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Biochemical and Molecular Actions of Nutrients

Quercetin Decreases Oxidative Stress, NF-B Activation, and iNOS Overexpression in Liver of Streptozotocin-Induced Diabetic Rats

Alexandre Simões Dias^*, Marilene Porawski, María Alonso^**, Norma Marroni^*,, Pilar S. Collado^** and Javier González-Gallego^**,1

^* Universidade Federal de Rio Grande do Sul, Porto Alegre, RS, Brasil; Universidade Luterana do Brasil, Canoas, RS, Brasil; and ^** Department of Physiology, University of León, 24071 León, Spain

¹To whom correspondence should be addressed. E-mail: dfijgg{at}unileon.es.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Increasing evidence in both experimental and clinical studies suggests that oxidative stress is involved in the pathogenesis and progression of diabetic tissue damage. This study investigated the protective effects of quercetin treatment on oxidative stress, nuclear factor (NF)-B activation and expression of inducible nitric oxide synthase (iNOS) in streptozotocin-induced diabetic rats. Male Wistar rats were divided into 4 groups: control rats, control rats treated daily with quercetin (150 µmol/kg, i.p.), untreated diabetic rats, and diabetic rats treated with quercetin. Diabetes was induced by a single i.p. injection of streptozotocin (70 mg/kg). Eight weeks later we measured TBARS and hydroperoxide-initiated chemiluminescence (QL) in liver as markers of oxidative stress, and activities of the antioxidant enzymes catalase, superoxide dismutase (SOD), and glutathione peroxidase, NF-B activation by an electrophoretic mobility shift assay and expression of IB kinases (IKK and IKKß), the inhibitor IB (IB and IBß), and iNOS by Western blot. The plasma glucose concentration was significantly increased in diabetic rats and was not changed by quercetin. Streptozotocin administration induced significant increases in hepatic TBARS concentration, QL, and SOD and catalase activities that were prevented by quercetin. Activation of NF-B, induction of IKK and iNOS protein levels, and increased degradation of IB were also observed in streptozotocin-treated rats. All of those effects were abolished by quercetin. These findings suggest that quercetin treatment, by abolishing the IKK/NF-B signal transduction pathway, may block the production of noxious mediators involved in the development of early diabetes tissue injury and in the evolution of late complications.

KEY WORDS: • diabetes • quercetin • oxidative stress • nuclear factor-B • nitric oxide

Diabetes mellitus is one of the most common endocrine metabolic disorders. In recent years, a large body of evidence suggested oxidative stress as a mechanism underlying insulin resistance, type I and type II diabetes, and diabetic complications (1). Hyperglycemia causes oxidative stress due to increased mitochondrial production of the superoxide anion (2), nonenzymatic glycation of proteins (3), and glucose autoxidation (4,5). FFA, which are elevated in diabetes and insulin resistance, may also contribute to the increased production of reactive oxygen species (ROS)² due to increased mitochondrial uncoupling and ß-oxidation (6,7). In addition, hyperglycemia and FFA-induced oxidative stress lead to the activation of stress-sensitive signaling pathways, including nuclear factor (NF)-B (8).

Hyperglycemia also favors, through the activation of NF-B, an increased expression of inducible nitric oxide synthase (iNOS), which is accompanied by increased generation of nitric oxide (9). Nitric oxide can react with superoxide to produce the strong oxidant peroxynitrite, which in turn can increase lipid peroxidation, protein nitration, and LDL oxidation, affecting many signal transduction pathways (10). Recent experimental evidence supports the idea of complex roles for nitric oxide, ROS, and peroxynitrite in the development of early diabetes tissue injury before the evolution of late complications (11).

The role of oxidative stress in insulin resistance and diabetes is clouded by the results of intervention studies with antioxidants, which are elusive or unsuccessful (12). Although studies of short duration appear to support an improved insulin sensitivity in insulin-resistant and diabetic patients (8) and a prevention of the progression of diabetic complications by vitamin C and vitamin E (13,14), long-duration clinical trials with classical antioxidants, in particular with vitamin E, did not demonstrate any maintained beneficial effect (12,15). However, although it may not be possible to completely reverse diabetic complications, antioxidants could be useful in preventing or attenuating the adverse effects of chronic hyperglycemia (7).

