The interaction of perfluorooctane sulfonate with hemoproteins and its relevance to molecular toxicology.

Perfluorooctane sulfonate (PFOS), a representative of perfluorinated compounds in industrial and commercial products, has posed a great threat to animals and humans via environmental exposure and dietary consumption. Herein, we investigated the effects of PFOS binding on the redox state and stability of two hemoproteins (hemoglobin (Hb) and myoglobin (Mb)). Fluorescence spectroscopy, circular dichroism and UV-vis absorption spectroscopy demonstrated that PFOS could induce the conformational changes of proteins along with the exposure of heme cavity and generation of hemichrome, which resulted in the increased release of free hemin. After that, free hemin liberated from hemoproteins led to reactive oxygen species formation, lipid peroxidation, cell membrane damage and loss of cell viability in vascular endothelial cells, while neither Hb nor Mb did show cytotoxicity. Chemical inhibitors of ferroptosis effectively mitigated hemin-caused toxicity, identifying the hemin-dependent ferroptotic cell death mechanisms. These data demonstrated that PFOS posed a potential threat of toxicity through a mechanism which involved its binding to hemoproteins, decreased oxygen transporting capacity, and increased hemin release. Altogether, our findings elucidate the binding mechanisms of PFOS with two hemoproteins, as well as possible risks on vascular endothelial cells, which would have important implications for the human and environmental toxicity of PFOS.

Serum albumin acted as an effective carrier to improve the stability of bioactive flavonoid.

The health-improving functions of bioactive flavonoids in vitro and in vivo are often limited by their low stability, which could be counteracted by the application of proteins as carriers of flavonoids. Clarification of the mechanism of protein-ligand interaction is crucial for the encapsulation of bioactive components. Herein, common plasma proteins [i.e., bovine serum albumin (BSA), human serum albumin (HSA), human immunoglobulin G (IgG) and fibrinogen (FG)] were compared for their binding characteristics to quercetin, the main component of flavonoids in human diet, in the absence and presence of free Cu2+ (an accelerator for flavonoids' instability) using multi-spectroscopic and computational methods. As a flexible open structure of proteins, both BSA and HSA were found to be the most promising carriers for quercetin and Cu2+ with an affinity on the order of 104 M-1. HSA-diligand complex (i.e., HSA-quercetin-Cu2+) was successfully generated when both quercetin and Cu2+ were added to the HSA solution. The stability and free radical scavenging activity of bioactive quercetin during incubation was promoted in the HSA-diligand complex relative to quercetin-Cu2+ complex. Quercetin/Cu2+ system could induce the formation of reactive oxygen species such as hydrogen peroxide (H2O2) and hydroxide radical (·OH), which were significantly suppressed upon HSA binding. Consistently, the cytotoxicity of the quercetin/Cu2+ system to endothelial cells was reduced in the HSA-diligand complex. These results demonstrate the possibility of developing serum albumin-based carriers for the protection of bioactive flavonoids in their nutritional application.

Serum albumin mitigated perfluorooctane sulfonate-induced cytotoxicity by affecting the cellular responses.

During the wide applications of perfluorinated materials such as perfluorooctane sulfonate (PFOS) in commercial and industrial products, the potential toxicity of these engineered compounds has attracted more and more attention. As a typical environmental pollutant, PFOS could preferentially bind to albumin protein in vivo. However, the role of protein-PFOS interactions in the cytotoxicity of PFOS was not stressed enough. Herein, we investigated the interactions of PFOS with human serum albumin (HSA, the most abundant protein in human plasma) using both experimental and theoretical approaches. It was demonstrated that PFOS could mainly bind to the Sudlow site I of HSA to generate HSA-PFOS complex through hydrogen bonds and van der Waals forces. Toxicity assays with endothelial cells illustrated that the binding of HSA could significantly attenuate the intracellular uptake and subcellular distribution of PFOS, thereby inhibiting the formation of reactive oxygen species and toxicity for those HSA-bound PFOS. Similarly, the presence of fetal bovine serum in the cell culture media greatly reduced PFOS-caused cytotoxicity. Conclusively, our study reveals that the binding of albumin protein to PFOS could mitigate its toxicity by the modulation of cellular responses. The formation of protein-complexed contaminants would significantly reduce the bioavailability of these chemicals and subsequently mitigate their environmental toxicology to the human health.

