. Moreover, the levels of nuclear Nrf2 and downstream heme oxygenase-1 (HO-1) and

NAD(P)H: quinone oxidoreductase 1 (NQO1) were elevated after PTE treatment. The PTE-induced elevation of nuclear Nrf2, as well as the increases in HO-1 and NQO1 levels, was abolished by Nrf2 siRNA. PTE treatment reduced the production of reactive oxygen species (ROS) and significantly enhanced the activities of the cellular anti-oxidants glutathione (GSH) and superoxide dismutase (SOD), indicating an attenuation of glutamate-induced oxidative stress. These changes in ROS and GSH and SOD activity were reversed by Nrf2 siRNA. Our results indicate that PTE treatment attenuates glutamate-induced oxidative stress injury in neuronal cells via the Nrf2 signaling pathway.

Copyright © 2016 Elsevier B.V. All rights reserved.

KEYWORDS: Glutamate; Neuroprotection; Nuclear factor erythroid 2 (NF-E2)-related factor 2 signaling; Oxidative stress; Pterostilbene

PMID: 27107941 DOI: 10.1016/j.brainres.2016.04.048

Drug Chem Toxicol.2017 Jan;40(1):36-46. doi: 10.3109/01480545.2016.1169542. Epub 2016 Apr 14.

A comparative assessment of the cytotoxicity and nitric oxide reducing ability of resveratrol, pterostilbene and piceatannol in transformed and normal mouse macrophages.

Adiabouah Achy-Brou CA1, Billack B1.

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The present study investigated the pharmacological effects of three stilbenoids, resveratrol (RES), pterostilbene (PTR) and piceatannol (PIC), in transformed and normal macrophages. Our first aim was to comparatively assess the cytotoxicity of RES, PTR and PIC in unstimulated transformed mouse macrophages (RAW 264.7 cells) and primary peritoneal macrophages (PMs) harvested from both wild type and Nrf2 (nuclear factor erythroid 2-related factor 2)-deficient female mice. Our second aim was to investigate whether the inhibitory effect of RES, PTR and PIC on nitric oxide (NO) release from stimulated PMs depends on the status of the transcription factor Nrf2. The rationale for investigating Nrf2 status was based upon recent reports showing that certain compounds (sulforaphane and linalool) suppress LPS-induced inflammation in an Nrf2-dependent manner. Cell viability studies confirmed our prior work in unstimulated RAW 264.7 cells, with cytotoxic potency decreasing in the order of PTR > PIC > RES. Unstimulated PMs, regardless of Nrf2 status, were less sensitive to stilbenes, requiring at least a threefold higher stilbene concentration to inhibit cell viability, with cytotoxic potency again decreasing in the order of PTR > PIC > RES. In studies focused on our second aim, IC50 values for NO inhibition (measured as [Formula: see text]) in wild type PMs were similar for all three stilbenes (∼10 μM). In Nrf2-deficient PMs, the IC50 for NO inhibition by PIC did not change; however, a rightward shift in the concentration effect curve was observed for both RES and PTR, indicating a role for Nrf2 in the suppression of LPS-induced [Formula: see text] accumulation by these particular stilbenes.

KEYWORDS: Nrf2; Resveratrol; anti-inflammatory effect of stilbenes; macrophages; piceatannol; pterostilbene; stilbenoids

PMID: 27079867 DOI: 10.3109/01480545.2016.1169542

Eur J Pharmacol.2016 Apr 15;777:9-16. doi: 10.1016/j.ejphar.2016.02.054. Epub 2016 Feb 24.

Anti-hyperlipidemic and anti-peroxidative role of pterostilbene via Nrf2 signaling in experimental diabetes.

Bhakkiyalakshmi E1, Sireesh D1, Sakthivadivel M2, Sivasubramanian S2, Gunasekaran P2, Ramkumar KM3.

