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ABSTRACT: Protein glutathionylation is a posttranslational modification of cysteine residues with glutathione in response to mild oxidative stress. Because 15-deoxy-Δ12,14-prostaglandin J(2) (15d-PGJ(2)) is an electrophilic prostaglandin that can increase glutathione (GSH) levels and augment reactive oxygen species (ROS) production, we hypothesized that it induces NF-κB-p65 glutathionylation and would exert anti-inflammatory effects. Herein, we show that 15d-PGJ(2) suppresses the expression of ICAM-1 and NF-κB-p65 nuclear translocation. 15d-PGJ(2) upregulates the Nrf2-related glutathione synthase gene and thereby increases the GSH levels. Consistent with this, Nrf2 siRNA molecules abolish the inhibition of p65 nuclear translocation in 15d-PGJ(2)-induced endothelial cells (ECs). ECs treated with GSSG show increased thiol modifications of p65 and also a block in TNFα-induced p65 nuclear translocation and ICAM-1 expression, but not in IκBα degradation. However, the overexpression of glutaredoxin 1 was found to be accompanied by a modest increase in NF-κB activity. Furthermore, we found that multiple cysteine residues in p65 are responsible for glutathionylation. 15d-PGJ(2) was observed to induce p65 glutathionylation and is suppressed by a GSH synthesis inhibitor, buthionine sulfoximine, by catalase, and by Nrf2 siRNA molecules. Our results thus indicate that the GSH/ROS-dependent glutathionylation of p65 is likely to be responsible for 15d-PGJ(2)-mediated NF-κB inactivation and for the enhanced inhibitory effects of 15d-PGJ(2) on TNFα-treated ECs.
Free radical biology & medicine 02/2012; 52(9):1844-53. · 5.42 Impact Factor
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ABSTRACT: To explore whether glutathione (GSH) increased through Nrf-2 activation is involved in the cytoprotective effects of carnosol in HepG2 cells.
Human hepatoma cell line HepG2 were exposed to rosemarry essential oil or carnosol. Cell viability was measured using an Alamar blue assay. The production of intracellular GSH was determined using monochlorobimane. The level of protein or mRNA was examined by Western blotting or RT-PCR, respectively.
Rosemarry essential oil (0.005%-0.02%) and carnosol (5 and 10 mol/L) increased the intracellular GSH levels and GSH synthesis enzyme subunit GCLC/GCLM expression. Rosemary essential oil and carnosol increased nuclear accumulation of Nrf2 and enhanced Nrf2-antioxidant responsive element (ARE)-reporter activity. Transfection of the treated cells with an Nrf2 siRNA construct blocks GCLC/GCLM induction. Furthermore, pretreatment of the HepG2 cells with essential oil and carnosol exerted significant cytoprotective effects against H(2)O(2) or alcohol. In TNFα-treated cells, the nuclear translocation and transcriptional activity of NF-κB was abolished for 12 h following carnosol pretreatment. Cotreatment with GSH also suppressed NF-κB nuclear translocation, whereas cotreatment with BSO, a GSH synthesis blocker, blocked the inhibitory effects of carnosol.
This study demonstrated that Nrf2 is involved in the cytoprotective effects by carnasol, which were at least partially mediated through increased GSH biosynthesis.
Acta Pharmacologica Sinica 01/2011; 32(1):62-9. · 1.95 Impact Factor
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ABSTRACT: The generation of NO by endothelial nitric oxide synthase (eNOS) plays a major role in maintaining cardiovascular homeostasis. The objective of our present study was to investigate the effects of the flavone compound, apigenin, on eNOS activity and elucidate the molecular mechanisms underlying these effects in endothelial cells (ECs).
Bovine artery endothelial cells (BAECs) were exposed in a serum-free medium to apigenin. Cell viability was measured using an Alamar blue assay. The production of intracellular NO was determined using DAF-2/DA. The level of protein was examined by Western blotting. The intracellular Ca(2+) was measured using a fluorescent dye, Fura 2-AM.
