Green Tea Prevents NAFLD by Modulation of miR-34a and miR-194 Expression in a High-Fat Diet Mouse Model.

BACKGROUND/AIMS: Nonalcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of metabolic syndrome. It is currently the most common chronic liver disease with complex pathogenesis and challenging treatment. Here, we investigated the hepatoprotective role of green tea (GT) and determined the involvement of miRNAs and its mechanism of action. METHODS: Male C57Bl/6 mice were fed with a high-fat diet for 4 weeks. After this period, the animals received gavage with GT (500 mg/kg body weight) over 12 weeks (5 days/week). HepG2 cell lines were transfected with miR-34a or miR-194 mimetics and inhibitors to validate the in vivo results or were treated with TNF-α to evaluate miRNA regulation. RESULTS: GT supplementation protects against NAFLD development by altering lipid metabolism, increasing gene expression involved in triglycerides and fatty acid catabolism, and decreasing uptake and lipid accumulation. This phenotype was accompanied by miR-34a downregulation and an increase in their mRNA targets Sirt1, Pparα, and Insig2. GT upregulated hepatic miR-194 by inhibiting TNF-α action leading to a decrease in miR-194 target genes Hmgcs/Apoa5. CONCLUSION: Our study identified for the first time that the beneficial effects of GT in the liver can be due to the modulation of miRNAs, opening new perspectives for the treatment of NAFLD focusing on epigenetic regulation of miR-34a and miR-194 as green tea targets.

Green tea polyphenol extract in vivo attenuates inflammatory features of neutrophils from obese rats.

PURPOSE: Our study aimed to evaluate whether obesity induced by cafeteria diet changes the neutrophil effector/inflammatory function and whether treatment with green tea extract (GT) can improve neutrophil function. METHODS: Male Wistar rats were treated with GT by gavage (12 weeks/5 days/week; 500 mg/kg of body weight), and obesity was induced by cafeteria diet (8 weeks). Neutrophils were obtained from the peritoneal cavity (injection of oyster glycogen). The following analyses were performed: phagocytic capacity, chemotaxis, myeloperoxidase activity (MPO), hypochlorous acid (HOCl), superoxide anion (O 2 (·-) ), hydrogen peroxide (H2O2), IL-1ß, IL-6 and TNFα, mRNA levels of inflammatory genes, calcium mobilisation, activities of antioxidant enzymes, hexokinase and G6PDH. RESULTS: Neutrophils from obese rats showed a significant decrease in migration capacity, H2O2 and HOCl production, MPO activity and O 2 (·-) production. Phagocytosis and CD11b mRNA levels were increased, while inflammatory cytokines release remained unmodified. mRNA levels of TLR4 and IκK were enhanced. Treatment of obese rats with GT increased neutrophil migration, MPO activity, H2O2, HOCl and O 2 (·-) production, whereas TNF-α and IL-6 were decreased (versus obese). Similar reductions in TLR4, IκK and CD11b mRNA were observed. Catalase and hexokinase were increased by obesity, while SOD and G6PDH were decreased. Treatment with GT reduced catalase and increased the GSH/GSSG ratio. CONCLUSION: In response to a cafeteria diet, we found a decreased chemotaxis, H2O2 release, MPO activity and HOCl production. We also showed a significant immunomodulatory effect of GT on the obese condition recovering some of these factors such H2O2 and HOCl production, also reducing the levels of inflammatory cytokines.

Green tea polyphenols change the profile of inflammatory cytokine release from lymphocytes of obese and lean rats and protect against oxidative damage.