Binding of serum albumin to perfluorooctanoic acid reduced cytotoxicity.

With the ubiquitous applications of perfluorinated compounds such as perfluorooctanoic acid (PFOA) in industrial and commercial products, the toxicity of these engineered materials in environmental and public health is received growing attention. As a typical organic pollutant, PFOA has been extensively found in wildlife and human bodies, and can preferentially bind to serum albumin in vivo. However, the importance of protein-PFOA interactions on the cytotoxicity of PFOA could not be stressed enough. In this study, we used both experimental and theoretical approaches, to investigate the interactions of PFOA with bovine serum albumin (BSA, the most abundant protein in blood). It was found that PFOA could mainly interact with Sudlow site I of BSA to form BSA-PFOA complex, in which van der Waals forces and hydrogen bonds played dominant roles. Moreover, the strong binding of BSA could greatly alter the cellular uptake and distribution of PFOA in human endothelial cells, and result in the decreases of reactive oxygen species formation and cytotoxicity for these BSA-coated PFOA. Consistently, the addition of fetal bovine serum into cell culture medium also significantly mitigated PFOA-induced cytotoxicity, which was attributed to the extracellular complexation between PFOA and serum proteins. Altogether, our study demonstrates that the binding of serum albumin to PFOA could reduce its toxicity by affecting the cellular responses.

Quercetin Attenuates Vascular Endothelial Dysfunction in Atherosclerotic Mice by Inhibiting Myeloperoxidase and NADPH Oxidase Function.

Myeloperoxidase (MPO) exhibits a unique property to use H2O2 to oxidize chloride and lead to the generation of a strong oxidant, hypochlorous acid (HOCl), which plays important roles in atherosclerosis. A lot of evidence indicates that quercetin, a natural polyphenol derived from human diet, effectively contributes to cardiovascular health. Herein, we found that dietary quercetin significantly inhibited vascular endothelial dysfunction and atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. Mechanistic studies revealed that dietary quercetin effectively suppressed the MPO level and activity in the vessels of ApoE-/- animals, and p47phox expression and NADPH oxidase activity were simultaneously attenuated after quercetin treatment. In vascular endothelial cells, NADPH oxidase was demonstrated to be the major source of H2O2 formation. Moreover, quercetin effectively attenuated MPO/H2O2-mediated HOCl production and toxicity to human vascular endothelial cells, and this compound was not toxic. The inhibitory effect on MPO activity was likely attributed to that quercetin significantly inhibited NADPH oxidase-derived H2O2 formation in human endothelial cells and could act as an effective mediator for MPO intermediates, subsequently preventing HOCl production by the MPO/H2O2 system. Collectively, it was suggested that quercetin effectively suppressed endothelial dysfunction in atherosclerotic vasculature through the reduction of MPO/NADPH oxidase-mediated HOCl production.

Binding of Quercetin to Hemoglobin Reduced Hemin Release and Lipid Oxidation.

The interactions between quercetin and bovine (or human) hemoglobin (Hb) were systematically investigated by fluorescence, UV-vis absorption spectroscopy, and molecular docking to demonstrate the structural mechanism by which quercetin affected the Hb redox state and stability. Quercetin could interact with the central cavity of the Hb molecule with one binding site to generate an Hb-quercetin complex, and the hydrophobic interaction played an important role in the formation of the complex. The binding constant for the Hb-quercetin complex at 298 K was observed to be 1.25 × 104 M-1. In addition, quercetin effectively inhibited Hb-induced lipid oxidation in liposomes or washed muscles, which was ascribed to the conversion to oxy-Hb and decreased hemin dissociation from met-Hb. Consistent with its lower abilities to bind Hb and scavenge free radicals, rutin (i.e., quercetin-3-rhamnosylglucsoside) did not significantly influence the redox state of Hb nor reduce hemin release from Hb, and subsequently, it less effectively inhibited Hb-induced lipid oxidation than quercetin. Altogether, the results herein provide novel insights into the antioxidant mechanism for quercetin and are beneficial to the application of natural quercetin in Hb-containing foods.