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Nuclear factor erythroid 2-related factor (Nrf2), a key transcription factor triggers the expression of antioxidant and detoxification genes thereby providing cellular protective functions against oxidative stress-mediated disorders. Recent research has identified that pharmacological activation of Nrf2 also regulates the largest cluster of genes associated with lipid metabolism. With this background, this paper highlights the anti-hyperlipidemic and anti-peroxidative role of pterostilbene (PTS), an Nrf2 activator, in streptozotocin (STZ)-induced diabetic model. PTS administration to diabetic mice for 5 weeks significantly regulated blood glucose levels through the elevation of insulin secretion. The circulatory and liver lipid profiles of total cholesterol (TC), triglycerides (TG) and non-esterified fatty acids (NEFA) were maintained to normal levels upon PTS treatment. Moreover, PTS administration also normalized the circulatory levels of very low-, low- and high densitylipoprotein cholesterols (VLDL-, LDL-, HDL-C) and also reduced lipid peroxidation in STZ-induced diabetic mice. In addition, Nrf2 and its downstream targets, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) enzyme activities and glutathione (GSH) levels were significantly elevated in liver tissues of diabetic mice upon PTS administration. Further, H&E staining of diabetic mouse liver showed collapse in hepatic microvesicles due to altered lipid metabolism. Both structural and functional alterations were attenuated by PTS indicating its role in diabetic dyslipidemia through Nrf2-mediated mechanism that could be considered as a promising therapeutic agent.

KEYWORDS: Diabetes; Dyslipidemia; Lipids; Nrf2; Pterostilbene; Streptozotocin

PMID: 26921755 DOI: 10.1016/j.ejphar.2016.02.054

Chem Res Toxicol.2016 Jan 19;29(1):47-57. doi: 10.1021/acs.chemrestox.5b00378. Epub 2016 Jan 7.

Pterostilbene Ameliorates Streptozotocin-Induced Diabetes through Enhancing Antioxidant Signaling Pathways Mediated by Nrf2.

Elango B, Dornadula S, Paulmurugan R1, Ramkumar KM.

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Nuclear factor erythroid 2-related factor 2 (Nrf2) remains a master regulator of cytoprotective and antioxidant genes. In this study, we investigated the antidiabetic role of pterostilbene (PTS) in streptozotocin (STZ)-induced diabetic model through Nrf2-mediated antioxidant mechanisms. The ability of PTS to activate Nrf2 in MIN6 cells was assessed by dissociation of the Nrf2-Keap1 complex at different time points and by expression of ARE-driven downstream target genes of Nrf2. Immunoblot experiments examining Nrf2 activation and phosphorylation indicated that it conferred cytoprotection against STZ-induced cellular damage. In STZ-induced diabetic mice, PTS administration significantly decreased blood glucose levels through the improvement of insulin secretion. In addition, we also observed insulin-positive cells with recovered islet architecture in the pancreas of STZ-induced diabetic mice after treatment with PTS. The activation of Nrf2 and expression of its downstream target genes were observed upon PTS treatment, thereby reducing oxidative damage to pancreas. Furthermore, PTS treatment significantly reverted the abundance of key glucose metabolism enzymes, such as hexokinase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, and fructose-1,6-bisphosphatase, to near-normal levels in liver tissue of STZ-induced diabetic mice. These results clearly indicate that PTS maintains glucose homeostasis, suggesting the possibility that it is a future candidate for use in diabetes management.

PMID: 26700463 DOI: 10.1021/acs.chemrestox.5b00378

Antioxid Redox Signal.2016 Jun 10;24(17):974-90. doi: 10.1089/ars.2015.6437. Epub 2016 Mar 15.

Pterostilbene Decreases the Antioxidant Defenses of Aggressive Cancer Cells In Vivo: A Physiological Glucocorticoids- and Nrf2-Dependent Mechanism.

Benlloch M1, Obrador E2, Valles SL2, Rodriguez ML2, Sirerol JA2, Alcácer J3, Pellicer JA2, Salvador R2, Cerdá C4, Sáez GT4,5, Estrela JM2.

