Effects of physiological quercetin metabolites on interleukin-1β-induced inducible NOS expression

Cytokines released by inflammatory cells around the pancreatic islets are implicated in the pathogenesis of diabetes mellitus. Specifically, interleukin-1β (IL-1β) is known to be involved in islet β-cell damage by activation of nuclear factor-κB (NF-κB)-mediated inducible nitric oxide synthase (iNOS) gene expression. Though most flavonoids are shown to have various beneficial effects, little is known about the anti-inflammatory effects of their metabolites. Therefore, we investigated the effects of quercetin and its metabolites quercetin 3'-sulfate, quercetin 3-glucuronide and isorhamnetin 3-glucuronide on IL-1β-stimulated iNOS gene expression in RINm5F β-cells. The nitrite level, iNOS protein and its mRNA expression levels and iNOS promoter activity were measured. In addition, IκBα protein phosphorylation, nuclear translocation of nuclear factor-κB (NF-κB) and NF-κB DNA binding activity were determined. Adenosine 5'-triphosphate disodium salt-induced insulin release was also measured. Quercetin significantly reduced IL-1β-induced nitrite production, iNOS protein and its mRNA expression levels, and it also inhibited IL-1β-induced IκBα phosphorylation, NF-κB activation and iNOS promoter activity. Additionally, quercetin significantly restored the inhibition of insulin secretion by IL-1β. Meanwhile, quercetin metabolites did not show any effect on IL-1β-induced iNOS gene expression and also on insulin secretion. Therefore, in terms of iNOS expression mechanism, dietary ingestion of quercetin is unlikely to show anti-inflammatory effects in rat islet β-cells exposed to IL-1β.     

Efficiency of absorption and metabolic conversion of quercetin and its glucosides in human intestinal cell line Caco-2

The efficiency of intestinal absorption and metabolic conversion of quercetin aglycone and its glucosides, quercetin-4'-O-beta-D-glucoside (Q4'G), quercetin-3-O-beta-D-glucoside (Q3G), and quercetin-3,4'-di-O-beta-D-glucoside (Q3,4'G), was estimated by using Caco-2 cell monolayers as an intestinal epithelial cell model. Aglycone was significantly lost from the apical side, resulting in the appearance of free and conjugated forms of quercetin and those of isorhamnetin in the cellular extracts. In the basolateral solution, the conjugated form of quercetin was predominant and increased with the elapse of incubation. As compared with quercetin aglycone, none of the quercetin glucosides were absorbed efficiently from apical side. However, Q4'G yielded conjugated quercetin and isorhamnetin in basolateral solution at higher amounts than Q3G or Q3,4'G. Lipophilicity of Q4'G was found to be higher than that of Q3G or Q3,4'G. This suggests that lipophilicity contributes to the relatively efficient absorption of Q4'G. It is likely that the occurrence of hydrolysis enhances the efficiency of intestinal absorption and metabolic conversion of dietary quercetin glucosides.     

Immune-regulation of the apolipoprotein A-I/C-III/A-IV gene cluster in experimental inflammation

Apolipoprotein A-IV is a member of the apo A-I/C-III/A-IV gene cluster. In order to investigate its hypothetical coordinated regulation, an acute phase was induced in pigs by turpentine oil injection. The hepatic expression of the gene cluster as well as the plasma levels of apolipoproteins were monitored at different time periods. Furthermore, the involvement of the inflammatory mediators' interleukins 1 and 6 and tumor necrosis factor in the regulation of this gene cluster was tested in cultured pig hepatocytes, incubated with those mediators and apo A-I/C-III/A-IV gene cluster expression at the mRNA level was measured. In response to turpentine oil-induced inflammation, a decreased hepatic apo A-IV mRNA expression was observed (independent of apo A-I and apo C-III mRNA) not correlating with the plasma protein levels. The distribution of plasma apo A-IV experienced a shift from HDL to larger particles. In contrast, the changes in apo A-I and apo C-III mRNA were reflected in their corresponding plasma levels. Addition of cytokines to cultured pig hepatocytes also decreased apo A-IV and apo A-I mRNA levels. All these results show that the down-regulation of apolipoprotein A-I and A-IV messages in the liver may be mediated by interleukin 6 and TNF-alpha. The well-known HDL decrease found in many different acute-phase responses also appears in the pig due to the decreased expression of apolipoprotein A-I and the enlargement of the apolipoprotein A-IV-containing HDL.     