Bovine Serum Albumin as a Potential Carrier for the Protection of Bioactive Quercetin and Inhibition of Cu(II) Toxicity.

Considering the protective ability of proteins and the potential toxicity of free Cu(II), it was proposed herein that the co-presence of protein could play an important role in suppressing the toxicity of free Cu(II) to the stability of bioactive quercetin if a flavonoid-protein-Cu(II) complex could be formed. In this study, the interaction between quercetin (a major flavonoid in the human diet) and bovine serum albumin (BSA) was investigated in the absence and presence of free Cu(II). The results demonstrated that both quercetin and free Cu(II) had a strong ability to quench the intrinsic fluorescence of BSA through a static procedure (i.e., formation of a BSA-monoligand complex). Site marker competitive experiments illustrated that the binding of both quercetin and Cu(II) to BSA mainly took place in subdomain IIA. The quenching process of free Cu(II) with BSA was easily affected by quercetin, and the increased binding capacity possibly resulted from the generation of a ternary quercetin-BSA-Cu(II) complex. The stability and free radical scavenging activity of bioactive quercetin during incubation was promoted in the BSA-diligand complex relative to a quercetin-Cu(II) complex. A quercetin-Cu(II) system could generate reactive oxygen species such as hydrogen peroxide (H2O2) and hydroxyl radicals (•OH), which were significantly inhibited upon BSA binding. Consistently, the cytotoxicity of the quercetin-Cu(II) system to endothelial cells was decreased in the BSA-diligand complex, where the co-presence of BSA played an important role. These results suggest the possibility and advantage of developing albumin-based carriers for the protection of bioactive components and suppression of Cu(II) toxicity in their biomedical and nutritional applications.

Myeloperoxidase Targets Apolipoprotein A-I for Site-Specific Tyrosine Chlorination in Atherosclerotic Lesions and Generates Dysfunctional High-Density Lipoprotein.

We previously demonstrated that apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), is an important target for myeloperoxidase (MPO)-catalyzed tyrosine chlorination in the circulation of subjects with cardiovascular diseases. Oxidation of apoA-I by MPO has been reported to deprive HDL of its protective properties. However, the potential effects of MPO-mediated site-specific tyrosine chlorination of apoA-I on dysfunctional HDL formation and atherosclerosis was unclear. Herein, Tyr192 in apoA-I was found to be the major chlorination site in both lesion and plasma HDL from humans with atherosclerosis, while MPO binding to apoA-I was demonstrated by immunoprecipitation studies in vivo. In vitro, MPO-mediated damage of lipid-free apoA-I impaired its ability to promote cellular cholesterol efflux by the ABCA1 pathway, whereas oxidation to lipid-associated apoA-I inhibited lecithin:cholesterol acyltransferase activation, two key steps in reverse cholesterol transport. Compared with native apoA-I, apoA-I containing a Tyr192 → Phe mutation was moderately resistant to oxidative inactivation by MPO. In high-fat-diet-fed apolipoprotein E-deficient mice, compared with native apoA-I, subcutaneous injection with oxidized apoA-I (MPO treated) failed to mediate the lipid content in aortic plaques while mutant apoA-I (Tyr192 → Phe) showed a slightly stronger ability to reduce the lipid content in vivo. Our observations suggest that oxidative damage of apoA-I and HDL involves MPO-dependent site-specific tyrosine chlorination, raising the feasibility of producing MPO-resistant forms of apoA-I that have stronger antiatherosclerotic activity in vivo.

Generation of a Bovine Serum Albumin-Diligand Complex for the Protection of Bioactive Quercetin and Suppression of Heme Toxicity.