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AIMS: Polyphenolic phytochemicals have anticancer properties. However, in mechanistic studies, lack of correlation with the bioavailable concentrations is a critical issue. Some reports had suggested that these molecules downregulate the stress response, which may affect growth and the antioxidant protection of malignant cells. Initially, we studied this potential underlying mechanism using different human melanomas (with genetic backgrounds correlating with most melanomas), growing in nude mice as xenografts, and pterostilbene (Pter, a natural dimethoxylated analog of resveratrol).

RESULTS: Intravenous administration of Pter decreased human melanoma growth in vivo. However, Pter, at levels measured within the tumors, did not affect melanoma growth in vitro. Pter inhibited pituitary production of the adrenocorticotropin hormone (ACTH), decreased plasma levels of corticosterone, and thereby downregulated the glucocorticoid receptor- and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent antioxidant defense system in growing melanomas. Exogenous corticosterone or genetically induced Nrf2 overexpression in melanoma cells prevented the inhibition of tumor growth and decreased antioxidant defenses in these malignant cells. These effects and mechanisms were also found in mice bearing different human pancreatic cancers. Glutathione depletion (selected as an antimelanoma strategy) facilitated the complete elimination by chemotherapy of melanoma cells isolated from mice treated with Pter.

INNOVATION: Although bioavailability-related limitations may preclude direct anticancer effects in vivo, natural polyphenols may also interfere with the growth and defense of cancer cells by downregulating the pituitary gland-dependent ACTH synthesis.

CONCLUSIONS: Pter downregulates glucocorticoid production, thus decreasing the glucocorticoid receptor and Nrf2-dependent signaling/transcription and the antioxidant protection of melanoma and pancreatic cancer cells. Antioxid. Redox Signal. 24, 974-990.

PMID: 26651028 PMCID: PMC4921902DOI: 10.1089/ars.2015.6437

Free Radic Biol Med.2015 Aug;85:1-11. doi: 10.1016/j.freeradbiomed.2015.03.027. Epub 2015 Apr 4.

Topical treatment with pterostilbene, a natural phytoalexin, effectively protects hairless mice against UVB radiation-induced skin damage and carcinogenesis.

Sirerol JA1, Feddi F2, Mena S1, Rodriguez ML1, Sirera P1, Aupí M1, Pérez S1, Asensi M3, Ortega A3, Estrela JM4.

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The aim of our study was to investigate in the SKH-1 hairless mouse model the effect of pterostilbene (Pter), a natural dimethoxy analog of resveratrol (Resv), against procarcinogenic ultraviolet B radiation (UVB)-induced skin damage. Pter prevented acute UVB (360 mJ/cm(2))-induced increase in skin fold, thickness, and redness, as well as photoaging-associated skin wrinkling and hyperplasia. Pter, but not Resv, effectively prevented chronic UVB (180 mJ/cm(2), three doses/week for 6 months)-induced skin carcinogenesis (90% of Pter-treated mice did not develop skin carcinomas, whereas a large number of tumors were observed in all controls). This anticarcinogenic effect was associated with (a) maintenance of skin antioxidant defenses (i.e., glutathione (GSH) levels, catalase, superoxide, and GSH peroxidase activities) close to control values (untreated mice) and (b) an inhibition of UVB-induced oxidative damage (using as biomarkers 8-hydroxy-2′-deoxyguanosine, protein carbonyls, and isoprostanes). The molecular mechanism underlying the photoprotective effect elicited by Pter was further evaluated using HaCaT immortalized human keratinocytes and was shown to involve potential modulation of the Nrf2-dependent antioxidant response.

KEYWORDS: Free radicals; Oxidative stress; Photocarcinogenesis; Phytochemicals; Polyphenols; Pterostilbene; Resveratrol; Skin damage; Stilbenes; UV radiation

PMID: 25845487 DOI: 10.1016/j.freeradbiomed.2015.03.027

Pharmacol Res.2015 Jan;91:104-14. doi: 10.1016/j.phrs.2014.10.004. Epub 2014 Oct 29.

