Berries in Microbiome-Mediated Gastrointestinal, Metabolic, and Immune Health.

PURPOSE OF REVIEW: Current research has shown that berry-derived polymeric substrates that resist human digestion (dietary fibers and polyphenols) are extensively metabolized in the gastrointestinal tract dominated by microbiota. This review assesses current epidemiological, experimental, and clinical evidence of how berry (strawberry, blueberry, raspberry, blackberry, cranberry, black currant, and grapes) phytochemicals interact with the microbiome and shape health or metabolic risk factor outcomes. RECENT FINDINGS: There is a growing evidence that the compositional differences among complex carbohydrate fractions and classes of polyphenols define reversible shifts in microbial populations and human metabolome to promote gastrointestinal health. Interventions to prevent gastrointestinal inflammation and improve metabolic outcomes may be achieved with selection of berries that provide distinct polysaccharide substrates for selective multiplication of beneficial microbiota or oligomeric decoys for binding and elimination of the pathogens, as well as phenolic substrates that hold potential to modulate gastrointestinal mucins, reduce luminal oxygen, and release small phenolic metabolites signatures capable of ameliorating inflammatory and metabolic perturbations. These mechanisms may explain many of the differences in microbiota and host gastrointestinal responses associated with increased consumption of berries, and highlight potential opportunities to intentionally shift gut microbiome profiles or to modulate risk factors associated with better nutrition and health outcomes.

Boysenberry and apple juice concentrate reduced acute lung inflammation and increased M2 macrophage-associated cytokines in an acute mouse model of allergic airways disease.

Bioactive compounds including anthocyanins and other polyphenols are associated with reduced lung inflammation and improved lung function in asthma and other lung diseases. This study investigated the effects of a Boysenberry and apple juice concentrate, high in cyanidin glycosides, ellagitannins, and chlorogenic acid, on a mouse model of allergic airways inflammation. Male C57BL/6J mice were orally gavaged with 2.5 mg/kg of total anthocyanins (TAC) from BerriQi® Boysenberry and apple juice concentrate (0.2 mg/kg human equivalent dose) or water control 1 hr before an acute intranasal ovalbumin (OVA) challenge and were gavaged again 2 days after the intranasal challenge. Consumption of BerriQi® Boysenberry and apple juice concentrate significantly decreased OVA-induced infiltrating eosinophils, neutrophils, and T cells in the lung, and mucous production. Quantification of gene expression for arginase (Arg1), chitinase 3-like 3 (Ym-1), found in inflammatory zone (Fizz1), which have been associated with an anti-inflammatory macrophage phenotype (M2), found significantly increased Arg1 expression in the lung in the Boysenberry and apple juice concentrate treatment group. There was also increased production of M2-associated cytokines C-X-C motif chemokine ligand (CXCL) 10 and C-C motif chemokine ligand (CCL) 4. These results suggest that consumption of BerriQi® Boysenberry and apple juice concentrate promoted a shift toward an anti-inflammatory environment within the lung leading to reduced immune cell infiltration and tissue damage.

Different immune and functional effects of urban dust and diesel particulate matter inhalation in a mouse model of acute air pollution exposure.

Increased global industrialization has increased air pollution resulting in 3 million annual deaths globally. Air pollutants could have different health effects, so specific models to identify the different immune effects are needed. The aim of this study was to determine the immune effects and lung function of acute exposure to two different pollution sources using a mouse model. Three intranasal challenges with either urban dust or diesel particulate matter resulted in significant (P < 0.001) immune cell infiltration into the lung, which was mostly because of an increased (P < 0.001) percentage of neutrophils. We found that exposure to either urban dust or diesel particulate matter significantly increased the lung tissue concentration of the neutrophil chemoattractant cytokine CXCL5 when compared with naïve controls. The urban dust challenge also significantly increased the concentration of the proinflammatory cytokine CCL20, but diesel particulate matter did not. The urban dust challenge significantly (P < 0.001) decreased tissue compliance and ability to stretch, and increased total airways constriction and lung tissue stiffness. In comparison, diesel particulate matter exposure slightly, but significantly (P = 0.022), increased tissue compliance and did not affect other lung function parameters. Although both urban dust and diesel particulate matter induced immune cell infiltration into the lung resulting in lung inflammation, their detrimental effects on cytokine production and lung function were quite different. This may be attributed to the variation in particulates that comprise these pollutants that directly interact with the lung tissue and consequently elicit a different functional response.