Quercetin's influence on exercise-induced changes in plasma cytokines and muscle and leukocyte cytokine mRNA.

Trained male cyclists (n = 40) ingested quercetin (Q; n = 20) (1,000 mg/day) or placebo (P; n = 20) supplements under randomized, double-blinded methods for 3 wk before and during a 3-day period in which subjects cycled for 3 h/day at approximately 57% maximal work rate. Blood samples were collected before and after each exercise session and assayed for plasma IL-6, IL-10, IL-1ra, IL-8, TNF-alpha, and monocyte chemoattractant protein 1, and leukocyte IL-10, IL-8, and IL-1ra mRNA. Muscle biopsies were obtained before and after the first and third exercise sessions and assayed for NF-kappaB and cyclooxygenase-2 (COX-2), IL-6, IL-8, IL-1beta, and TNF-alpha mRNA. Postexercise increases in plasma cytokines did not differ between groups, but the pattern of change over the 3-day exercise period tended to be lower in Q vs. P for IL-8 and TNF-alpha (P = 0.094 for both). mRNA increased significantly postexercise for each cytokine measured in blood leukocyte and muscle samples. Leukocyte IL-8 and IL-10 mRNA were significantly reduced in Q vs. P (interaction effects, P = 0.019 and 0.012, respectively) with no other leukocyte or muscle mRNA group differences. Muscle NF-kappaB did not increase postexercise and did not differ between Q and P. Muscle COX-2 mRNA increased significantly postexercise but did not differ between Q and P. In summary, 1 g/day quercetin supplementation by trained cyclists over a 24-day period diminished postexercise expression of leukocyte IL-8 and IL-10 mRNA, indicating that elevated plasma quercetin levels exerted some effects within the blood compartment. Quercetin did not, however, influence any of the muscle measures, including NF-kappaB content, cytokine mRNA, or COX-2 mRNA expression across a 3-day intensified exercise period.     

Quercetin metabolites inhibit MMP-2 expression in A549 lung cancer cells by PPAR-γ associated mechanisms

Our previous study demonstrated that quercetin-metabolite-enriched plasma (QP) but not quercetin itself upregulates peroxisome proliferator-activated receptor gamma (PPAR-γ) expression to induce G₂/M arrest in A549 cells. In the present study, we incubated A549 cells with QP as well as quercetin-3-glucuronide (Q3G) and quercetin-3′-sulfate (Q3′S), two major metabolites of quercetin, to investigate the effects of quercetin metabolites on cell invasion and migration, the possible mechanisms and the role of PPAR-γ. We also compared the effects of QP with those of quercetin and troglitazone (TGZ), a PPAR-γ ligand. The results showed that QP significantly suppressed cell invasion and migration, as well as matrix metalloproteinases (MMPs)-2 activity and expression in a dose-dependent manner. The effects of 10% QP on those parameters were similar to those of 10 μM quercetin and 20 μM TGZ. However, QP and TGZ rather than quercetin itself increased the expressions of nm23-H1 and tissue inhibitor of metalloproteinase (TIMP-2). Furthermore, we demonstrated that Q3G and Q3′S also inhibited the protein expression of MMP-2. GW9662, a PPAR-γ antagonist, significantly diminished such an effect of Q3G and Q3′S. Silencing PPAR-γ expression in A549 cells also significantly diminished the suppression effect of Q3G and Q3′S on MMP-2 expression. Taken together, our study demonstrated that QP inhibited cell invasion and migration through nm23-H1/TIMP-2/MMP-2 associated mechanisms. The upregulation of PPAR-γ by quercetin metabolites such as Q3G and Q3′S could play an important role in the effects of QP.     