As an abundant protein in milk and blood serum, bovine serum albumin (BSA) contains various sites to bind a lot of bioactive components, generating BSA-monoligand complex. Demonstration of the interaction between BSA and bioactive components (such as heme, flavonoids) is important to develop effective carrier for the protection of bioactive ligands and to reduce cytotoxicity of heme. Herein, the bindings of BSA to quercetin and/or heme were investigated by multispectroscopic and molecular docking methods. The fluorescence of protein was significantly quenched by both quercetin and heme in a static mode (i.e., generation of BSA-ligand complex). Although quercetin had lower affinity to protein than heme, the interactions of both compounds with protein did locate in site I (i.e., subdomain IIA). BSA-diligand complex was successfully generated after the coaddition of quercetin and heme. The cytotoxicity of free heme to endothelial cells was reduced in the BSA-diligand complex relative to that of heme or BSA-monoligand complex, while the stability of bioactive quercetin was promoted in the complex relative to free flavonoid. The complex provided a better inhibition on the cytotoxicity of heme than BSA-monoligand complex, in which the copresence of quercetin played a vital role.

Quercetin Attenuated Myeloperoxidase-Dependent HOCl Generation and Endothelial Dysfunction in Diabetic Vasculature.

Myeloperoxidase (MPO)-dependent hypochlorous acid (HOCl) generation plays crucial roles in diabetic vascular complications. As a natural polyphenol, quercetin has antioxidant properties in various diabetic models. Herein, we investigated the therapeutic mechanism for quercetin on MPO-mediated HOCl generation and endothelial dysfunction in diabetic vasculature. In vitro, the presence of MPO could amplify high glucose-induced endothelial dysfunction which was significantly inhibited by the NADPH oxidase inhibitor, HOCl or H2O2 scavengers, revealing the contribution of MPO/H2O2/HOCl to vascular endothelial injury. Furthermore, quercetin effectively inhibited MPO/high glucose-mediated HOCl generation and cytotoxicity to vascular endothelial cells. The inhibitive effect on MPO activity was related to the fact that quercetin reduced high glucose-induced H2O2 generation in endothelial cells and directly acted as a competitive substrate for MPO, thus limiting MPO/H2O2-dependent HOCl production. Moreover, quercetin could attenuate HOCl-caused endothelial dysfunction in endothelial cells and isolated aortas. In vivo, dietary quercetin significantly inhibited aortic endothelial dysfunction in diabetic mice, while this compound simultaneously suppressed vascular MPO expression and activity. Therefore, it was demonstrated herein that quercetin inhibited endothelial injury in diabetic vasculature via suppression of MPO/high glucose-dependent HOCl formation.

Quercetin Inhibited Endothelial Dysfunction and Atherosclerosis in Apolipoprotein E-Deficient Mice: Critical Roles for NADPH Oxidase and Heme Oxygenase-1.

NADPH oxidase-dependent superoxide (O2·-) production and oxidative stress play important roles in endothelial dysfunction and atherosclerosis. Herein, we investigated the potential effects of dietary quercetin, a flavonoid derived in the diet from vegetables and fruit, on vascular endothelial function and atherosclerosis in the high-fat diet (HFD)-fed apolipoprotein E-deficient (ApoE-/-) mice. Dietary quercetin treatment significantly suppressed endothelial dysfunction and aortic atherosclerosis in HFD-fed ApoE-/- mice (P < 0.05, all cases). Mechanistic studies demonstrated that dietary quercetin significantly attenuated p47phox expression and inhibited NADPH oxidase-derived oxidative stress in the aortas of HFD-fed ApoE-/- mice, while the expression and activity of antioxidant enzyme heme oxygenase-1 (HO-1) was enhanced after quercetin treatment (P < 0.05, all cases). In vitro, it was found that quercetin significantly attenuated NADPH oxidase-derived O2·- formation (75 ± 5.6% for quercetin versus 100 ± 6.0% for the control group, P < 0.01) in endothelial cells through induction of HO-1. In addition, the favorable effects of quercetin on oxidant (i.e., H2O2)-induced endothelial dysfunction could be eliminated by tin protoporphyrin IX (an HO-1 inhibitor) or HO-1-specific siRNA. Our results demonstrated the critical roles of NADPH oxidase and HO-1 for the indirect antioxidant properties of quercetin in vascular diseases.

Dietary nitrate attenuated endothelial dysfunction and atherosclerosis in apolipoprotein E knockout mice fed a high-fat diet: A critical role for NADPH oxidase.