Progress in the understanding of the pathology of allergic asthma and the potential of fruit proanthocyanidins as modulators of airway inflammation.

Allergic asthma is a chronic inflammatory lung disease characterized by sensitization of the airways, and the development of immunoglobulin E antibodies, to benign antigens. The established pathophysiology of asthma includes recurrent lung epithelial inflammation, excessive mucus production, bronchial smooth muscle hyperreactivity, and chronic lung tissue remodeling, resulting in reversible airflow restriction. Immune cells, including eosinophils and the recently characterized type 2 innate lymphoid cells, infiltrate into the lung tissue as part of the inflammatory response in allergic asthma. It is well established that a diet high in fruits and vegetables results in a reduction of the risk of developing inflammatory diseases. Secondary plant metabolites, such as proanthocyanidins which are found in apples, blackcurrants, boysenberries, cranberries, and grapes, have shown promising results in reducing or preventing allergic asthma airway inflammation. Recent evidence has also highlighted the importance of microbiome-mediated metabolism of plant polyphenols in modulating the immune system. In this review, we will discuss advances in our understanding of the pathophysiology of allergic asthma, including the role of the microbiome in lung immune function, and how proanthocyanidins modulate the airway inflammation. We will highlight the potential of dietary proanthocyanidins to impact on allergic asthma and the immune system.

Blackcurrant anthocyanins modulate CCL11 secretion and suppress allergic airway inflammation.

CCL11, a chemokine, is linked to the early development of airways eosinophilia in allergic asthma. Therefore, CCL11 production is a target for abrogating eosinophilic-driven airway inflammation. Blackcurrants are high in compounds that regulate inflammation, particularly anthocyanins. In this study, we investigated the effect of oral blackcurrant supplementation on allergen-induced eosinophilia and CCL11 production; we also profiled key compounds in blackcurrants that were linked to this effect. Ten milligram per kilogram (total anthocyanins) of a commercially available, anthocyanin-rich New Zealand "Ben Ard" blackcurrant extract ("Currantex 30") attenuated ovalbumin-induced inflammation, eosinophilia (by 52.45 ± 38.50%), and CCL11 production (by 48.55 ± 28.56%) in a mouse model of acute allergic lung inflammation. Ten blackcurrant polyphenolic extracts were also found to suppress CCL11 secretion by stimulated human lung epithelial cells in vitro. Correlation analysis identified potential blackcurrant polyphenolic anthocyanin constituents specifically delphinidins and cyanidins, involved in CCL11 suppression. Our findings show oral supplementation with New Zealand blackcurrant is effective in reducing lung inflammation, and highlight the potential benefit of developing cultivars with specific polyphenolic profiles for the creation of functional foods with desirable biological activity.

Boysenberry ingestion supports fibrolytic macrophages with the capacity to ameliorate chronic lung remodeling.

Lung fibrosis negatively impacts on lung function in chronic asthma and is linked to the development of profibrotic macrophage phenotypes. Epidemiological studies have found that lung function benefits from increased consumption of fruit high in polyphenols. We investigated the effect of boysenberry consumption, in both therapeutic and prophylactic treatment strategies in a mouse model of chronic antigen-induced airway inflammation. Boysenberry consumption reduced collagen deposition and ameliorated tissue remodeling alongside an increase in the presence of CD68+CD206+arginase+ alternatively activated macrophages in the lung tissue. The decrease in tissue remodeling was associated with increased expression of profibrolytic matrix metalloproteinase-9 protein in total lung tissue. We identified alternatively activated macrophages in the mice that consumed boysenberry as a source of the matrix metalloproteinase-9. Oral boysenberry treatment may moderate chronic tissue remodeling by supporting the development of profibrolytic alternatively activated macrophages expressing matrix metalloproteinase-9. Regular boysenberry consumption therefore has the potential to moderate chronic lung remodeling and fibrosis in asthma and other chronic pulmonary diseases.