Isorhamnetin-induced anti-adipogenesis is mediated by stabilization of β-catenin protein

AimsPrevious studies have shown that isorhamnetin has anti-adipogenic effects in mouse 3T3-L1 cells. This study was conducted to elucidate the inhibitory mechanisms of isorhamnetin during adipogenic differentiation of human adipose tissue-derived stem cells (hAMSCs).Main methodsThe effect of isorhamnetin on adipogenic differentiation of hAMSCs was quantified by Oil Red O staining and a triglyceride assay. In addition, real-time PCR and Western blot were used to determine the expression of adipogenesis-related genes.Key findingsIsorhamnetin inhibited the adipocyte differentiation of hAMSCs. Additionally, when the effects of Wnt antagonists that promote adipogenesis were evaluated, isorhamnetin was found to down-regulate the mRNA levels of sFRP1 and Dkk1, but had no effect on the mRNA levels of sFRP2, sFRP3, sFRP4 and Dkk3. Isorhamnetin also inhibited the expression of Wnt receptor and co-receptor genes. Furthermore, isorhamnetin increased the protein levels of β-catenin, an effector molecule of Wnt signaling, but had no effect on the mRNA levels of β-catenin. The phosphorylation level of GSK 3β was also increased by isorhamnetin. These results were confirmed by the fact that the expression of c-myc, cyclin D1 and PPARδ, which are target genes of β-catenin, was upregulated by isorhamnetin. Moreover, isorhamnetin reduced the mRNA expression levels of C/EBPα and PPARγ, which are known to be inhibited by c-myc or by cyclin D1 and PPARδ, respectively.SignificanceOur results indicate that isorhamnetin inhibits the adipogenic differentiation of hAMSCs and that its mechanisms are mediated by the stabilization of β-catenin.     

Treatment with Isorhamnetin Protects the Brain Against Ischemic Injury in Mice

Ischemic stroke is a major cause of morbidity and mortality, yet lacks effective neuroprotective treatments. The aim of this work was to investigate whether treatment with isorhamnetin protected the brain against ischemic injury in mice. Experimental stroke mice underwent the filament model of middle cerebral artery occlusion with reperfusion. Treatment with isorhamnetin or vehicle was initiated immediately at the onset of reperfusion. It was found that treatment of experimental stroke mice with isorhamnetin reduced infarct volume and caspase-3 activity (a biomarker of apoptosis), and improved neurological function recovery. Treatment of experimental stroke mice with isorhamnetin attenuated cerebral edema, improved blood–brain barrier function, and upregulated gene expression of tight junction proteins including occludin, ZO-1, and claudin-5. Treatment of experimental stroke mice with isorhamnetin activated Nrf2/HO-1, suppressed iNOS/NO, and led to reduced formation of MDA and 3-NT in ipsilateral cortex. In addition, treatment of experimental stroke mice with isorhamnetin suppressed activity of MPO (a biomarker of neutrophil infiltration) and reduced protein levels of IL-1β, IL-6, and TNF-α in ipsilateral cortex. Furthermore, it was found that treatment of experimental stroke mice with isorhamnetin reduced mRNA and protein expression of NMDA receptor subunit NR1 in ipsilateral cortex. In conclusion, treatment with isorhamnetin protected the brain against ischemic injury in mice. Isorhamnetin could thus be envisaged as a countermeasure for ischemic stroke but remains to be tested in humans.     

Downregulation of connexin32 protein and gap-junctional intercellular communication by cytokine-mediated acute-phase response in immortalized mouse hepatocytes