This study aimed to investigate whether green tea polyphenols (GT) modulate some functional parameters of lymphocytes from obese rats. Male Wistar rats were treated with GT by gavage (12 weeks/5 days/week; 500 mg/kg of body weight) and obesity was induced by cafeteria diet (8 weeks). Lymphocytes were obtained from mesenteric lymph nodes for analyses. In response to the cafeteria diet we observed an increase in activity of the metabolic enzyme hexokinase, ROS production, MnSOD, CuZnSOD and GR enzyme activities and proliferation capacity of the cells (baseline), whereas IL-10 production was decreased. Obese rats treated with GT decreased cell proliferation (under ConA stimulation). Hexokinase and G6PDH activity, ROS production and MnSOD, CuZnSOD, GPx and GR enzymes remained increased, accompanied by an increase in Nrf2 mRNA level. There was a decrease in pro-inflammatory IL-2, IL-6, IL-1ß, TNF-α cytokines that were accompanied by a decrease in the mRNA level of TRL4 while IL-10 production was increased in obese rats treated with GT. GT treatment of lean rats showed similar results to that of obese rats treated with GT, indicating that the effects of GT are independent of diet. Foxp3 and IRF4 mRNA levels were increased by GT. In conclusion, cafeteria diet modulated the function of lymphocytes from lymph nodes, increasing ROS production and decreasing anti-inflammatory IL-10, which could contribute to the inflammatory state in obesity. GT reduced ROS production, improving the redox status and reducing pro-inflammatory cytokine production by lymphocytes, suggesting that GT treatment may be driving lymphocytes to a more anti-inflammatory than pro-inflammatory microenvironment.

Green tea catechins alone or in combination alter functional parameters of human neutrophils via suppressing the activation of TLR-4/NFκB p65 signal pathway.

The purpose of this study was to evaluate the potential of a mixture containing the four main catechins found in green tea, as well it separately, as modulators of the functional parameters of human neutrophils. The cells were obtained from peripheral blood of healthy individuals isolated and cultured with a mix: 30 µM of EGCG, 3 µM of EGC, 2 µM of ECG and 1.4 µM of EC, as well as each one alone. We evaluated the cytotoxicity of catechins, production of several reactive oxygen species (ROS), antioxidant enzymes (SOD, CAT, GPx and GR), Nrf2, TLR4/IKK/NFκB, CD11b mRNA levels, intracellular calcium release, chemotactic and phagocytic capacity, myeloperoxidase (MPO), and G6PDH activities, hypochlorous acid (HOCl) and pro-inflammatory cytokines release, protein levels of TLR4, p38 MAPK, iNOS and p-65 NFκB. The actions of the catechins were evidenced by the reduction in inflammatory parameters, including the suppression of TLR4, NFκB and iNOS protein expression, decreased release of TNF-α, IL-1ß and IL-6, migration capacity, MPO activity and HOCl production and the suppression of ROS, nitric oxide and peroxynitrite production, while inducing antioxidant enzyme activities and Nrf2 mRNA levels, phagocytic capacity and calcium release. Our results demonstrate that catechins present marked immunomodulatory actions, either alone or in combination.

Effects of chronic resveratrol supplementation in military firefighters undergo a physical fitness test--a placebo-controlled, double blind study.

PURPOSE: The purpose of this study was to determine the plasma metabolic response and certain indicators of oxidative stress (antioxidant system and oxidative stress biomarkers) in plasma and erythrocytes of Brazilian military firefighters supplemented or not with resveratrol (RES) for 90 days (100 mg/day). The analyses were performed before and after a typical physical fitness test (FT) used to induce oxidative stress. METHODS/RESULTS: In this placebo-controlled double-blinded study, we observed that RES supplementation did not present hepatic consequences compared with the placebo group following analysis of AST, ALT and GGT plasma activities. Plasma glucose and triglycerides levels were increased after the FT in firefighters supplemented with RES but were not elevated at baseline. Neither total nor cholesterol fractions were modified by RES supplementation. CK levels were increased after the firefighters performed the FT; however, no differences were determined between the placebo and RES groups. Ferric-reducing ability of plasma as well as uric acid was increased after the FT, but was not modified by RES supplementation. Plasma oxidative stress biomarkers, such as thiol content, 8-isoprostane and 8OHdG, showed no modifications, while IL-6 and TNF-α were decreased in the RES group after the FT. Among antioxidant enzyme activities determined in erythrocytes from the firefighters, only GPx activity was reduced by RES supplementation both before and after the FT. CONCLUSION: In summary, the most pronounced effect of RES supplementation is its anti-inflammatory effect, which reduced IL-6 and TNF-α level. The FT applied to Brazilian military firefighters was not sufficient to challenge the antioxidant defense systems, and, therefore, 100mg of RES for three months did not induce significant effects.

Fucoxanthin in association with vitamin C acts as modulators of human neutrophil function.