Role of miR-146a in regulation of the acute inflammatory response to monosodium urate crystals.

OBJECTIVES: MicroRNAs (miRNA) are small non-coding RNAs that function as post-transcriptional repressors of gene expression. We hypothesised that miRNA regulate gene expression of proinflammatory cytokines in response to monosodium urate (MSU) crystals. METHODS: We stimulated human monocytic THP-1 cells with MSU crystals and examined miRNA and proinflammatory cytokine gene expression. The effects of miR-146a overexpression were examined by transfecting THP-1 cells with miR-146a precursor. miR-146a expression was examined in the urate peritonitis model, in peripheral blood mononuclear cells from people with gout and control participants, and in gouty tophus samples. RESULTS: MSU crystals increased miR-146a expression in THP-1 cells, but not other miRNA implicated in interleukin (IL)-1ß regulation. Overexpression of miR-146a expression reduced MSU crystal-induced IL-1ß, tumour necrosis factor-α (TNFα), monocyte chemoattractant protein-1 (MCP-1) and IL-8 gene expression. In the urate peritonitis model, reduced miR-146a expression was observed during the acute inflammatory response to MSU crystal injection. In people with intercritical gout, peripheral blood mononuclear cells expressed significantly higher levels of miR-146a, compared with normouricaemic and hyperuricaemic control participants and those with acute gout flares. Expression of miR-146a was also observed in all tophus samples. CONCLUSIONS: Collectively, these data suggest that miR-146a is a transcriptional brake that is lost during the acute inflammatory response to MSU crystals.

Brief report: Granulocyte-macrophage colony-stimulating factor drives monosodium urate monohydrate crystal-induced inflammatory macrophage differentiation and NLRP3 inflammasome up-regulation in an in vivo mouse model.

OBJECTIVE: To determine the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of inflammatory macrophages in an in vivo model of monosodium urate monohydrate (MSU) crystal-induced inflammation. METHODS: C57BL/6J mice were treated with either clodronate liposomes to deplete peritoneal macrophages or GM-CSF antibody and were then challenged by intraperitoneal injection of MSU crystals. Peritoneal lavage fluid was collected, and cellular infiltration was determined by flow cytometry. Purified resident and MSU crystal-recruited monocyte/macrophages were stimulated ex vivo with MSU crystals. The interleukin-1ß (IL-1ß) levels in lavage fluids and ex vivo assay supernatants were measured. GM-CSF-derived and macrophage colony-stimulating factor (M-CSF)-derived macrophages were generated in vitro from bone marrow cells. Protein expression of IL-1ß, caspase 1, NLRP3, and ASC by in vitro- and in vivo-generated monocyte/macrophages was analyzed by Western blotting. RESULTS: Depletion of resident macrophages lowered MSU crystal-induced IL-1ß and GM-CSF levels in vivo as well as IL-1ß production by MSU crystal-recruited monocytes stimulated ex vivo. GM-CSF neutralization in vivo decreased MSU crystal-induced IL-1ß levels and neutrophil infiltration. MSU crystal-recruited monocyte/macrophages from GM-CSF-neutralized mice expressed lower levels of the macrophage marker CD115 and produced less IL-1ß following ex vivo stimulation. These monocytes exhibited decreased expression of NLRP3, pro/active IL-1ß, and pro/active caspase 1. In vitro-derived GM-CSF-differentiated macrophages expressed higher levels of NLRP3, pro/active IL-1ß, and pro/active caspase 1 compared to M-CSF-differentiated macrophages. CONCLUSION: GM-CSF plays a key role in the differentiation of MSU crystal-recruited monocytes into proinflammatory macrophages. GM-CSF production may therefore contribute to the exacerbation of inflammation in gout.