Apigenin significantly induced NO production after 6h of treatment. This production was inhibited by pretreatment with the eNOS inhibitor, N(ω)-nitro l-arginine methyl ester (L-NAME). However, treatment with apigenin did not alter the eNOS protein levels but induced a sustained activation of eNOS Ser(1179) phosphorylation. Apigenin was further found to activate ERK1/2, JNK and Akt over various time courses in ECs. Treatment with specific PI3-kinase inhibitors significantly inhibited the increases in NO production and phosphorylation. In contrast, the inhibition of (ERK)1/2, JNK and p38 had no influence on NO production. In addition, apigenin stimulates an increase in the cytosolic Ca(2+) concentration. Apigenin-induced eNOS Ser(1179) phosphorylation and NO production are calcium-dependent, as pretreatment with extracellular or intracellular Ca(2+) chelators inhibits these processes.
Apigenin-induced calcium-dependent activation of eNOS is primarily mediated via phosphatidylinositol 3-kinase- and Akt pathways, and occurs independently of the eNOS protein content.
Life sciences 10/2010; 87(23-26):743-9. · 2.56 Impact Factor
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ABSTRACT: The increased adhesion of monocytes to injured endothelial layers is a critical early event in atherogenesis. Under inflammatory conditions, there is increased expression of specific cell adhesion molecules on activated vascular endothelial cells, which increases monocyte adhesion. In our current study, we demonstrate a putative mechanism for the anti-inflammatory effects of carnosol, a diterpene derived from the herb rosemary. Our results show that both carnosol and rosemary essential oils inhibit the adhesion of TNFalpha-induced monocytes to endothelial cells and suppress the expression of ICAM-1 at the transcriptional level. Moreover, carnosol was found to exert its inhibitory effects by blocking the degradation of the inhibitory protein IkappaBalpha in short term pretreatments but not in 12 h pretreatments. Our data show that carnosol reduces IKK-beta phosphorylation in pretreatments of less than 3 h. In TNFalpha-treated ECs, NF-kappaB nuclear translocation and transcriptional activity was abolished by up to 12 h of carnosol pretreatment and this was blocked by Nrf-2 siRNA. The long-term inhibitory effects of carnosol thus appear to be mediated through its induction of Nrf-2-related genes. The inhibition of ICAM-1 expression and p65 translocation is reversed by HO-1 siRNA. Carnosol also upregulates the Nrf-2-related glutathione synthase gene and thereby increases the GSH levels after 9 h of exposure. Treating ECs with a GSH synthesis inhibitor, BSO, blocks the inhibitory effects of carnosol. In addition, carnosol increases p65 glutathionylation. Hence, our present findings indicate that carnosol suppresses TNFalpha-induced singling pathways through the inhibition of IKK-beta activity or the upregulation of HO-1 expression. The resulting GSH levels are dependent, however, on the length of the carnosol pretreatment period.
Toxicology and Applied Pharmacology 05/2010; 245(1):21-35. · 4.45 Impact Factor
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ABSTRACT: Reversible protein glutathionylation is an important posttranslational modification that provides protection against oxidation. In endothelial cells (ECs), cinnamaldehyde is an electrophilic compound that can increase the intracellular glutathione (GSH) levels or reactive oxygen species (ROS) production depending on the treatment duration. ECs treated with GSH and H(2)O(2) show increased sulfhydryl modifications of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), which are responsible for NF-kappaB inactivation, and also a block in TNF-alpha-induced p65 nuclear translocation and inter-cellular adhesion molecule-1 (ICAM-1) expression. In our current study, we find that cinnamaldehyde induces p65 glutathionylation and inhibits TNF-alpha-induced p65 nuclear translocation and ICAM-1 expression within 12 h of treatment. Our analyses also reveal that p65 glutathionylation is suppressed by a GSH synthesis inhibitor, buthionine sulfoximine (BSO), and we further observed that the inhibitory effects of p65 nuclear translocation and ICAM-1 expression are also suppressed by BSO. NF-E2-related factor-2 small interfering RNA (siRNA) molecules not only inhibit glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) induction and increases in GSH but also abolish cinnamaldehyde-induced p65 glutathionylation and its inhibitory effects. The gene expression and activity of glutaredoxin-1 (Grx-1), which catalyzes the formation of protein-glutathione mixed disulfides (protein-SSG), were also found to be increased after cinnamaldehyde treatment. A knock down of endogenous Grx-1 by siRNA or pretreatment with an inhibitor of Grx-1 activity, CdCl(2), abolishes p65-SSG formation. In addition, Grx-1 siRNA blocks the inhibition of p65 nuclear translocation and ICAM-1 expression, suggesting that this enzyme is involved in the cinnamaldehyde-mediated NF-kappaB inhibition. Our current results thus indicate that the GSH/Grx-1-dependent glutathionylation of p65 is likely to be responsible for cinnamaldehyde-mediated NF-kappaB inactivation and for the enhanced inhibitory effects of cinnamaldehyde upon TNF-alpha-treated ECs.