In the present study, we have analyzed the direct effects of cytokines, which mediate the acute-phase response in liver, on connexin expression and gap-junctional intercellular communication in immortalized MHSV12 mouse hepatocytes. When these cells were stimulated for 24 h with interleukin 1 and interleukin 6, the amount of connexin26 (Cx26) mRNA increased together with β?fibrinogen mRNA, as expected for this positive acute-phase gene. In contrast, connexin32 (Cx32) mRNA expression was not affected under these conditions. Indirect immunfluorescence revealed a drastic decrease in Cx32 signals, whereas slightly more Cx26 signals were found. Stronger stimulation with interleukin 1 and tumor necrosis factor α gave a dose-dependent increase in steady state levels of Cx26 and β-fibrinogen mRNA, but no further change in Cx32 mRNA level was seen. However, when Cx32 protein was analyzed on immunoblots, we found a 5-fold decrease in expression even at low cytokine doses that did not affect Cx32 mRNA expression. Under these conditions, cell to cell transfer of Lucifer yellow, microinjected into immortalized hepatocytes, was decreased by 70%, suggesting that intercellular communication through Cx32 channels was partially inhibited earlier than other genetic alterations characteristic of the acute-phase response. Thus, the major hepatic gap junction protein was largely downregulated at the beginning of the experimental inflammatory reaction, but about 30% of gap-junctional intercellular communication was maintained. This suggests that, during the acute-phase response, the second hepatic Cx26 protein may compensate in part for the downregulation of the Cx32 protein.     

Acetate reduces microglia inflammatory signaling in vitro

Acetate supplementation increases brain acetyl‐CoA and histone acetylation and reduces lipopolysaccharide (LPS)‐induced neuroglial activation and interleukin (IL)‐1β expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen‐activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor‐kappa B (NF‐κB) in primary and BV‐2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS‐induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL‐1β, IL‐6, and tumor necrosis factor‐alpha (TNF‐α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL‐6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor‐β1 (TGF‐β1) and both IL‐4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2–4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS‐induced elevation of NF‐κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non‐histone protein acetylation.     

Synergistic chemoprotective mechanisms of dietary phytoestrogens in a select combination against prostate cancer

Combination of dietary phytoestrogens with diverse molecular mechanisms may enhance their anticancer efficacy at physiological concentrations, as evidenced in epidemiological studies. A select combination of three dietary phytoestrogens containing 8.33 μM each of genistein (G), quercetin (Q) and biochanin A (B) was found to be more potent in inhibiting the growth of androgen-responsive prostate cancer cells (LNCaP) as well as DU-145 and PC-3 prostate cancer cells in vitro than either 25 μM of G, B or Q or 12.5+12.5 μM of G+Q, Q+B or G+B. Subsequent mechanistic studies in PC-3 cells indicated that the action of phytoestrogens was mediated both through estrogen receptor (ER)-dependent and ER-independent pathways as potent estrogen antagonist ICI-182780 (ICI, 5 μM) could not completely mask the synergistic anticancer effects, which were sustained appreciably in presence of ICI. G+Q+B combination was significantly more effective than individual compounds or their double combinations in increasing ER-β, bax (mRNA expression); phospho-JNK, bax (protein levels); and in decreasing bcl-2, cyclin E, c-myc (mRNA expression); phospho-AKT, phospho-ERK, bcl-2, proliferating cell nuclear antigen (protein levels) in PC-3 cells. Phytoestrogens also synergistically stimulated caspase-3 activity. Our findings suggest that selectively combining anticancer phytoestrogens could significantly increase the efficacy of individual components resulting in improved efficacy at physiologically achievable concentrations. The combination mechanism of multiple anticancer phytochemicals may be indicative of the potential of some vegetarian diet components to elicit chemopreventive effects against prostate cancer at their physiologically achievable concentrations, in vivo.     

Antioxidant activity and metabolite profile of quercetin in vitamin-E-depleted rats