The effect of diet-induced obesity on the inflammatory phenotype of non-adipose-resident macrophages in an in vivo model of gout.

OBJECTIVE: Gout is strongly associated with obesity. The aim of this study was to determine if obesity altered the inflammatory phenotype of non-adipose tissue-resident macrophages in response to the gout-causing agent monosodium urate (MSU) crystals. METHODS: C57BL/6J mice were fed a high-fat diet for 12 weeks. Resident peritoneal macrophages were stimulated ex vivo with MSU crystals (200 µg/ml for 18 h) and the supernatants were collected. Mice were challenged with MSU crystals in vivo (3 mg, intraperitonal) and the peritoneal lavage fluid was collected (8 and 16 h). Cytokine and chemokine levels were analysed by multiplex bead array and peritoneal cell populations were analysed by flow cytometry. RESULTS: Peritoneal macrophages from obese mice produced elevated background levels of IL-6, monocyte chemoattractant protein 1 (MCP-1) and keratinocyte-derived cytokine (KC) that decreased following MSU crystal stimulation ex vivo. MSU-induced IL-1ß production was higher for macrophages from obese mice compared with controls. High background levels of IL-6, MCP-1, KC and GM-CSF, but not IL-1ß, were measured in the peritoneal fluid of unchallenged obese mice. MSU crystal challenge in vivo raised IL-1ß levels equally in both control and obese mice, whereas elevated background levels of IL-6, MCP-1, KC and GM-CSF levels dropped in obese mice. There was a consistent trend towards lower numbers of naive peritoneal resident macrophages and MSU-recruited monocytes and neutrophils in obese mice. CONCLUSION: Obesity induces a background pro-inflammatory environment orchestrated by non-adipose tissue-resident macrophages. However, this may not automatically translate into exacerbation of MSU crystal-induced inflammation in gout.

Blackcurrant cultivar polyphenolic extracts suppress CCL26 secretion from alveolar epithelial cells.

Eosinophil recruitment to the airways is a characteristic feature of allergic asthma. Eotaxins are potent chemokines that regulate the recruitment of eosinophils to sites of inflammation. Of these, CCL26 is linked to persistent eosinophil recruitment in the later phase of an allergic response. We evaluated the effectiveness of 10 different blackcurrant cultivar polyphenolic extracts in suppressing CCL26 secretion in stimulated human alveolar epithelial cells. Correlation analysis to identify the potential blackcurrant composition constituent(s) involved in CCL26 suppression and the effects of the four major anthocyanins present in blackcurrants to validate results was conducted. All blackcurrant polyphenolic extracts suppressed CCL26 secretion by lung alveolar cells; however, differential efficacy was observed, which was attributed to their cultivar-specific polyphenolic composition profiles. We identified that the ratio of concentrations of delphinidin glycosides to cyanidin glycosides in the blackcurrant cultivars was an important determinant in influencing CCL26 suppression in lung cells. Our findings support the potential use of blackcurrants or blackcurrant-derived foods/ingredients in managing lung inflammation and the development of specific cultivars as functional foods/ingredients with beneficial biological activities.

An efficient single prime protocol for the induction of antigen-induced airways inflammation.

There are a number of mouse models of allergic airway inflammation used to delineate various aspects of asthma. One of the hurdles with using mice is their natural resistance to developing a Th2 allergic response. This is often overcome by double priming with the allergen and an adjuvant. Here we report on an efficient 11day, single antigen/alum priming protocol that is sufficient to sensitise mice for the development of Th2-mediated inflammation in the lung following antigen challenge.

Bradykinin receptor 2 extends inflammatory cell recruitment in a model of acute gouty arthritis.