Toxicological Sciences 03/2010; 116(1):151-63. · 4.65 Impact Factor
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ABSTRACT: Ganoderma tsugae is a medicinal fungus with several biological activities. It has long been used as a folk remedy for the promotion of health and longevity in China and other oriental countries. Here, a bioactive fraction of G. tsugae was progressively purified to be enriched in the activity of cytoprotective enzymes. The highest bioactivity was detected in the 20% EtOH-precipitated fraction, which was prepared from submerged fermentation filtrate of G. tsugae. Following further purification by gel filtration chromatography and acetone extraction, the most bioactive fraction, F5-2, was identified as a peptidoglycan-like compound. Extracts of G. tsugae (F5-2) induced heme oxygenase-1 (HO-1) and thioredoxin reductase-1 (TrxR1) expression in endothelial cells by increasing NF-E2-related factor-2 (Nrf2) nuclear translocation. Pretreatment with F5-2 increased intracellular glutathione (GSH) and protected against H(2)O(2), suggesting that induction of these antioxidant enzymes is important in protection against oxidative stress. Hence the bioactive peptidoglycan-like compound from G. tsugae might protect endothelial cells.
Bioscience Biotechnology and Biochemistry 09/2009; 73(8):1757-63. · 1.28 Impact Factor
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ABSTRACT: Resveratrol is a natural polyphenol and possesses many biological functions such as anti-inflammatory activity and protection against atherosclerosis and myocardial infraction. Parkinson's disease is a common progressive neurodegenerative disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is the most useful neurotoxin to induce Parkinsonism. The present study was carried out to elucidate the neuroprotective effect and possible mechanism of resveratrol on MPTP-induced striatal neuron loss. Sixty adult Balb/c mice were divided into four groups: sham operation, MPTP treatment (30 mg/kg, i.p.), MPTP combined with resveratrol administration (20 mg/kg, i.v.), and resveratrol treatment alone. Microdialysis and high-performance liquid chromatography were used to analyze dihydroxybenzoic acid (DHBA) that reflected the hydroxyl radical level. In the present study, we found MPTP chronic administration significantly induced motor coordination impairment in mice. After MPTP administration, the hydroxyl radical levels in substantia nigra were also significantly elevated and animals displayed severe neuronal loss. Resveratrol administration significantly protected mice from MPTP-induced motor coordination impairment, hydroxyl radical overloading, and neuronal loss. Our results demonstrated that resveratrol could elicit neuroprotective effects on MPTP-induced Parkinsonism through free radical scavenging.
