Sulforaphane inhibits blue light-induced inflammation and apoptosis by upregulating the SIRT1/PGC-1α/Nrf2 pathway and autophagy in retinal pigment epithelial cells.

The present study elucidated mechanisms through which sulforaphane (SFN) protects retinal pigment epithelial (RPE) cells from blue light-induced impairment. SFN could activate the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increase the expression of the heme oxygenease-1 (HO-1) gene and production of glutathione. SFN reduced blue light-induced oxidative stress, and effectively activated cytoprotective components including Nrf-2, HO-1, thioredoxin-1, and glutathione. The protective effect of SFN on blue light-induced injury was blocked by the Nrf2 inhibitor ML385, suggesting that the SFN-induced Nrf2 pathway is involved in the cytoprotective effect of SFN. SFN inhibited intercellular adhesion molecule-1 expression induced by TNF-α or blue light, suggesting the anti-inflammatory activity of SFN. The inhibitory effect of SFN was associated with the blocking of NF-κB p65 nuclear translocation in blue light-exposed RPE cells. SFN protected RPE cells from blue light-induced interruption of the mitochondrial membrane potential and reduction of the Bcl-2/Bax ratio and cleaved caspase-3 and PARP-1 expression, suggesting the antiapoptotic activity of SFN. SFN alone or together with blue light exposure increased the expression of the autophagy-related proteins LC3BII and p62. An autophagy inhibitor, 3-MA, inhibited the protective effect of SFN on blue light-induced cell damage. SFN increased sirtuin-1 (SIRT1) expression; however, treatment with blue light induced peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) expression. Our study results demonstrated that SFN exerts its protective effect under blue light exposure by maintaining the Nrf2-related redox state and upregulating SIRT1 and PGC-1α expression and autophagy.

Carbon monoxide­releasing molecules protect against blue light exposure and inflammation in retinal pigment epithelial cells.

The most common cause of vision loss among the elderly is age­related macular degeneration (AMD). The aim of the present study was to investigate the potential cytoprotective and anti­inflammatory effects of carbon monoxide­releasing molecules (CORMs), and their ability to activate the expression of nuclear factor erythroid 2­related factor 2 (Nrf2)­related genes in human retinal pigment epithelium (RPE) cells, as well as the inhibition of endothelial cell migration. It was first determined that CORM2 and CORM3 suppressed blue light­induced cell damage. In addition, a decrease in the level of cleaved poly(ADP­ribose) polymerase 1 protein and dissipation of mitochondrial membrane potential were considered to reflect the anti­apoptotic activity of CORMs. Furthermore, CORM2 induced Nrf­2 activation and the expression of the Nrf2­related genes heme oxygenase­1 and glutamate­cysteine ligase. Pretreatment with CORM2 abolished the blue light­induced increase in oxidative stress, suggesting that CORM2­induced antioxidant activity was involved in the cytoprotection against blue light. It was also demonstrated that CORMs markedly suppressed tumor necrosis factor (TNF)α­induced intercellular adhesion molecule­1 expression. Moreover, it was further observed that CORMs exert their inhibitory effects through blocking nuclear factor­κB/p65 nuclear translocation and IκBα degradation in TNFα­treated RPE cells. It was observed that CORM2, but not CORM3, protected against oxidative stress­induced cell damage. CORMs abolished vascular endothelial growth factor­induced migration of endothelial cells. The findings of the present study demonstrated the cytoprotective, antioxidant and anti­inflammatory effects of CORMs on RPE cells and anti­angiogenic effects on endothelial cells, suggesting the potential clinical application of CORMs as anti­AMD agents.

Peanut arachidin-1 enhances Nrf2-mediated protective mechanisms against TNF-α-induced ICAM-1 expression and NF-κB activation in endothelial cells.