Nitric oxide (NO) deficiency and NADPH oxidase plays key roles in endothelial dysfunction and atherosclerotic plaque formation. Recent evidence demonstrates that nitrate-nitrite-NO pathway in vivo exerts beneficial effects upon the cardiovascular system. We aimed to investigate the effects of dietary nitrate on endothelial function and atherosclerosis in apolipoprotein E knockout (ApoE-/-) mice fed a high-fat diet. It was shown that dietary nitrate significantly attenuated aortic endothelial dysfunction and atherosclerosis in ApoE-/- mice. Mechanistic studies revealed that dietary nitrate significantly improved plasma nitrate/nitrite, inhibited vascular NADPH oxidase activity and oxidative stress in ApoE-/- mice, while xanthine oxidoreductase (XOR) expression and activity was enhanced in ApoE-/- mice in comparison with wide type animals. These beneficial effects of nitrate in ApoE-/- mice were abolished by PTIO (NO scavenger) and significantly prevented by febuxostat (XOR inhibitor). In the presence of nitrate, no further effect of apocynin (NADPH oxidase inhibitor) was observed, suggesting NADPH oxidase as a possible target. In vitro, NO donor significantly inhibited NADPH oxidase activity in vascular endothelial cells via the induction of heme oxygenase-1. Altogether, boosting this nitrate-nitrite-NO signaling pathway resulted in the decreases of vascular NADPH oxidase-derived oxidative stress and endothelial dysfunction, and consequently protected ApoE-/- mice against atherosclerosis. These findings may have novel nutritional implications for the preventive and therapeutic strategies against vascular endothelial dysfunction in atherosclerotic disease.

Phosphine-Free Ru-Catalyzed Regio- and Stereoselective Addition of Benzoic Acids to Trifluoromethylated Alkynes toward Facile Access to Trifluoromethyl Group-Substituted (E)-Enol Esters.

A combination of ruthenium catalyst with silver salt and copper salt was proved to be a highly efficient protocol for the direct addition reaction of benzoic acids with unsymmetrical trifluoromethylated internal alkynes. Diverse trifluoromethyl group-substituted (E)-enol esters were readily obtained for a broad substrate scope in moderate to good yields with excellent regio- and stereoselectivities under mild reaction conditions.

Supplementation of dietary nitrate attenuated oxidative stress and endothelial dysfunction in diabetic vasculature through inhibition of NADPH oxidase.

The metabolic disorders in diabetes, which are usually accompanied by oxidative stress and impaired nitric oxide (NO) bioavailability, increase the risk of detrimental cardiovascular complications. Herein, we investigated the therapeutic potential of dietary nitrate, which is found in high content in green leafy vegetables, on vascular oxidative stress and endothelial dysfunction in diabetic mice induced by high-fat diet and streptozotocin injection. Dietary nitrate in drinking water fuelled a nitrate-nitrite-NO pathway, which inhibited vascular oxidative stress, endothelial dysfunction and many features of metabolic syndrome in diabetic mice. These beneficial effects of nitrate on diabetic mice were abolished by PTIO (NO scavenger) treatment and significantly prevented by febuxostat (xanthine oxidoreductase inhibitor), demonstrating the central importance of NO in bioactivation of nitrate. The favorable effects of nitrate were not further influenced by apocynin (NADPH oxidase inhibitor), suggesting NADPH oxidase as a possible target. In high glucose-incubated vascular endothelial cells, NO donor attenuated oxidative stress and endothelial dysfunction via the inhibition of NADPH oxidase, where a heme oxygenase-1 (HO-1)-dependent mechanism was demonstrated for the antioxidant abilities of NO. Altogether, boosting this nitrate-nitrite-NO signaling pathway resulted in the decreases of NADPH oxidase-derived oxidative stress, endothelial dysfunction and metabolic disorders in diabetic vasculature. These findings may have novel implications for the preventive strategy against diabetes-induced vascular dysfunction and associated complications.

Formation of a bovine serum albumin diligand complex with rutin for the suppression of heme toxicity.