Journal of Agricultural and Food Chemistry 08/2008; 56(16):6910-3. · 2.82 Impact Factor
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ABSTRACT: The production of adhesion molecules and subsequent attachment of leukocytes to endothelial cells (ECs) are critical early events in atherogenesis. These adhesion molecules thus play an important role in the development of this disease. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of cinnamaldehyde, a Cinnamomum cassia Presl-specific diterpene. In our current study, we have examined the effects of both cinnamaldehyde and extracts of C. cassia on cytokine-induced monocyte/human endothelial cell interactions. We find that these compounds inhibit the adhesion of TNFalpha-induced monocytes to endothelial cells and suppress the expression of the cell adhesion molecules, VCAM-1 and ICAM-1, at the transcriptional level. Moreover, in TNFalpha-treated ECs, the principal downstream signal of VCAM-1 and ICAM-1, NF-kappaB, was also found to be abolished in a time-dependent manner. Interestingly, cinnamaldehyde exerts its anti-inflammatory effects by blocking the degradation of the inhibitory protein IkappaB-alpha, but only in short term pretreatments, whereas it does so via the induction of Nrf2-related genes, including heme-oxygenase-1 (HO-1), over long term pretreatments. Treating ECs with zinc protoporphyrin, a HO-1 inhibitor, partially blocks the anti-inflammatory effects of cinnamaldehyde. Elevated HO-1 protein levels were associated with the inhibition of TNFalpha-induced ICAM-1 expression. In addition to HO-1, we also found that cinnamaldehyde can upregulate Nrf2 in nuclear extracts, and can increase ARE-luciferase activity and upregulate thioredoxin reductase-1, another Nrf2-related gene. Moreover, cinnamaldehyde exposure rapidly reduces the cellular GSH levels in ECs over short term treatments but increases these levels after 9 h exposure. Hence, our present findings indicate that cinnamaldehyde suppresses TNF-induced singling pathways via two distinct mechanisms that are activated by different pretreatment periods.
Toxicology and Applied Pharmacology 07/2008; 229(2):161-71. · 4.45 Impact Factor
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ABSTRACT: Many polyphenols have been found to increase endothelial nitric oxide (NO) production. In our present study, we investigated the effects of 1-alpha-O-galloylpunicalagin upon endothelial nitric oxide synthase (eNOS) activity in endothelial cells (ECs). Both 1-alpha-O-galloylpunicalagin and punicalagin induced NO production in a dose-dependent manner in ECs. Despite having similar chemical structures, punicalagin induced lower levels of NO production than 1-alpha-O-galloylpunicalagin. After 1-alpha-O-galloylpunicalagin addition, a rise in the intracellular Ca(2+) concentration preceded NO production. The Ca(2+) ionophore A23187 stimulated eNOS phosphorylation and augmented NO production. Pretreatment with Ca(2+) chelators inhibited 1-alpha-O-galloylpunicalagin-induced eNOS phosphorylation and NO production. Treatment with 1-alpha-O-galloylpunicalagin did not alter the eNOS protein levels but, unlike punicalagin, induced a sustained activation of eNOS Ser(1179) phosphorylation. 1-alpha-O-galloylpunicalagin was also found to activate ERK1/2, JNK and Akt in ECs. Moreover, simultaneous treatment of these cells with specific phosphatidylinositol-3-kinase inhibitors significantly inhibited the observed increases in eNOS activity and phosphorylation levels. In contrast, the inhibition of (ERK)1/2, JNK and p38 had no influence on eNOS Ser(1179) phosphorylation. Our present results thus indicate that the 1-alpha-O-galloylpunicalagin-induced calcium-dependent activation of eNOS is primarily mediated via a phosphatidylinositol 3-kinase/Akt-dependent increase in eNOS activity, and occurs independently of the eNOS protein content.
Molecular Nutrition & Food Research 05/2008; 52(10):1162-71. · 4.30 Impact Factor
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ABSTRACT: Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables. We here report that SFN is a potent inhibitor of LPS-induced monocyte adhesion, and also blocks the gene expression of the adhesion molecule, ICAM-1, at non-toxic concentrations. Downstream of ICAM-1, NF- kappaB activity was also found to be abolished in a dose-and time-dependent by SFN in LPS-treated endothelial cells (ECs). SFN exerts its suppressive effects on NF- kappaB activity in these cells by preventing the degradation of IkappaB-alpha. Interestingly, the inhibition of P65 translocation and IkappaB-alpha degradation was reversed slightly after 12 hours pretreatment. The intracellular GSH levels in SFN-treated ECs were observed to be reduced, the time course coincident with the suppression of P65 translocation and IkappaB-alpha degradation. NAC and GSH reverse the inhibitory effects of SFN upon p65 translocation and IkappaB-alpha degradation when preincubated with this agent. Furthermore, the use of BSO to decrease intracellular GSH levels further enhanced the effects of SFN. These data thus suggest that the anti-inflammatory mechanisms of SFN are dependent upon intracellular glutathione level.