Flavonoids are phenolic phytochemicals; they are important constituents of the nonenergetic part of the human diet and are thought to promote optimal health, partly via their antioxidant effects in protecting cellular components against ROS (16). Quercetin (3,5,7,3'4'-pentahydroxy flavon) is one of the most widely distributed flavonoids, present in fruit, vegetables, and many other dietary sources (17). This compound was reported to scavenge superoxide in ischemia-reperfusion injury (18), to protect against oxidative stress induced by UV light (19), spontaneous hypertension (20), secondary biliary cirrhosis (21), and bacterial lipopolysaccharide (22), and to inhibit angiogenesis (23), carcinogenesis, (24) and portal hypertensive gastropathy (25). At doses of 50 and 80 mg/kg, quercitin significantly lowered plasma TBARS and lipid hydroperoxides when given to rats with streptozotocin-induced diabetes for 45 d (26,27). A very recent report indicates that quercetin is also able to partially prevent serum nitric oxide increases in streptozotocin-treated rats (28).

The liver is the main organ of oxidative and detoxifying processes, as well as free radical reactions; in many diseases, biomarkers of oxidative stress are elevated in the liver at an early stage (29). Because liver is subjected to ROS-mediated injury in diabetes (30), our experiments were performed to investigate the potential protective effects of quercetin treatment on liver oxidative stress, NF-B activation, and iNOS expression in an experimental model of chronic hyperglycemia. We used a dose of quercetin likely to be achieved in humans (25,26) and previously reported to induce maximal beneficial effects in different liver diseases (21,25).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and experimental procedure. Male Wistar rats (Panlab) were caged at 24°C, with a 12-h light:dark cycle and free access to food (standard diet for rats Panlab A04³) and water until the time of experiments. The rats were randomly divided into 4 groups. In 2 groups, diabetes was induced by a single i.p. injection of streptozotocin (70 mg/kg body weight; Sigma Chemical) in freshly prepared 10 mmol/L sodium citrate, pH 4.5. Five days after the streptozotocin injection, plasma glucose concentration was measured using tail vein blood samples obtained from rats after overnight food deprivation. A plasma glucose level > 14 mmol/L was considered indicative of diabetes. The experimental groups comprised the normal control group (C: n = 8); normal rats treated daily with quercetin (CQ: 150 µmol/kg body weight suspended given immediately before i.p., administration in 500 µL of a 0.2% Tween aqueous solution for 8 wk, n = 8). The diabetic groups were untreated (D: n = 8) or treated with quercetin (DQ: n = 8). Quercetin treatment was initiated 5 d after the administration of streptozotocin. Rats in both the C and DC groups were administered 500 µL/d of vehicle, i.p. for 8 wk.

The rats were killed by exsanguination 8 wk after administration of streptozotocin. Blood samples were centrifuged at 1800 x g for 15 min to obtain plasma and the livers were excised, weighed, and immediately frozen at –70°C. All experiments were performed in accordance with the NIH guidelines (31) and consent was provided by the Ethical Committee of the University of León.

    Plasma glucose concentration. Blood plasma glucose was measured spectrophotometrically at a wavelength of 505 nm using a standard assay kit (GOD-PAP, Sigma Chemical).

    Hepatic markers of oxidative stress. The amount of aldehydic products generated by lipid peroxidation was quantified by the TBA reaction (32) using 3 mg of protein/sample. Spectrophotometric absorbance was determined in the supernatant at 535 nm. Results were referred to as TBARS. Hydroperoxide-initiated chemiluminescence (QL) (33) was measured by a liquid scintillation counter in the out-of-coincidence mode.