Serum albumin binds avidly to heme to form heme-serum albumin complex and can protect against the potentially toxic effects of heme. Rutin is a glycoside of the bioflavonoid quercetin with various protective effects due to its antioxidant ability. Clarification of the interaction mechanisms between serum albumin and bioactive components (such as heme and flavonoid) is important to develop effective carriers for encapsulation of heme and suppression of its toxicity. In this study, bindings of bovine serum albumin (BSA) to heme and/or rutin were investigated by experimental and molecular docking techniques. The fluorescence of BSA was quenched by both heme and rutin in static mode (i.e. formation of BSA-monoligand complexes), which was confirmed by Stern-Volmer calculations. Although heme showed higher affinity to BSA than rutin, the interactions of both components with BSA did locate within subdomain IIA (site I). BSA-diligand complexes were successfully formed after the simultaneous addition of heme and rutin. Bioactive rutin in the BSA-diligand complex still kept strong free radical scavenging activity compared to free rutin or BSA-monoligand complex. Hydrogen peroxide (H2O2)-induced heme degradation and free iron release was inhibited upon BSA binding and further decreased in BSA-diligand complexes. Consistently, the cytotoxicity of heme and oxidative stress in endothelial cells was decreased in the BSA-diligand complexes relative to those of heme or BSA-heme complex, where the co-presence of rutin played an important role. These results suggest the possibility and advantage of developing BSA-based carriers for the suppression of heme toxicity in their biomedical applications.

Formation of a bovine serum albumin diligand complex with rutin and single-walled carbon nanotubes for the reduction of cytotoxicity.

Carbon nanotubes (CNTs) are extensively used in the area of biotechnology and biomedicine, and the binding of proteins to CNTs plays an important role in the potential toxicity of nanomaterials. Rutin is a glycoside of the bioactive quercetin with various health-improving effects due to its antioxidant ability. Demonstration of the interaction between serum albumin and bioactive components is important to design effective carriers for the suppression of CNTs' toxicity. In this study, bindings of bovine serum albumin (BSA) to single-walled CNTs and/or rutin were investigated by fluorescence and molecular docking techniques. The fluorescence of BSA was significantly quenched by both CNTs and rutin in static mode, which was confirmed by the Stern-Volmer calculations. Although rutin showed higher affinity to protein than CNTs, the interactions of both components with BSA did mainly locate within subdomain IIA (site I). BSA-diligand complexes were successfully formed after the simultaneous addition of CNTs and rutin. Bioactive rutin in the BSA-diligand complex still kept strong free radical scavenging activity compared to free rutin or BSA-monoligand complex. Consistently, the cytotoxicity of CNTs and reactive oxygen species formation in endothelial cells was reduced in the BSA-diligand complexes relative to those of BSA-CNTs corona or CNTs alone, where the co-presence of rutin played an important role. These findings suggest the possibility and advantage of designing BSA-based carriers for the suppression of CNTs' toxicity in their biomedical applications.

Quercetin, but not rutin, attenuated hydrogen peroxide-induced cell damage via heme oxygenase-1 induction in endothelial cells.

Oxidative stress plays an important role in the pathogenesis of cardiovascular disease. Quercetin, a naturally occurring flavonoid presents in plants and human diet, has been reported to exert antioxidant properties in vivo and in vitro. The upregulation of antioxidant enzyme heme oxygenase-1 (HMOX1) in endothelial cells is considered to be beneficial in cardiovascular disease. In this work, we tested whether quercetin might suppress hydrogen peroxide (H2O2)-induced cell damage in endothelial cells by augmenting this cellular antioxidant defense. It was found that quercetin upregulated HMOX1 expression to protect endothelial cells against oxidative stress, and the protective effects of quercetin on H2O2-induced endothelial cell damage (such as loss of cell viability and reduction of nitric oxide) could be abolished by the specific small-interfering RNA against HMOX1 expression or HMOX1 activity inhibitor. In addition, the activation of ERK/Nrf2 signaling pathway was critical to the upregulation of HMOX1 induced by quercetin. Consistent with its non-effective ability to induce HMOX1, rutin (the glycoside of quercetin) showed less protective effects on H2O2-induced cell damage than quercetin. Therefore, quercetin could attenuate oxidative stress-induced endothelial cell damage at least partly through ERK/Nrf2/HMOX1 pathway. Our results also suggested a novel mechanism for the anti-oxidant property of quercetin and might explain in part the protective cardiovascular effects of diets rich in these compounds.

Quercetin suppressed NADPH oxidase-derived oxidative stress via heme oxygenase-1 induction in macrophages.