INTRODUCTION: Neutrophils provide the first line of defense of the innate immune system by phagocytosing, killing and digesting bacteria and fungi. During this process, neutrophils produce reactive oxygen species (ROS), which in excess, can damage the cells themselves and surrounding tissues. The carotenoid fucoxanthin (Fc) has been studied concerning its antioxidant and anti-inflammatory actions. Vitamin c (Vc) also demonstrates potent antioxidant action. This study aimed to evaluate the effect of Fc (2 µM) in association with Vc (100 µM) on functional parameters of human neutrophils in vitro. MATERIALS AND METHODS: We evaluated the migration and phagocytic capacity, intracellular calcium mobilization, ROS production (O2(·)⁻, H2O2, HOCl), myeloperoxidase activity, profile of antioxidant enzymes, phosphorylation of p38 MAPK and p65 NFκB subunit, GSH/GSSG ratio and release of pro-inflammatory cytokines (TNF-α and IL-6) in neutrophils under different stimuli. RESULTS: We verified an increase in phagocytic capacity for all treatments, together with an increase in intracellular calcium only in cells treated with Fc and Fc + Vc. ROS production was reduced by all treatments, although Vc was a better antioxidant than Fc. Phosphorylation of the p-65 subunit of NFκB was reduced in cells treated with Fc + Vc and release of TNF-α and IL-6 was reduced by all treatments. These findings indicate that the regulation of inflammatory cytokines by neutrophils is not exclusively under the control of the NFκB pathway. Fc reduced the activity of some antioxidant enzymes, whereas Vc increased GR activity and the GSH/GSSG ratio. CONCLUSION: In conclusion, the results presented in this study clearly show an immunomodulatory effect of the carotenoid fc alone or in combination with Vc on the function of human neutrophils.

Changes in lymphocyte oxidant/antioxidant parameters after carbonyl and antioxidant exposure.

During normal B- and T-cell life, processes including activation, proliferation, signaling pathways and apoptosis are markedly dependent on ROS generation. However, these cells can also suffer the effect of oxidant overproduction. Thus, the purpose of the present study was to examine the possible pro-oxidant effects of MGO/high glucose and antioxidant effects of astaxanthin associated with vitamin C on some oxidative and antioxidant parameters of human lymphocytes in vitro. Lymphocytes from healthy subjects were treated with 20mM of glucose and 30 µM MGO followed or not by the addition of the antioxidants astaxanthin (2 µM) and vitamin C (100 µM) for up to 24h. We examined superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase (G6PDH) activities, GSH/GSSG ratio and total thiol and carbonyl content. Oxidative parameters included superoxide anion, hydrogen peroxide and nitric oxide production. The association of astaxanthin and vitamin C proved to be a powerful antioxidant in human lymphocytes as showed by the marked reduction in superoxide anion, and hydrogen peroxide production as well as increased GSH content, GSH/GSSG ratio, GPx and GR activities. The antioxidant association showed to be more potent than their individual application. High glucose and methylglyoxal did not promote oxidative stress in human lymphocytes, since neither the oxidative parameters nor the antioxidant defense system was altered. According to these results, new therapies with the association of astaxanthin and vitamin C may be helpful to improve the immune function of patients with exacerbated production of ROS.

Combination of astaxanthin and fish oil supplementation alters antioxidant enzyme profile of dental pulp tissue.

AIM: To evaluate the effect of administration of astaxanthin (ASTA) and fish oil (FO) on enzymatic antioxidant parameters of dental pulp tissue from healthy rats. METHODOLOGY: Thirty-two healthy Wistar rats were divided into four groups: untreated control, ASTA-treated (1 mg kg(-1) body weight), FO-treated (10 mg eicosapentaenoic acid per kg BW and 7 mg docosahexaenoic acid per kg BW) and FO plus ASTA-treated. A prophylactic dose was administered in each group daily by gavage, 5 days a week, for 45 days. After treatment, the rats were killed and all incisor dental pulps were removed. Superoxide dismutase (SOD), catalase, glutathione (GSH) peroxidase and reductase activities were determined. Data were compared by anova and the Tukey's post-test ( P  < 0.05). RESULTS: Treatment with FO, ASTA and FO plus ASTA caused a reduction in SOD and GSH reductase activities in dental pulp tissue compared to untreated control rats ( P  < 0.05). ASTA partially stimulated catalase activity. CONCLUSIONS: The preventive administration of ASTA and FO changed the enzymatic antioxidant system of dental pulp tissue, possibly by controlling oxidative stress.