    Hepatic antioxidant enzyme activities. Frozen liver from each rat was homogenized in ice-cold phosphate buffer (KCl 140 mmol/L, phosphate 20 mmol/L, pH 7.4) and centrifuged at 14,000 x g for 10 min. Catalase (EC 1.11.1.6) activity was determined by measuring the exponential disappearance of H₂O₂ at 240 nm and was expressed as U/g protein (34). The assay of cytosolic glutathione peroxidase (EC 1.11.1.19) was carried out according to Flohe and Guntzler (35). Cumene hydroperoxide was used as the substrate, and 1 U of enzymatic activity was defined as the amount of protein that oxidizes 1 µmol of reduced NADPH/min. Cytosolic superoxide dismutase (SOD; EC 1.15.1.1) was assayed according to Misra and Fridovich (36) at 30°C. The rate of autooxidation of epinephrine, which is progressively inhibited by increasing amounts of SOD in the homogenate, was monitored spectrophotometrically at 560 nm. The amount of enzyme that inhibits epinephrine autooxidation at 50% of the maximum inhibition was defined as 1 U of SOD activity.

    Western blot. Protein extraction and Western blotting were performed as described (25). Membranes were probed with polyclonal anti-IB kinase (IKK), anti-IKK/ß, anti-IB, anti-IBß, or anti-iNOS antibodies (Santa Cruz Biotechnology). Bound primary antibody was detected with horseradish peroxidase-conjugated anti-rabbit antibody (DAKO) by chemiluminescence. The density of the specific IKK, IKKß, inhibitor of NF-B (IB), IBß, and iNOS bands was quantitated with an imaging densitometer.

    Electrophoretic mobility shift assay. Nuclear extracts were prepared from liver lysates as described previously (37). Activation of transcription factor NF-B was examined using consensus oligonucleotides of NF-B (5'-AGT TGA GGG GAC TTT CCC AGG C-3'). Probes were labeled by T4 polynucleotide kinase. Binding reactions included 10 µg of nuclear extracts in incubation buffer [50 mmol/L Tris-HCl pH 7.5, 200 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L mercaptoethanol, 20% glycerol and 1 µg poly (dI-dC)]. After 15 min on ice, the labeled oligonucleotide (10,000 dpm) was added and the mixture incubated for 20 min at room temperature. For competition studies, 3.5 pmol of unlabeled NF-B oligonucleotide (competitor) or 3.5 pmol of labeled NF-B oligonucleotide mutate (noncompetitor) were mixed 15 min before the incubation with the labeled oligonucleotide. The mixture was electrophoresed through a 6% polyacrylamide gel for 90 min at 220 V. The gel was then dried and autoradiographed at –70°C overnight. Signals were analyzed densitometrically.

    Statistical analysis. Means and SEM were calculated. Data were analyzed using a 2 (diabetic and nondiabetic rats) x 2 (quercetin-treated and quercetin-untreated rats) ANOVA. Post hoc comparisons were carried out using the Newman Keuls test. Statistical significance was set at P < 0.05. SPSS+ version 13.0 statistical software was used.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Blood glucose. Glucose concentration in the blood plasma of streptozotocin-treated rats was significantly higher (2-fold) than in the normal control group and was not affected by quercetin treatment (Table 1).

    Hepatic antioxidant enzymes. SOD activity was 34% greater in liver of diabetic rats compared with controls, and quercetin treatment of diabetic rats reduced SOD to a level less than that of controls. Catalase activity was significantly altered by diabetes (+130% vs. C), and this effect was prevented by quercetin treatment. Glutathione peroxidase activity was not affected by either streptozotocin or quercetin (Table 1).

    Hepatic NF-B activation. Quercetin did not affect NF-B binding activity in controls. Experimental diabetes markedly induced NF-B (+71% vs. C), an effect that was abolished by quercetin treatment (Fig. 1).

View larger version (25K): [in this window] [in a new window]   FIGURE 1 Effect of streptozotocin-induced diabetes and quercetin on NF-B activation in rat liver. Specific binding was verified by the addition of unlabeled (cold) oligonucleotide (competitor, C–) or labeled oligonucleotide mutate (noncompetitor, C+). (A) A representative EMSA. (B) Values are mean ± SEM, n = 8. ANOVA: Q, 0.025; Q x D, 0.001. Means without a common letter differ, P < 0.05.