The aim of this study was determine the effect of bradykinin receptor antagonism on MSU crystal-induced chemokine production and leukocyte recruitment. Mice were injected intraperitoneally with monosodium urate (MSU) crystals ± bradykinin B1- or B2 receptor antagonists, Des-Arg-HOE-140 and HOE-140, respectively. MSU crystal-induced chemokine production and leukocyte recruitment in the peritoneum were measured over 24h and B1 and B2 receptor expression on leukocytes and peritoneal membrane was determined by flow cytometry and fluorescence microscopy. Data analysis showed that only B2 receptor antagonism decreased monocyte and neutrophil infiltration 24 h post MSU crystal administration. Decreased leukocyte infiltration was associated with reduced monocyte (CCL2) chemokine levels. MSU crystal-induced damage to the surrounding visceral membrane was also attenuated in the presence of B2 receptor antagonism. Together, these data show that bradykinin receptor 2 plays a role in maintaining MSU crystal-induced leukocyte infiltration and membrane permeability and identify the B2 receptor as a potential therapeutic target for managing inflammation in gout.

Tin protoporphyrin provides protection following cerebral hypoxia-ischemia: involvement of alternative pathways.

The contribution of heme oxygenase (HO)-linked pathways to neurodegeneration following cerebral hypoxia-ischemia (HI) remains unclear. We investigated whether HO modulators affected HI-induced brain damage and explored potential mechanisms involved. HI was induced in 26-day-old male Wistar rats by left common carotid artery ligation, followed by exposure to a humidified atmosphere of 8% oxygen for 1 hr. Tin protoporphyrin (SnPP; an HO inhibitor), ferriprotoporphyrin (FePP; an HO inducer), or saline was administered intraperitoneally once daily from 1 day prior to HI until sacrifice at 3 days post-HI. SnPP reduced (P < 0.05) infarct volume compared with saline-treated animals, but FePP had no effect on brain injury. SnPP did not significantly inhibit HO activity at 3 days post-HI, but SnPP increased (P < 0.001) total nitric oxide synthase (NOS) activity compared with HI + saline. Both inducible NOS and cyclooxygenase activities were attenuated (P < 0.05) by SnPP, whereas mitochondrial complex I and V activities were augmented (P < 0.05) by SnPP. SnPP had no effect on NMDA receptor currents. Overall, like other HO inhibitors, SnPP produced many nonselective effects, such as attenuation of inflammatory enzymes and increased mitochondrial respiratory function, which were associated with a protective response 3 days post-HI.

Remote ischemic preconditioning stimulus decreases the expression of kinin receptors in human neutrophils.

BACKGROUND: Remote ischemic preconditioning (RIPC) has been shown to reduce ischemic-reperfusion injury and is induced by brief forearm ischemia. Kinins are known to be involved in RIPC and act via the G protein coupled B1 and B2 receptors. Interaction of the kinins with their respective receptors causes receptor internalization, thereby reducing the potential for further activation. This may be critical for the protective effect of RIPC and if so, we hypothesized, would significantly decrease the expression of kinin receptors on the surface of neutrophils. METHODS: The study was performed on five healthy human volunteers. The left forearm was rendered ischemic for three 5-min periods, each separated by 5 min of reperfusion. Three venous blood samples were taken from the right arm, one before and two after RIPC. Neutrophil isolation, immunofluorescence labeling, and confocal microscopy were performed. Mean pixel intensity data were generated using a fixed circular area of interest (AOI, 40×40 µm). For every image, the AOI was placed over a cell and the mean pixel intensity was recorded. The mean intensity was expressed as pixel×10(2)/µm(2) and presented as mean±SEM. Immunofluorescence at the different time points was compared by one way analysis of variance with Bonferroni's post-hoc test. A P value<0.05 was considered significant. RESULTS: The mean pixel intensity for kinin B1 receptors was decreased at 24 h after RIPC compared with both baseline and 15 min after RIPC (P<0.001). Similarly, the intensity for B2 receptor labeling on neutrophils was significantly decreased 24 h after RIPC compared with the baseline value (P<0.001). CONCLUSIONS: RIPC decreases expression of kinin receptors on circulating human neutrophils. Reduction in kinin surface receptors suggests internalization of receptors and is consistent with the concepts of kinin receptor activation and their role in RIPC.