The emerging role of redox-sensitive Nrf2-Keap1 pathway in diabetes.

Bhakkiyalakshmi E1, Sireesh D2, Rajaguru P3, Paulmurugan R4, Ramkumar KM5.

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The pathogenic processes involving in the development of diabetes range from autoimmune destruction of pancreatic β-cells with consequent insulin deficiency to abnormalities that result in resistance to insulin action. The major contributing factor for excessive β-cell death includes oxidative stress-mediated mitochondrial damage, which creates an imbalance in redox homeostasis. Yet, β-cells have evolved adaptive mechanisms to endure a wide range of stress conditions to safeguard its potential functions. These include ‘Nrf2/Keap1’ pathway, a key cellular defense mechanism, to combat oxidative stress by regulating phase II detoxifying and antioxidant genes. During diabetes, redox imbalance provokes defective Nrf2-dependent signaling and compromise antioxidant capacity of the pancreas which turnout β-cells to become highly vulnerable against various insults. Hence, identification of small molecule activators of Nrf2/Keap1 pathway remains significant to enhance cellular defense to overcome the burden of oxidative stress related disturbances. This review summarizes the molecular mechanism behind Nrf2 activation and the impact of Nrf2 activators in diabetes and its complications.

KEYWORDS: Antioxidants; Bardoxolone methyl (PubChem CID: 400769); Cinnamaldehyde (PubChem CID: 637511); Curcumin (PubChem CID: 969516); Diabetes; Epigallocatechin gallate (PubChem CID: 65064); MG-132 (PubChem CID: 462382); Magnesium Lithospermate B (PubChem CID: 6438135); Nrf2 activators; Nrf2–Keap1 pathway; Oxidative stress; Pterostilbene (PubChem CID: 5281727); Resveratrol (PubChem CID: 445154); Sulforaphane (PubChem CID: 5350); tert-Butylhydroquinone (PubChem CID: 16043)

PMID: 25447793 DOI: 10.1016/j.phrs.2014.10.004

J Pharmacol Sci.2014;126(3):216-29. Epub 2014 Oct 21.

Involvement of the Nrf2 pathway in the regulation of pterostilbene-induced apoptosis in HeLa cells via ER stress.

Zhang B1, Wang XQ, Chen HY, Liu BH

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Among the various cancer cell lines, HeLa cells were found to be sensitive to pterostilbene (Pte), a compound that is enriched in small fruits such as grapes and berries. However, the mechanism involved in the cytotoxicity of Pte has not been fully characterized. Using biochemical and free radical biological experiments in vitro, we identified the pro-apoptotic profiles of Pte and evaluated the level of redox stress-triggered ER stress during HeLa cell apoptosis. The data showed a strong dose-response relationship between Pte exposure and the characteristics of HeLa apoptosis in terms of changes in apoptotic morphology, DNA fragmentation, and activated caspases in the intrinsic apoptotic pathway. During drug exposure, alterations in the intracellular redox homeostasis that favor oxidation were necessary to cause ER stress-related apoptosis, as demonstrated by enzymatic and non-enzymatic redox modulators. A statistically significant and dose-dependent increase (P < 0.05) was found with regard to the unique expression levels of Nrf2/ARE downstream target genes in HeLa cells undergoing late apoptosis, levels that were restored with anti-oxidant application with the Pte treatment. Our research demonstrated that Pte trigged ER stress by redox homeostasis imbalance, which was negatively regulated by a following activation of Nrf2.

PMID: 25341683

Food Chem Toxicol.2014 Oct;72:303-11. doi: 10.1016/j.fct.2014.07.038. Epub 2014 Aug 8.

The berry constituents quercetin, kaempferol, and pterostilbene synergistically attenuate reactive oxygen species: involvement of the Nrf2-ARE signaling pathway.