    Hepatic IKK and IB protein levels.

View larger version (17K): [in this window] [in a new window]   FIGURE 2 Effect of streptozotocin-induced diabetes and quercetin on Western blot analysis of IKK and IB proteins in rat liver. Total cellular protein was separated on 12% SDS-polyacrylamide gels and blotted with anti-IKK or anti-IB antibodies. (A) A representative Western blot photograph. (B) Values are means ± SEM, n = 8. ANOVA: Q, 0.025; Q x D, 0.001. Means without a common letter differ, P < 0.05.

View larger version (16K): [in this window] [in a new window]   FIGURE 3 Effect of streptozotocin-induced diabetes and quercetin on Western blot analysis of iNOS protein in rat liver. Total cellular protein was separated on 12% SDS-polyacrylamide gels and blotted with anti-iNOS antibodies. (A) A representative Western blot photograph. (B) Values are means ± SEM, n = 8. ANOVA: IKK: D, 0.010; Q, 0.011; Q xD, 0.042. IB: D, 0.010; Q, 0.032; Q x D, 0.034. Means without a common letter differ, P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Increased levels of TBARS, an end product of lipoperoxidation, were found previously in the liver of streptozotocin-induced diabetic rats (40,41). Both changes in the concentration of TBARS and the hydroperoxide-initiated QL confirm this finding, indicating increased overall oxidative stress in diabetic rats. Results from the present study also suggest an amelioration of oxidative stress by quercetin, coinciding with the decreased plasma levels of hydroperoxides and TBARS recently reported by other authors (27).

Oxidative stress is the result of a redox imbalance between the generation of ROS and the compensatory response from the endogenous antioxidant network. There is no consensus concerning changes in the activities of antioxidant enzymes of different organs in diabetic rats. Although some studies measuring activities of SOD, catalase, and glutathione peroxidase in diabetes mellitus showed reductions in the levels of these enzymes (28,42–44), other authors reported increased activities in streptozotocin-induced diabetic rats (40,45–47). These apparently contradictory results could be due to tissue specificity, variation in severity and duration of the disease, or other experimental conditions (37). In the present study, catalase and SOD activities increased in diabetic rats. The increase in catalase activity may be a compensatory response for an increase in endogenous H₂O₂ production in diabetic liver because insulin deficiency promotes the ß-oxidation of fatty acids with resulting H₂O₂ formation (48). The increase in SOD activity could be due to its induction by increased production of superoxide, and H₂O₂ was reported to act as an inducer of tissue SOD (49). The increases in both SOD and catalase activities may thus be an adaptive response for increased oxidative stress in the liver tissue; quercetin, by scavenging ROS, prevents the elevation of those antioxidant enzyme activities in diabetic rat liver. Our data are consistent with those previously described for the effect of -lipoic acid (39) or the flavonoid-like compound, caffeic acid phenethyl ester (40), although they differ from the report (27) that both catalase and SOD activities are reduced in erythrocytes of diabetic animals and normalized after 45 d of quercetin administration.

The molecular mechanisms whereby oxidative stress contributes to organ damage and to the development of diabetic complications are undefined. In a variety of tissues, hyperglycemia and elevated FFA result in the generation of ROS, leading to increased oxidative stress. In the absence of an appropriate compensatory response from the endogenous antioxidant network, the system becomes overwhelmed (redox imbalance), leading to the activation of stress-sensitive signaling pathways, such as NF-B, and others. In bovine endothelial cells, it was shown that exposure to hyperglycemia initially increased intracellular production of ROS and activated NF-B (50). It was also found that -lipoic acid blocks NF-B activation in patients with type 2 diabetes (51). In addition, -phenyl-tert-butylnitrone, a spin-trapping agent that reacts with free radical species, significantly reduces the severity of hyperglycemia in both alloxan- and streptozotocin-induced diabetes, coinciding with inhibiting activation of NF-B (52), and both activation of NF-B and elevation in oxidative stress are reduced in rats fed a diet supplemented with multiple antioxidants for up to 14 mo (53). These data indicate that activation of NF-B is an initial signaling event that leads to cellular dysfunction and damage (2) and our finding, extended to liver tissue, that quercetin is able to reduce both oxidative stress and NF-B activation, would support the suggestion that oxidative stress is the initial change induced by high glucose, followed by activation of other pathways.