Dietary antioxidants interact in a dynamic fashion, including recycling and sparing one another, to decrease oxidative stress. Limited information is available regarding the interrelationships in vivo between quercetin and vitamin E. We investigated the antioxidant activity and metabolism of quercetin (Q) in 65 F-344 rats (n=13 per group) randomly assigned to the following vitamin E (VE)-replete and -deficient diets: (a) VE replete (30 mg alpha-tocopherol acetate/kg diet) control ad libitum (C-AL), (b) VE replete pair fed (C-PF), (c) VE replete+5.0 g Q/kg diet (R-VE+5Q), (d) VE deplete (<1 mg/kg total tocopherols)+5.0 g Q/kg diet (D-VE+5Q) and (e) D-VE. After 12 weeks, blood and tissue were collected for measurement of plasma vitamin E, quercetin and its metabolites, serum pyruvate kinase (PK), plasma protein carbonyls, malondialdehyde (MDA) and oxygen radical absorbance capacity. D-VE diets decreased serum alpha-tocopherol and increased PK activity in a time-dependent manner. The D-VE diet increased plasma protein carbonyls but did not affect MDA. Dietary quercetin supplementation increased quercetin and its metabolites in plasma and liver but did not affect D-VE-induced changes in plasma alpha-tocopherol, PK or protein carbonyls. Plasma isorhamnetin and its disposition in muscle were enhanced by the D-VE diet, as compared to the R-VE diet. Conversely, tamarixetin disposition in muscle was decreased by the D-VE diet. Thus, quercetin did not slow vitamin E decline in vivo; neither did it provide antioxidant activity in vitamin-E-depleted rats. However, vitamin E status appears to enhance the distribution of isorhamnetin into the circulation and its disposition in muscle.     

Proinflammatory adipocytokines induce TIMP-1 expression in 3T3-L1 adipocytes

Tissue inhibitor of metalloproteinase (TIMP)-1 is an adipocyte-secreted protein upregulated in obesity which promotes adipose tissue development. Furthermore, the proinflammatory adipocytokines tumor necrosis factor alpha (TNFalpha) and interleukin (IL)-6 induce insulin resistance, and plasma concentrations are increased during weight gain. In the current study, the impact of TNFalpha and IL-6 on TIMP-1 mRNA and protein expression was determined in 3T3-L1 adipocytes. Interestingly, TNFalpha and IL-6 induced TIMP-1 protein secretion more than 3- and 2-fold, respectively. Furthermore, TIMP-1 mRNA was upregulated in a time- and dose-dependent fashion. Inhibitor experiments suggested that nuclear factor kappaB and p 44/42 mitogen-activated protein kinase are involved in both, basal and adipocytokine-induced TIMP-1 expression. Moreover, the thiazolidinedione troglitazone partly reversed TNFalpha- but not IL-6-induced TIMP-1 synthesis. Taken together, we demonstrate that TIMP-1 expression is selectively upregulated in fat cells by proinflammatory adipocytokines and might play a role in maintaining adipose tissue mass in obesity.     

Synovial Fibroblasts Promote the Expression and Granule Accumulation of Tryptase via Interleukin-33 and Its Receptor ST-2 (IL1RL1)

A characteristic feature of tissue resident human mast cells (MCs) is their hTryptase-β-rich cytoplasmic granules. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-β, and we have shown that this tetramer-forming tryptase has beneficial roles in innate immunity but adverse roles in inflammatory disorders like experimental arthritis. Because the key tissue factors that control tryptase expression in MCs have not been identified, we investigated the mechanisms by which fibroblasts mediate the expression and granule accumulation of mMCP-6. Immature mouse bone marrow-derived MCs (mBMMCs) co-cultured with fibroblast-like synoviocytes (FLS) or mouse 3T3 fibroblasts markedly increased their levels of mMCP-6. This effect was caused by an undefined soluble factor whose levels could be increased by exposing FLS to tumor necrosis factor-α or interleukin (IL)-1β. Gene expression profiling of mBMMCs and FLS for receptor·ligand pairs of potential relevance raised the possibility that IL-33 was a sought after fibroblast-derived factor that promotes tryptase expression and granule maturation via its receptor IL1RL1/ST2. MCs lacking IL1RL1 exhibited defective fibroblast-driven tryptase accumulation, whereas recombinant IL-33 induced mMCP-6 mRNA and protein accumulation in wild-type mBMMCs. In agreement with these data, synovial MCs from IL1RL1-null mice exhibited a marked reduction in mMCP-6 expression. IL-33 is the first factor shown to modulate tryptase expression in MCs at the mRNA and protein levels. We therefore have identified a novel pathway by which mesenchymal cells exposed to inflammatory cytokines modulate the phenotype of local MCs to shape their immune responses.