Carbonyl stress and a combination of astaxanthin/vitamin C induce biochemical changes in human neutrophils.

The purpose of the present study was to find out whether co-treatment of human neutrophils with high glucose and methylglyoxal (MGO) can alter the biochemical parameters of human neutrophils. We also examined if astaxanthin associated with vitamin C can improve those biochemical parameters. Neutrophils from healthy subjects were treated with 20mM of glucose and 30 µM MGO followed or not by the addition of the antioxidants astaxanthin (2 µM) and vitamin C (100 µM). MGO/high glucose treatment reduced the phagocytic capacity and the G6PDH, total/SOD and GR activities. Additionally, there was an increase in the activity of myeloperoxidase (MPO) with consequent increase in the hypochlorous acid production, CAT activity and in the release of IL-6 cytokine without changes in intracellular calcium mobilization. Our study also shows that the association of astaxanthin with vitamin C greatly improved neutrophil phagocytic capacity, decreasing all reactive oxygen species measured, pro-inflammatory IL-1ß and TNF-α release, MPO activity and HClO production. The combination of astaxanthin with vitamin C alone has more antioxidant and anti-inflammatory effects than when they were in the presence of MGO/high glucose. Injury to the function of neutrophils due to high glucose and methylglyoxal appears not to involve oxidative stress or calcium release. The association of antioxidants astaxanthin and vitamin C promoted a significant improvement in the function of neutrophils and in the redox status.

Oxidative stress in human lymphocytes treated with fatty acid mixture: role of carotenoid astaxanthin.

Fatty acids (FA) have been shown to alter leukocyte function, and depending on concentration and type, they can modulate both inflammatory and immune responses. Astaxanthin (ASTA) is a carotenoid that shows notable antioxidant properties. In the present study we propose to evaluate the oxidative stress in human lymphocytes induced by a FA mixture and the possible protective role of ASTA. The present study showed that the FA mixture at 0.3mM caused a marked increase in the production of superoxide anion, hydrogen peroxide and nitric oxide, which was accompanied by an increase in total-SOD activity, in TBARS levels and a reduction of catalase activity and content of GSH and free thiol groups. The FA mixture also promoted an increase in intracellular Ca(2+) mobilization and in the proliferative capacity of B-lymphocytes. The addition of ASTA (2 µM) partially decreased the ROS production and TBARS levels and increased the levels of free thiol groups. ASTA decreased the proliferative capacity of cells treated with FA but not by reducing intracellular calcium concentration. Based on these results we can conclude that ASTA can partially prevent oxidative stress in human lymphocytes induced by a fatty acid mixture, probably by blenching/quenching free radical production.

In vivo astaxanthin treatment partially prevents antioxidant alterations in dental pulp from alloxan-induced diabetic rats.

AIM: To evaluate the effect of astaxanthin on antioxidant parameters of dental pulp from diabetic rats. The hypothesis tested was that supplementation of diabetic rats with astaxanthin might eliminate, or at least attenuate, the defect in their antioxidative status. METHODOLOGY: Wistar rats (n=32) were divided into four groups: untreated control, treated control, untreated diabetic and treated diabetic rats. A prophylactic dose of astaxanthin (20 mg kg(-1) body weight) was administered daily by gavage for 30 days. On day 23, diabetes was induced by injection of alloxan (60 mg kg(-1) body weight). After 7 days of diabetes induction, the rats were killed, and pulp tissue from incisor teeth removed. Superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and reductase activities were determined. Data were compared by anova and the Newman-Keuls test (P<0.05). RESULTS: Diabetes caused a reduction in SOD, GPx and reductase activity in dental pulp tissue. Astaxanthin had no effect on SOD and catalase activities; however, it stimulated GPx in control and diabetic rats. CONCLUSIONS: Diabetes altered the antioxidant system in dental pulp tissue; astaxanthin partially improved the diabetic complications.