Cerebral heme oxygenase 1 and 2 spatial distribution is modulated following injury from hypoxia-ischemia and middle cerebral artery occlusion in rats.

The regional and cellular distribution of heme oxygenase (HO)-1 and -2 following cerebral ischemia has not been ascertained. Employing the transient middle cerebral artery occlusion (MCAO) and hypoxia-ischemia (HI) models of unilateral brain injury, the aim was to elucidate immunolocalization of HO-1 and HO-2. Animals were sacrificed 3 days post-ischemia and immunohistochemistry and Western blotting were utilized to determine HO-1 and HO-2 expression. In the ipsilateral hemisphere following HI, HO-1 immunoreactivity was significantly upregulated in many neuronal and glial populations (including the cortex, hippocampus and thalamus). HO-1 was also detected in macrophages/microglia within the infarct. In addition to widespread neuronal HO-2 labelling, HO-2 was also expressed in vascular endothelial cells. Inflammatory cells within the infarct of MCAO and HI animals were surprisingly immunoreactive for HO-2, but only HI animals had significantly elevated HO-2 protein expression in the ipsilateral hemisphere. This may be due to the presence of global hypoxia in the HI model which can upregulate vascular endothelial growth factor and subsequent proliferation of endothelial cells. This report of HO-2 protein expression upregulation following HI coupled with an increase in HO-1 immunoreactivity suggests that this response may be implicated in reducing cell death or repairing damage induced by cerebral ischemia.

Kallikreins, kininogens and kinin receptors on circulating and synovial fluid neutrophils: role in kinin generation in rheumatoid arthritis.

OBJECTIVES: Neutrophils traffic into and have the capacity to generate kinins in SF of RA patients. The aim of this study was to assess the expression of kallikreins, kininogens and kinin receptors in circulating and SF neutrophils, as well as synovial tissue of RA patients, and to assess kinin generation in SF. METHODS: Neutrophils were isolated from blood and SF of RA patients and blood of healthy volunteers. Expression of kallikreins, kininogens and kinin receptors in neutrophils and synovial tissue was assessed by immunocytochemistry using specific antibodies, with visualization by brightfield and confocal microscopy. Levels of basal and generated kinins in SF of RA patients were measured by ELISA. RESULTS: Kinin labelling was significantly reduced, indicating the loss of the kinin moiety from kininogen on circulating (P < 0.001) and SF neutrophils (P < 0.05) of RA patients. Immunolabelling of tissue kallikrein was also decreased, whereas kinin B(1) and B(2) receptor expression was increased in circulating and SF neutrophils of RA patients. Immunolabelling of kallikreins and kinin receptor proteins was similar in RA and normal synovial tissues. The basal kinin level in SF of RA patients was 5.7 +/- 6.1 ng/ml and the mean concentration of kinins generated in vitro was 80.6 +/- 56.3 ng/ml. The capacity for kinin generation was positively correlated with measures of disease activity. CONCLUSIONS: Kallikrein-kinin proteins on neutrophils play an important role in kinin generation and the pathophysiology of RA. Specific kallikrein and kinin receptor antagonists may be useful as IA therapies for inflamed joints.

Epigenetic regulation of human epithelial cell cancers.