Arachidin-1 [trans-4-(3-methyl-1-butenyl)-3,5,3',4'-tetrahydroxystilbene] is a polyphenol produced by peanut kernels during germination. The aim of the present study was to investigate the mechanism underlying the anti-inflammatory effect of arachidin-1 in endothelial cells (ECs). The results of cell adhesion and western blotting assays demonstrated that arachidin-1 attenuated tumor necrosis factor (TNF)-α-induced monocyte/EC adhesion and intercellular adhesion molecule-1 (ICAM-1) expression. Arachidin-1 was demonstrated to exert its inhibitory effects by the attenuation of TNF-α-induced nuclear factor-κB (NF-κB) nuclear translocation and inhibitor of κB-α (IκBα) degradation. Furthermore, arachidin-1 upregulated nuclear factor-E2-related factor-2 (Nrf-2), a known mediator of phase II enzyme expression, and increased the transcriptional activity of antioxidant response element. Transfection of ECs with Nrf-2 siRNA blocked the inhibitory effect of arachidin-1 on ICAM-1 expression, NF-κB nuclear translocation and IκBα degradation. In addition, arachidin-1 induced the expression of the phase II enzymes thioredoxin-1, thioredoxin reductase-1, heme oxygenase-1, glutamyl-cysteine synthetase and glutathione S-transferase. Following arachidin-1 pretreatment, the H2O2-induced generation of reactive oxygen species was reduced. Therefore, the present results indicate that arachidin-1 suppresses TNF-α-induced inflammation in ECs through the upregulation of Nrf-2-related phase II enzyme expression.

Effect of acetic acid on ethanol production by Zymomonas mobilis mutant strains through continuous adaptation.

BACKGROUND: Acetic acid is a predominant by-product of lignocellulosic biofuel process, which inhibits microbial biocatalysts. Development of bacterial strains that are tolerant to acetic acid is challenging due to poor understanding of the underlying molecular mechanisms. RESULTS: In this study, we generated and characterized two acetic acid-tolerant strains of Zymomonas mobilis using N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-acetate adaptive breeding. Two mutants, ZMA-142 and ZMA-167, were obtained, showing a significant growth rate at a concentration of 244 mM sodium acetate, while the growth of Z. mobilis ATCC 31823 were completely inhibited in presence of 195 mM sodium acetate. Our data showed that acetate-tolerance of ZMA-167 was attributed to a co-transcription of nhaA from ZMO0117, whereas the co-transcription was absent in ATCC 31823 and ZMA-142. Moreover, ZMA-142 and ZMA-167 exhibited a converstion rate (practical ethanol yield to theorical ethanol yield) of 90.16% and 86% at 195 mM acetate-pH 5 stress condition, respectively. We showed that acid adaptation of ZMA-142 and ZMA-167 to 146 mM acetate increased ZMA-142 and ZMA-167 resulted in an increase in ethanol yield by 32.21% and 21.16% under 195 mM acetate-pH 5 stress condition, respectively. CONCLUSION: The results indicate the acetate-adaptive seed culture of acetate-tolerant strains, ZMA-142 and ZMA-167, could enhance the ethanol production during fermentation.

Lycopene inhibits NF-κB activation and adhesion molecule expression through Nrf2-mediated heme oxygenase-1 in endothelial cells.

The endothelial expression of cell adhesion molecules plays a leading role in atherosclerosis. Lycopene, a carotenoid with 11 conjugated double bonds, has been shown to have anti-inflammatory properties. In the present study, we demonstrate a putative mechanism for the anti-inflammatory effects of lycopene. We demonstrate that lycopene inhibits the adhesion of tumor necrosis factor α (TNFα)-stimulated monocytes to endothelial cells and suppresses the expression of intercellular cell adhesion molecule-1 (ICAM-1) at the transcriptional level. Moreover, lycopene was found to exert its inhibitory effects by blocking the degradation of the inhibitory protein, IκBα, following 6 h of pre-treatment. In TNFα-stimulated endothelial cells, nuclear factor-κB (NF-κB) nuclear translocation and transcriptional activity were abolished by up to 12 h of lycopene pre-treatment. We also found that lycopene increased the intracellular glutathione (GSH) level and glutamate-cysteine ligase expression. Subsequently, lycopene induced nuclear factor-erythroid 2 related factor 2 (Nrf2) activation, leading to the increased expression of downstream of heme oxygenase-1 (HO-1). The use of siRNA targeting HO-1 blocked the inhibitory effects of lycopene on IκB degradation and ICAM-1 expression. The inhibitory effects of lycopene thus appear to be mediated through its induction of Nrf2-mediated HO-1 expression. Therefore, the findings of the present study indicate that lycopene suppresses the activation of TNFα-induced signaling pathways through the upregulation of Nrf2-mediated HO-1 expression.

Carbon monoxide releasing molecule induces endothelial nitric oxide synthase activation through a calcium and phosphatidylinositol 3-kinase/Akt mechanism.