Under normal physiologic conditions, NF-B forms a complex with its inhibitors, the IBs ( or ß), and is maintained in the cytosol in this inactive state. NF-B can be freed from its inhibitors through the direct action of protein kinases, the IKKs that form a complex consisting of the catalytic subunits IKK and IKKß and the regulatory subunit IKK (54). Activation of the IKK complex leads to the phosphorylation of the IBs, thus targeting them for polyubiquitination and degradation by the 26S proteosome complex. Freed from its inhibitor, NF-B enters the nucleus and transactivates NF-B–responsive genes (55,56). Interestingly, some flavonoids were reported to inhibit NF-B through the activation of IKK (57), and quercetin potently inhibits both IKK and IKKß in vitro (58). Results from the present study indicate that in streptozotocin diabetic rats, quercetin decreases IB degradation by inhibiting upregulation of members of the IKK complex. Effects on the IKK/IB cascade in turn contribute to inhibition of NF-B activation.

One major consequence of the activation of stress-sensitive signaling pathways is the generation of gene products such as nitric oxide that cause cellular damage and are ultimately responsible for the late complications of diabetes Enhanced nitric oxide production may contribute to the hyperfiltration and microalbuminuria that characterize early diabetic nephropathy (59). Diabetes was previously reported to lead to increased activity and expression of liver iNOS in streptozotocin-induced diabetic rats (60) and nitric oxide levels are significantly elevated in diabetic liver at a very early stage (29). In this situation, nitric oxide may react with ROS such as the superoxide radical to yield the highly reactive oxidant species peroxynitrite, leading to more aggressive oxidative and nitrosative stress (61). In the present investigation, quercetin-induced suppression of the release of NF-B by preventing the degradation of IB was accompanied by downregulation of the expression of iNOS, confirming results in other experimental models such as lipopolysaccharide-activated macrophage cells (62,63) and supporting the previous report that elevations of nitric oxide and nitrostyrosine levels in the retina of diabetic rats are reduced by administration of multiple antioxidants in parallel with an inhibition of NF-B activation (53).

In summary, the results presented here show that administration of quercetin inhibits oxidative stress, NF-B activation, and iNOS overexpression in liver of streptozotocin-diabetic rats. Quercetin treatment, by abolishing the IKK/NF-B signal transduction pathway, might block the production of noxious mediators involved in the development of early injury and in the evolution of late complications in different tissues affected by chronic hyperglycemia. Although inhibition by quercetin and other antioxidants of specific pathways that are activated as a consequence of increased oxidative stress and glucose flux may not be sufficient to completely reverse diabetic complications once they have been established, further investigation is warranted to determine whether it may be possible to slow down the progression, or prevent the onset of complications by preemptive therapy before the development of tissue damage. Because the in vivo activity of quercetin depends on its bioavailability, which varies among foods (64), the effectiveness of this flavonoid when it is consumed as a part of the human diet and the required level of dietary intake also warrant further studies. Considering that quercetin inhibits the activation of genes under NF-B control, another important goal of futures studies will be determination of which antioxidants are more effective at preventing NF-B activation, along with the identification of the molecular sites of action.

	FOOTNOTES

 
² Abbreviations used: C, control; CQ, quercetin treatment; D, diabetes; DQ, diabetes + quercetin treatment; EMSA, electrophoretic mobility shift assay; IB, inhibitor of NFB; IKK, IB kinase; iNOS, inducible nitric oxide synthase; NF, nuclear factor; QL, chemiluminescence; ROS, reactive oxygen species; SOD, superoxide dismutase.

³ The composition of the standard diet was: 15.4% protein, 2.9% fat, 60.5% carbohydrate, 3.9% fiber, 5.3% minerals, and 12% water.

Manuscript received 10 June 2005. Initial review completed 28 June 2005. Revision accepted 11 July 2005.

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