Phenanthrene decreases neutrophil function by disrupting intracellular redox balance.

The aim of the present work was to evaluate whether the treatment of human neutrophils with phenanthrene (PHN) can alter the phagocytic and microbicidal capacity of these cells by causing a disruption in redox balance. Peripheral neutrophils from healthy subjects were treated for up to 24 h with increasing concentrations of phenanthrene. Phagocytic/microbicidal activities, antioxidant enzymes, oxidative lesions (thiobarbituric acid-reactive substances and protein thiol and carbonyl groups) and redox signaling compounds (intracellular Ca(2+), superoxide, hydrogen peroxide and nitric oxide) were monitored on neutrophils exposed to 10 microg PHN ml(-1). Cell viability decreased abruptly at PHN concentrations above 10 microg ml(-1) (LC50 = 20.86 +/- 0.51 microg ml(-1) and p-sigmoidal slope = 19.88 +/- 10.11). Phagocytic and microbicidal capacities were decreased by 60 and 82%, respectively. Substantial increases in total-/Mn-SOD, catalase, glutathione peroxidase and glutathione reductase activities (by 61, 15, 87, 245 and 70%, respectively) matched the oxidative injury obtained in TBARS (2.5-fold higher) and protein thiol (54% lower). Diminished productions of superoxide by 18% and hydrogen peroxide by 29% were observed in association to exacerbated calcium (27%) and nitric oxide (63%) levels. The data indicate that phenanthrene might be associated with substantial reduction in human neutrophil functions due to severe intracellular redox imbalances.

Diabetes induces apoptosis in lymphocytes.

The occurrence of DNA fragmentation in lymphocytes obtained from alloxan-induced diabetic rats and diabetic patients was investigated. A high proportion of apoptotic lymphocytes in diabetic states may explain the impaired immune function in poorly controlled diabetic patients. Rat mesenteric lymph node lymphocytes were analysed for DNA fragmentation by using flow cytometry and agarose gel, and for chromatin condensation by Hoescht 33342 staining under different situations. Immediately after being obtained, the proportion of lymphocytes with fragmented DNA was twofold higher in alloxan-induced diabetic rats than in cells from control rats. After 48 h in culture, the occurrence of DNA fragmentation was also higher (81%) in cells from diabetic rats. Hoescht staining and fragmented DNA visualized in agarose gel were also higher in lymphocytes from alloxan-induced diabetic rats than in control cells. To investigate if this phenomenon also occurs in humans, blood lymphocytes from 14 diabetic subjects were examined. Similar results to those of rat lymphocytes were found in cells from diabetic patients immediately after being obtained and after 48 h in culture. The high occurrence of apoptosis in lymphocytes was accompanied by a reduced number of blood-circulating lymphocytes in diabetic patients. The involvement of low insulinaemia for the occurrence of apoptosis in lymphocytes was also examined. Insulin treatment markedly reduced the proportion of lymphocytes with fragmented DNA in alloxan-induced diabetic rats.

Diabetes causes marked changes in lymphocyte metabolism.

An enhanced susceptibility to infections is well known to occur in a poorly controlled diabetic state. Since glucose and glutamine are essential for lymphocyte function, we investigated whether their metabolism is changed in lymphocytes obtained from mesenteric lymph nodes of alloxan-induced diabetic rats (40 mg/kg body weight). The activities of hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase (G6PDH), citrate synthase and phosphate-dependent glutaminase were determined. Decarboxylation of metabolites [U-14C]-, [1-14C]- and [6-14C]-glucose, [1-14C]- and [2-14C]-pyruvic acid, [U-14C]-palmitic acid and [U-14C]-glutamine was evaluated in incubated lymphocytes isolated from mesenteric lymph nodes. The measurements were carried out in cells following three experimental protocols: (1) lymphocytes freshly obtained from control and alloxan-induced diabetic rats, (2) lymphocytes from insulin-treated (2 U/rat per day) diabetic rats and (3) lymphocytes obtained from control and diabetic rats and cultured in the presence of insulin (1 mU/ml) for 6 h. The activities of hexokinase, G6PDH and citrate synthase were decreased by the diabetic state, whereas that of phosphofructokinase was raised. Decarboxylation of [U-14C]- and [6-14C]-glucose, [1-14C]- and [2-14C]-pyruvate and [U-14C]-glutamine were also decreased in lymphocytes from diabetic rats, whereas [U-14C]-palmitic acid decarboxylation was increased. Insulin administration in vivo or added to the culture medium reversed the changes observed in freshly obtained lymphocytes. Alloxan-induced diabetes did change lymphocyte metabolism and this may be an important mechanism leading to impairment of lymphocyte function.