Saw CL1, Guo Y2, Yang AY2, Paredes-Gonzalez X2, Ramirez C3, Pung D4, Kong AN5.

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Quercetin, kaempferol, and pterostilbene are abundant in berries. The anti-oxidative properties of these constituents may contribute to cancer chemoprevention. However, their precise mechanisms of action and their combinatorial effects are not completely understood. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates anti-oxidative stress enzymes and Phase II drug metabolizing/detoxifying enzymes by binding to antioxidant response element (ARE). This study aimed to investigate the anti-oxidative stress activities of quercetin, kaempferol, and pterostilbene individually and in combination, as well as the involvement of the Nrf2-ARE signaling pathway. Quercetin, kaempferol, and pterostilbene all exhibited strong free-radical scavenging activity in the DPPH assay. The MTS assay revealed that low concentration combinations we tested were relatively non-toxic to HepG2-C8 cells. The results of the DCFH-DA assay and combination index (CI) indicated that quercetin, kaempferol, and pterostilbene attenuated intracellular reactive oxygen species (ROS) levels when pretreated individually and had synergistic effects when used in combination. In addition, the combination treatment significantly induced ARE and increased the mRNA and protein expression of Nrf2-regulated genes. Collectively, our study demonstrated that the berry constituents quercetin, kaempferol, and pterostilbene activated the Nrf2-ARE signaling pathway and exhibited synergistic anti-oxidative stress activity at appropriate concentrations.

KEYWORDS: Antioxidant response element (ARE); Kaempferol; Nuclear factor (erythroid-derived 2)-like 2 (Nrf2); Pterostilbene; Quercetin; Reactive oxygen species

PMID: 25111660 DOI: 10.1016/j.fct.2014.07.038

Br J Pharmacol.2014 Apr;171(7):1747-57. doi: 10.1111/bph.12577.

Therapeutic potential of pterostilbene against pancreatic beta-cell apoptosis mediated through Nrf2. 

Bhakkiyalakshmi E1, Shalini D, Sekar TV, Rajaguru P, Paulmurugan R, Ramkumar KM.

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BACKGROUND AND PURPOSE: Nuclear factor erythroid 2-related factor 2 (Nrf2) is considered to be a ‘master regulator’ of the antioxidant response as it regulates the expression of several genes including phase II metabolic and antioxidant enzymes and thus plays an important role in preventing oxidative stress-mediated disorders, including diabetes. In this study, for the first time, we investigated the protective properties of a naturally available antioxidant, pterostilbene (PTS), against pancreatic beta-cell apoptosis and the involvement of Nrf2 in its mechanism of action.

EXPERIMENTAL APPROACH: Immunoblotting and quantitative reverse transcriptase (qRT)-PCR analysis were performed to identify PTS-mediated nuclear translocation of Nrf2 protein and the following activation of target gene expression, respectively, in INS-1E cells. In addition, an annexin-V binding assay was carried out to identify the apoptotic status of PTS-treated INS-1E cells, while confirming the anti-apoptotic potential of Nrf2 by qRT-PCR analysis of the expressions of both pro- and anti-apoptotic genes.

KEY RESULTS: PTS induced significant activation of Nrf2, in dose– and time-dependent manner, in streptozotocin-treated INS-1E rat pancreatic beta-cells. Furthermore, PTS increased the expression of target genes downstream of Nrf2, such as heme oxygenase 1 (HO1), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), that confer cellular protection. PTS also up-regulated the expression of anti-apoptotic gene, Bcl-2, with a concomitant reduction in pro-apoptotic Bax and caspase-3 expression.

CONCLUSION AND IMPLICATIONS: Collectively, our findings indicate the therapeutic potential of Nrf2 activation by PTS as a promising approach to safeguard pancreatic beta-cells against oxidative damage in diabetes.

© 2014 The British Pharmacological Society.