Cancer is the second leading cause of death in industrialized countries, with epithelial cell cancers (carcinomas) representing approximately 85% of all diagnosed cancers. The 5-year survival rate for many carcinomas remains low, highlighting the requirement for improved diagnosis and more effective therapies. Epigenetic modifications that do not involve changes in the DNA sequence, but result in changes in gene expression, are rapidly being realized as important in carcinogenesis. Evidence is emerging that DNA methylation, histone modification and alternative mRNA splicing are involved in various human epithelial cell cancers, and diagnostic and therapeutic strategies based on these epigenetic phenomena are under investigation. This review provides an overview of studies demonstrating the importance of epigenetic regulation of gene expression in the diagnosis, progression and response to treatment of human carcinomas. The use of therapeutic agents to reverse these epigenetic changes, either as single treatments or in combination with other therapies, is also discussed.

(-)-Epigallocatechin gallate as an intervention for the acute treatment of cerebral ischemia.

This study examined the neuroprotective effects and possible hepatotoxicity of (-)-epigallocatechin gallate (EGCG) in a rat model of transient focal cerebral ischemia. Male Sprague-Dawley rats (265-295 g) were treated with either 50 mg kg(-1) of EGCG or saline, i.p., immediately post-ischemia and every day thereafter, in a middle cerebral artery occlusion model of stroke. Sacrifice occurred 72 h post-ischemia and 2,3,5-triphenyltetrazolium chloride staining was used to quantify neuronal infarction. Hepatotoxicity was determined by taking blood samples for plasma alanine aminotransferase (ALT) activity. Spleen, kidney, liver and testes wet weights were also recorded. Total infarct volume was significantly (P<0.05) reduced in the EGCG-treated group as compared to controls. Analysis of the mean infarct area showed a significant (P<0.05) decrease in slices 6 and 7 in the EGCG-treated group. No significant differences were found in organ weights or ALT levels between treatment groups. Our findings, in part, validate and extend previous observations illustrating that 50 mg kg(-1), i.p. EGCG is non-toxic and neuroprotective. However, we also found that EGCG treatment appreciably increased (>50%) the number of animals that developed an intracerebral hemorrhage. We therefore conclude that 50 mg kg(-1) EGCG is not a viable intervention for the acute treatment of cerebral ischemia, as it is likely to increase the risk of intracerebral hemorrhaging.

Neuroprotective effects of (-)-epigallocatechin gallate following hypoxia-ischemia-induced brain damage: novel mechanisms of action.

(-)-Epigallocatechin gallate (EGCG) is a potent antioxidant that is neuroprotective against ischemia-induced brain damage. However, the neuroprotective effects and possible mechanisms of action of EGCG after hypoxia-ischemia (HI) have not been investigated. Therefore, we used a modified "Levine" model of HI to determine the effects of EGCG. Wistar rats were treated with either 0.9% saline or 50 mg/kg EGCG daily for 1 day and 1 h before HI induction and for a further 2 days post-HI. At 26-days-old, both groups underwent permanent left common carotid artery occlusion and exposure to 8% oxygen/92% nitrogen atmosphere for 1 h. Histological assessment showed that EGCG significantly reduced infarct volume (38.0+/-16.4 mm(3)) in comparison to HI + saline (99.6+/-15.6 mm(3)). In addition, EGCG significantly reduced total (622.6+/-85.8 pmol L-[(3)H]citrulline/30 min/mg protein) and inducible nitric oxide synthase (iNOS) activity (143.2+/-77.3 pmol L-[(3)H]citrulline/30 min/mg protein) in comparison to HI+saline controls (996.6+/-113.6 and 329.7+/-59.6 pmol L-[(3)H]citrulline/30 min/mg protein for total NOS and iNOS activity, respectively). Western blot analysis demonstrated that iNOS protein expression was also reduced. In contrast, EGCG significantly increased endothelial and neuronal NOS protein expression compared with HI controls. EGCG also significantly preserved mitochondrial energetics (complex I-V) and citrate synthase activity. This study demonstrates that the neuroprotective effects of EGCG are, in part, due to modulation of NOS isoforms and preservation of mitochondrial complex activity and integrity. We therefore conclude that the in vivo neuroprotective effects of EGCG are not exclusively due to its antioxidant effects but involve more complex signal transduction mechanisms.