Effect of dietary fat on lymphocyte proliferation and metabolism.

The effect of diets enriched with fat containing different fatty acids on glucose and glutamine metabolism of mesenteric lymph nodes lymphocytes, spleen, and thymus and lymphocyte proliferation was examined. The following fat-rich diets were tested: (1) standard chow (CC); (2) medium chain saturated fatty acids (MS)--coconut fat oil; (3) long chain saturated fatty acids (LS)--cocoa butter; (4) monounsaturated fatty acids (MU)--canola oil (n-9); (5) polyunsaturated fatty acids (PU)--soybean oil (n-6). Of the fat-rich diets tested, MS was the one to present the least pronounced effect. Lymphocyte proliferation was reduced by LS (64 per cent), MU (55 per cent), and PU (60 per cent). Hexokinase activity was enhanced in lymph node lymphocytes by PU (67 per cent), in the spleen by MS (42 per cent), and in the thymus by PU (30 per cent). This enzyme activity was reduced in the spleen (33 per cent) by LS and MU (35 per cent). In the thymus, this enzyme activity was reduced by LS (26 per cent) and MU (13 per cent). Maximal phosphate-dependent glutaminase activity was raised in lymphocytes by MS (70 per cent) and MU (20 per cent). This enzyme activity, however, was decreased in lymphocytes by PU (26 per cent), in the spleen by LS (15 per cent), and in the thymus by MU (44 per cent). Citrate synthase activity was increased in lymphocytes by MU (35 per cent), in the spleen by LS (56 per cent) and MU (68 per cent), and in the thymus by LS (42 per cent). This enzyme activity was decreased in lymphocytes by PU (24 per cent) only. [U-14C]-Glucose decarboxylation was raised by all fat-rich diets; MS (88 per cent). LS (39 per cent), MU (33 per cent), and PU (50 per cent), whereas [U-14C]-glutamine decarboxylation was increased by LS (53 per cent) and MU (55 per cent) and decreased by MS (17 per cent). The results presented indicate that the reduction in lymphocyte proliferation due to LS, LU and PU could well be a consequence of changes in glucose and glutamine metabolism.

Dietary fats alter the activity and expression of glucose-6-phosphate dehydrogenase in rat lymphoid cells and tissues.

The effect of diets enriched with fat containing different fatty acids on the activity and expression of the glucose-6-phosphate dehydrogenase (EC 1.1.1.49) of mesenteric lymph nodes lymphocytes and intraperitoneal macrophages was examined. Measurements of the enzyme were also performed using spleen, thymus and liver for comparison. The following fat rich diets containing a variety of fatty acids were used: 1-standard chow (CC); 2-medium chain saturated fatty acids (MS)-coconut fat-oil; 3-long chain saturated fatty acids (LS)-cocoa butter; 4-monounsaturated fatty acids (MU)-canola oil (n-9); 5-polyunsaturated fatty acids (PU)-soybean oil (n-6). Of the fat-rich diets tested, MS had the least effect. The G6PDH activity of lymphocytes was reduced by all the fat-rich diets; 16% for MS, 38% for LS, and 54% for MU. Similarly, the enzyme activity was reduced in macrophages; 35%, 86%, and 73%, for LS, MU, and PU, respectively. In contrast, the fat-rich diets elevated G6PDH activity in the lymphoid organs; by 42% in the spleen due to LS and by 131%, 35%, and 56% in the thymus due to LS, MU, and PU, respectively. Fat-rich diets decreased the activity of G6PDH in liver; 42%, 68%, and 39% for MS, MU, and PU, respectively. Some of the changes in G6PDH activity induced by the fat-rich diets occur through the mechanisms of mRNA abundance.