KEYWORDS: Nrf2; apoptosis; diabetes; pancreatic beta cells; pterostilbene; streptozotocin

PMID: 24417315 PMCID: PMC3966753DOI: 10.1111/bph.12577

Anal Chem.2013 Aug 6;85(15):7542-9. doi: 10.1021/ac401569j. Epub 2013 Jul 23.

Reporter protein complementation imaging assay to screen and study Nrf2 activators in cells and living animals. 

Ramkumar KM1, Sekar TV, Foygel K, Elango B, Paulmurugan R.

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NF-E2-related factor-2 (Nrf2) activators promote cellular defense mechanism and facilitate disease prevention associated with oxidative stress. In the present study, Nrf2 activators were identified using cell-based luciferase enzyme fragment complementation (EFC) assay, and the mechanism of Nrf2 activation was studied by molecular imaging. Among the various Nrf2 activators tested, pterostilbene (PTS) showed effective Nrf2 activation, as seen by luminometric screening, and validation in a high throughput-intact cell-imaging platform. Further, PTS increased the expression of Nrf2 downstream target genes, which was confirmed using luciferasereporter driven by ARE-NQO1 and ARE-GST1 promoters. Daily administration of PTS disturbed Nrf2/Keap1 interaction and reduced complemented luciferase signals in HEK293TNKS mouse tumor xenografts. This study reveals the potentials of Nrf2 activators as chemosensitizing agents’ for therapeutic intervention in cancer treatment. Hence, the validated assay can be used to evaluate the identified activators preclinically in small animal models by noninvasive molecular imaging approach.

PMID: 23826874 PMCID: PMC3759980DOI: 10.1021/ac401569j

J Agric Food Chem.2011 Mar 23;59(6):2725-33. doi: 10.1021/jf2000103. Epub 2011 Feb 28.

Pterostilbene is more potent than resveratrol in preventing azoxymethane (AOM)-induced colon tumorigenesis via activation of the NF-E2-related factor 2 (Nrf2)-mediated antioxidant signaling pathway.

Chiou YS1, Tsai ML, Nagabhushanam K, Wang YJ, Wu CH, Ho CT, Pan MH.

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Inflammatory bowel diseases have been a risk factor of colorectal cancer (CRC). The reactive oxygen species (ROS) generated by inflammatory cells create oxidative stress and contribute to neoplastic transformation, proliferation, and even metastasis. Previously, resveratrol (RS) and pterostilbene (PS) had been reported to prevent chemical-induced colon carcinogenesis by anti-inflammatory and pro-apoptotic properties. In this study, we investigated whether RS and PS could prevent the azoxymethane (AOM)-induced colon tumorigenesis via antioxidant action and to explore possible molecular mechanisms. Male BALB/c mice were injected with AOM (5 mg/kg of body weight) with or without RS or PS, and at the end of the protocol, all of the mice were euthanized and colons were analyzed. Administrations of PS can be more effective than RS in reducing AOM-induced formation of aberrant crypt foci (ACF), lymphoid nodules (LNs), and tumors. We also find that PS is functioning more effectively than RS to reduce nuclear factor-κB (NF-κB) activation by inhibiting the phosphorylation of protein kinase C-β2 (PKC-β2) and decreasing downstream target gene expression, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and aldose reductase (AR) in mouse colon stimulated by AOM. Moreover, administration of RS and PS for 6 weeks significantly enhanced expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1) and glutathione reductase (GR), via activation of NF-E2-related factor 2 (Nrf2) signaling. When the above findings are taken together, they suggest that both stilbenes block cellular inflammation and oxidative stress through induction of HO-1 and GR, thereby preventing AOM-induced colon carcinogenesis. In comparison, PS was a more potent chemopreventive agent than RS for the prevention of colon cancer. This is also the first study to demonstrate that PS is a Nrf2 inducer and AR inhibitor in the AOM-treated colon carcinogenesis model.

PMID: 21355597 DOI: 10.1021/jf2000103