Vascular Pharmacology 02/2008; 48(1):54-61. · 1.99 Impact Factor
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ABSTRACT: Chalcone, an alpha,beta-unsaturated flavonoid, possesses anti-inflammatory properties. In our present study, we have demonstrated chalcone inhibited IL-6- and LPS-induced ICAM-1 gene expression. In adhesion assay, chalcone reduced the LPS-induced adhesion of THP-1 cells to endothelial cells (ECs). Chalcone was found to abrogate the activation of STAT3 and NF-kappaB in a dose- and time-dependent manner, in IL-6- and LPS-treated ECs. Other flavonoids, quercetin and cyanidin, which lack alpha,beta-unsaturated carbonyl group, showed weaker or no inhibitory effect on both IL-6-induced STAT3 phosphorylation and LPS-induced p65 translocation. However, the electrophilic compounds curcumin and crotonaldehyde, which also contain an alpha,beta-unsaturated carbonyl moiety, mimic the inhibitory effects of chalcone with different efficiencies. In addition, N-acetyl-L-cysteine (NAC) could reverse the inhibition of STAT3 phosphorylation when preincubated with chalcone. The use of buthionine sulfoximine (BSO) to decrease intracellular GSH levels further enhanced the effects of chalcone. On the other hand, in ECs treated with BSO only no abrogation of IL-6-induced STAT3 phosphorylation was observed. We also found that chalcone could reduce the GSH level in vitro. Furthermore, the cellular GSH levels were rapidly reduced after 25 microM chalcone treatment. Following 6 h exposure, however, chalcone treatment rescued the GSH levels in ECs, coincident with the inhibition of STAT3 and NF-kappaB activation. In contrast, chalcone induced expression of thioredoxin reductase and heme-oxygenase genes after prolonged treatment. Furthermore, chalcone upregulated the levels of the transcription factor Nrf2 in nuclear extracts and increased antioxidant response element (ARE)-luciferase activity and thioredoxin reductase promoter activity. Hence, our present findings indicate that chalcone suppresses both IL-6- and LPS-induced signaling pathways through the thiol-dependent intracellular redox state. In addition, chalcone may provide distinct cytoprotective effects at different durations of pretreatment.
Life Sciences 04/2007; 80(15):1420-30. · 2.53 Impact Factor
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ABSTRACT: Piceatannol is an anti-inflammatory and anti-proliferative plant-derived stilbene. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme to activate by various phytochemicals. In this study, we examined the ability of piceatannol to upregulate HO-1 expression in endothelial cells. We found piceatannol at micromolar (10-50 microM) concentrations dramatically increased HO-1 protein levels in a time-dependent manner. Piceatannol was similarly potent in the induction of HO-1 as hemin, arsenate, and 15d-PGJ2, and was more potent than some other phytochemicals including curcumin, EGCG, baicalein, and quercetin. In contrast, the similar chemical structure compounds, trans-stilbene, stilbene oxide, and resveratrol had no HO-1-inducing effects, suggesting a critical role for the hydroxyl groups in HO-1 induction. No cytotoxicity and superoxide production was observed after 10-50 microM piceatannol treatments. Piceatannol-mediated HO-1 induction was abrogated in the presence of N-acetylcysteine and glutathione, but not by other antioxidants. Induction of HO-1 by piceatannol was further enhanced by using buthionine sulfoximine. In addition, we determined that tyrosine kinase was involved in the induction of HO-1 by using tyrosine kinase inhibitors, herbimycin A, erbstatin, and genistein; in contrast, no significant changes in the pretreatment of PI3 kinase or MAP kinase inhibitors was determined. HO-1 induction was blocked by the protein kinase C inhibitors calphostin C, rottlerin, and long PMA pretreatment, whereas conventional PKC inhibitors, Go6976, and Ca2+ chelator BAPTA/AM, had no effect. Elevated HO-1 protein levels were associated with the inhibition of tumor necrosis factor-alpha (TNFalpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression. Treating ECs with zinc protoporphyrin, an HO-1 inhibito blocked the anti-inflammatory effect of piceatannol. In summary, this study identified piceatannol as a novel phytochemical inducer of HO-1 expression and identified the mechanisms involved in this process.
Pharmacological Research 03/2006; 53(2):113-22. · 4.44 Impact Factor