The production of nitric oxide (NO) by endothelial NO synthase (eNOS) plays a major role in maintaining vascular homeostasis. This study elucidated the potential role of carbon monoxide (CO)-releasing molecules (CORMs) in NO production and explored the underlying mechanisms in endothelial cells. We observed that 25µM CORM-2 could increase NO production and stimulate an increase in the intracellular Ca2+ level. Furthermore, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetra acetic acid caused CORM-2-induced NO production, which was abolished by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetraacetoxy-methyl ester (BAPTA-AM), indicating that intracellular Ca2+ release plays a major role in eNOS activation. The inhibition of the IP3 receptor diminished the CORM-2-induced intracellular Ca2+ increase and NO production. Furthermore, CORM-2 induced eNOS Ser1179 phosphorylation and eNOS dimerization, but it did not alter eNOS expression. CORM-2 (25µM) also prolonged Akt phosphorylation, lasting for at least 12h. Pretreatment with phosphatidylinositol 3-kinase inhibitors (wortmannin or LY294002) inhibited the increases in NO production and phosphorylation but did not affect eNOS dimerization. CORM-2-induced eNOS Ser1179 phosphorylation was intracellularly calcium-dependent, because pretreatment with an intracellular Ca2+ chelator (BAPTA-AM) inhibited this process. Although CORM-2 increases intracellular reactive oxygen species (ROS), pretreatment with antioxidant enzyme catalase and N-acetyl-cysteine did not abolish the CORM-2-induced eNOS activity or phosphorylation, signifying that ROS is not involved in this activity. Hence, CORM-2 enhances eNOS activation through intracellular calcium release, Akt phosphorylation, and eNOS dimerization.

Lycopene inhibits ICAM-1 expression and NF-κB activation by Nrf2-regulated cell redox state in human retinal pigment epithelial cells.

AIMS: Age-related macular degeneration (AMD) is one of the most common diseases leading to blindness in elderly people. The progression of AMD may be prevented through anti-inflammation and antioxidation in retinal pigment epithelium (RPE) cells. Lycopene, a carotenoid, has been shown to possess both antioxidative and anti-inflammatory properties. This research was conducted to detail the mechanisms of these effects of lycopene-treated RPE cells. MAIN METHODS: We exposed ARPE-19 cells to TNFα after pretreatment with lycopene, and measured monocyte adhesion, ICAM-1 expression, NF-κB nuclear translocation, and transcriptional activity. Cell viability was assayed with Alamar Blue. The cell redox state was tested by glutathione (GSH) and reactive oxygen species (ROS) levels. The importance of the Nrf2 pathway was tested in nuclear translocation, promoter reporter assay, and siRNA. KEY FINDINGS: Lycopene could reduce TNF-α-induced monocyte adhesion and H2O2- induced cell damage in RPE cells. Furthermore, lycopene inhibits ICAM-1 expression and abolishes NF-κB activation for up to 12h in TNFα-treated RPE cells. Lycopene upregulates Nrf2 levels in nuclear extracts and increases the transactivity of antioxidant response elements. The use of Nrf2 siRNA blocks the inhibitory effect of lycopene in TNF-α-induced ICAM-1 expression and NF-κB activation. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the de novo synthesis of GSH. We found that lycopene increases intracellular GSH levels and GCL expression. Following lycopene treatment, TNF-α-induced ROS production was abolished. SIGNIFICANCE: The Nrf2-regulated antioxidant property plays a pivotal role in the anti-inflammatory mechanism underlying the inhibition of NF-κB activation in lycopene-treated ARPE-19 cells.

Carbon monoxide induces heme oxygenase-1 to modulate STAT3 activation in endothelial cells via S-glutathionylation.

IL-6/STAT3 pathway is involved in a variety of biological responses, including cell proliferation, differentiation, apoptosis, and inflammation. In our present study, we found that CO releasing molecules (CORMs) suppress IL-6-induced STAT3 phosphorylation, nuclear translocation and transactivity in endothelial cells (ECs). CO is a byproduct of heme degradation mediated by heme oxygenase (HO-1). However, CORMs can induce HO-1 expression and then inhibit STAT3 phosphorylation. CO has been found to increase a low level ROS and which may induce protein glutathionylation. We hypothesized that CORMs increases protein glutathionylation and inhibits STAT3 activation. We found that CORMs increase the intracellular GSSG level and induce the glutathionylation of multiple proteins including STAT3. GSSG can inhibit STAT3 phosphorylation and increase STAT3 glutathionylation whereas the antioxidant enzyme catalase can suppress the glutathionylation. Furthermore, catalase blocks the inhibition of STAT3 phosphorylation by CORMs treatment. The inhibition of glutathione synthesis by BSO was also found to attenuate STAT3 glutathionylation and its inhibition of STAT3 phosphorylation. We further found that HO-1 increases STAT3 glutathionylation and that HO-1 siRNA attenuates CORM-induced STAT3 glutathionylation. Hence, the inhibition of STAT3 activation is likely to occur via a CO-mediated increase in the GSSG level, which augments protein glutathionylation, and CO-induced HO-1 expression, which may enhance and maintain its effects in IL-6-treated ECs.