NADPH oxidase-derived superoxide (O2.-) generation and oxidative stress is usually considered as an important factor to the pathogenesis of inflammatory diseases. Quercetin, widely known for their anti-oxidant and anti-inflammatory properties in vitro and in vivo, is recently identified to induce expression of antioxidant enzyme heme oxygenase-1 (HO-1). Previous studies suggest that HO-1 induction and/or subsequent HO-1 end product generation in vitro and in vivo may suppress NADPH oxidase-derived oxidative stress. In this study, we tested whether quercetin might modulate NADPH oxidase activity in macrophages via induction of HO-1. In RAW264.7 macrophages, quercetin significantly attenuated NADPH oxidase-derived O2.- generation via a HO-1-dependent mechanism. Mechanistically, the protective effects of quercetin were (1) linked to increased expression of HO-1 in the presence or absence of lipopolysaccharide (LPS), (2) similar to that observed with the NADPH oxidase inhibitor apocynin, and (3) could be abolished by the specific small-interfering RNA against HO-1 expression or HO-1 activity inhibitor tin protoporphyrin. The induction of HO-1 by quercetin was associated with the nuclear accumulation of Nrf2 and downregulation of Keap1, a negative regulator of Nrf2. In addition, this flavonoid also inhibited the overproduction of nitric oxide and inflammatory cytokines in LPS-stimulated macrophages via simultaneous induction of HO-1 expression. In agreement with the observations in macrophages, pretreatment with quercetin significantly alleviated LPS-induced inflammation in mice which was concomitant with decreased NADPH oxidase activity and increased HO-1 expression. Our results suggested that quercein could modulate NADPH oxidase-derived O2.- production in macrophages at least partly through HO-1 induction. Suppression of NADPH oxidase-dependent oxidative stress may represent a novel mechanism underlying the anti-oxidant and anti-inflammatory properties of quercetin/HO-1 pathway.

NADPH oxidase is a primary target for antioxidant effects by inorganic nitrite in lipopolysaccharide-induced oxidative stress in mice and in macrophage cells.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and oxidative stress is usually considered as an important factor to the pathogenesis of various diseases. Inorganic nitrite, previously viewed as a harmful substance in our diet or inert metabolites of endogenous NO, is recently identified as an important biological NO reservoir in vasculature and tissues. Stimulation of a nitrite-NO pathway shows organ-protective effects on oxidative stress and inflammation, but the mechanisms or target are not clear. In this study, the hypothesis that inorganic nitrite attenuated lipopolysaccharide (LPS)-induced oxidative stress in mice and in macrophage cells by modulating NADPH oxidase activity and NO bioavailability were investigated. We showed that nitrite treatment, in sharp contrast with the worsening effect of NO synthases inhibition, significantly attenuated aortic oxidative stress, endothelial dysfunction and mortality in LPS-induced shock in mice. Mechanistically, protective effects of nitrite were abolished by NO scavenger and xanthine oxidase inhibitor, and inhibition of NADPH oxidase with apocynin attenuated LPS-induced oxidative stress similar to that of nitrite. In the presence of nitrite, no further effect of apocynin was observed, suggesting NADPH oxidase as a possible target. In LPS-activated macrophage cells, nitrite reduced NADPH oxidase activity and oxidative stress and these effects of nitrite were also abolished by NO scavenger and xanthine oxidase inhibitor, where xanthine oxidase-mediated reduction of nitrite attenuated NADPH oxidase activity in activated macrophages via a NO-dependent mechanism. In conclusion, these novel findings position NADPH oxidase in the inflammatory vasculature as a prime target for the antioxidant effects of inorganic nitrite, and open a new direction to modulate the inflammatory response.

Iridium-Catalyzed Regioselective Synthesis of Trifluoromethylated Isocoumarins through Annulation of Benzoic Acids with Trifluoromethylated Alkynes.

An unprecedented Ir-catalyzed oxidative coupling of benzoic acids with trifluoromethylated alkynes was successfully developed to provide diverse trifluoromethylated isocoumarins in moderate to good yields. This new practical procedure was highlighted by mild reaction conditions, broad substrate scope, good regioselectivity, high efficiency, and easy operation.