CO-releasing molecules and increased heme oxygenase-1 induce protein S-glutathionylation to modulate NF-κB activity in endothelial cells.

Protein glutathionylation is a protective mechanism that functions in response to mild oxidative stress. Carbon monoxide (CO) can increase the reactive oxygen species concentration from a low level via the inhibition of cytochrome c oxidase. We therefore hypothesized that CO would induce NF-κB-p65 glutathionylation and then show anti-inflammatory effects. In this study, we found that CO-releasing molecules suppress TNFα-induced monocyte adhesion to endothelial cells (ECs) and reduce ICAM-1 expression. Moreover, CO donors were further found to exert their inhibitory effects by blocking NF-κB-p65 nuclear translocation, but do so independent of IκBα degradation, in TNFα-treated ECs. In addition, p65 protein glutathionylation represents the response signal to CO donors and is reversed by the reducing agent dithiothreitol. Thiol modification of the cysteine residue in the p65 RHD region was required for the CO-modulated NF-κB activation. The suppression of p65 glutathionylation by a GSH synthesis inhibitor, BSO, and by catalase could also attenuate TNFα-induced p65 nuclear translocation and ICAM-1 expression. CO donors induce Nrf2 activation and Nrf2 siRNA suppresses CO-induced p65 glutathionylation and inhibition. Furthermore, we found that the CO donors induce heme oxygenase-1 (HO-1) expression, which increases p65 glutathionylation. In contrast, HO-1 siRNA attenuates CO donor- and hemin-induced p65 glutathionylation. Our results thus indicate that the glutathionylation of p65 is likely to be responsible for CO-mediated NF-κB inactivation and that the HO-1-dependent pathway may prolong the inhibitory effects of CO donors upon TNFα treatment of ECs.

The glutathionylation of p65 modulates NF-κB activity in 15-deoxy-Δ¹²,¹4-prostaglandin J2-treated endothelial cells.

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.

Upregulation of NF-E2-related factor-2-dependent glutathione by carnosol provokes a cytoprotective response and enhances cell survival.

AIM: To explore whether glutathione (GSH) increased through Nrf-2 activation is involved in the cytoprotective effects of carnosol in HepG2 cells. METHODS: 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. RESULTS: 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. CONCLUSION: This study demonstrated that Nrf2 is involved in the cytoprotective effects by carnasol, which were at least partially mediated through increased GSH biosynthesis.

Calcium- and phosphatidylinositol 3-kinase/Akt-dependent activation of endothelial nitric oxide synthase by apigenin.

AIMS: 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). MAIN METHODS: 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. KEY FINDINGS: 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. SIGNIFICANCE: 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.

The glutaredoxin/glutathione system modulates NF-kappaB activity by glutathionylation of p65 in cinnamaldehyde-treated endothelial cells.

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.

Dual mechanisms of NF-kappaB inhibition in carnosol-treated endothelial cells.

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.

Isolating a cytoprotective compound from Ganoderma tsugae: effects on induction of Nrf-2-related genes in endothelial cells.

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.

Neuroprotective effects of resveratrol on MPTP-induced neuron loss mediated by free radical scavenging.

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.

Tannin 1-alpha-O-galloylpunicalagin induces the calcium-dependent activation of endothelial nitric-oxide synthase via the phosphatidylinositol 3-kinase/Akt pathway in endothelial cells.

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.

Sulforaphane inhibition of monocyte adhesion via the suppression of ICAM-1 and NF-kappaB is dependent upon glutathione depletion in endothelial cells.

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.

Cinnamaldehyde inhibits the tumor necrosis factor-alpha-induced expression of cell adhesion molecules in endothelial cells by suppressing NF-kappaB activation: effects upon IkappaB and Nrf2.

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.

Chalcone inhibits the activation of NF-kappaB and STAT3 in endothelial cells via endogenous electrophile.

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.