Hae Young Chung, PhD, College of Pharmacy, Pusan National University, Gumjung-gu, Busan 609–735, Korea. Tel.: 82-51-510-2814; fax: 82-51-518-2821; e-mail: email@example.com
Nuclear factor-κB (NF-κB), a redox-sensitive transcription factor, plays an important role in the aging process. Thus, developing and identifying specific components that modulate NF-κB without adverse side-effects would be of major importance. Hesperetin, a flavanone abundant in citrus fruits, has a variety of pharmacological properties being antioxidant, cholesterol-lowering, and anti-inflammatory.
In this study, we investigated how hesperetin fed to 6- and 24-month-old rats modulates NF-κB in their kidneys. Results showed that hesperetin suppressed NF-κB activation and related gene expressions. An even more interesting finding is that hesperetin suppressed NF-κB through four signal transduction pathways, NIK/IKK, ERK, p38, and JNK. Further evidence showed the remarkable efficacy of hesperetin to suppress the translocation of Trx/Ref-1, indicating its beneficial effect on the redox status. The most significant findings of the current study report new information on the use of hesperetin as a potential anti-aging agent.
Aging is a natural and inevitable part of the life process, characterized by gradual declines of physiological functions that ultimately lead to morbidity and mortality. With advancing age, oxidative alterations accumulate in DNA, proteins and other cellular components, including antioxidative defense systems, leading to functional deficits and many age-related degenerative diseases (Harman, 1973; Yu, 1996). The progressive increase of oxidative stress during aging not only causes oxidative damage to cellular macromolecules, but also modulates the pattern of gene expression through functional alterations of transcription factors (Lavrosky et al., 2000).
NF-κB (nuclear factor kappa B) is an oxidative stress responsive and redox-sensitive transcription factor that regulates the expression of a variety of genes involved in immune function, inflammatory responses, cell proliferation, and apoptosis (Luo et al., 2003). IκB is an inhibitor of NF-κB and is phosphorylated, ubiquitinylated and ultimately degraded by the proteasome system upon stimulation, hence NF-κB is activated. Following stimulation, NF-κB-inducing kinase (NIK) activates the IκB kinase (IKK) complex, which consists of IKKα, IKKβ, and IKKγ. Activation of the IKK complex is mediated through phosphorylation of either IKKα or IKKβ, and leads to the phosphorylation of the N-terminal Ser-32 and Ser-36 of IκBα. Several proinflammatory stimuli that induce COX-2, iNOS, VCAM-1, and ICAM-1 expression activate the mitogen-activated protein kinase (MAPK) pathway (Luo et al., 2003). Four distinct MAPK subfamilies have been identified in mammalian cells: the extracellular signal-regulated kinase (ERK), the c-Jun N-teminal kinase/stress-activated protein kinase (JNK/SAPK), and the p38. The MAPK subfamililes regulate NF-κB (Choi et al., 2004).
There are two levels of modulation of these transcription factor activities by redox status: induction and translocation of the transcription factor and transcription factor DNA-binding activity (Galter et al., 1994; Schenk et al., 1994; Sen & Packer, 1996; Hirota et al., 1997; Jin et al., 1997). Both of these processes can be modulated by ROS and redox proteins such as thioredoxin (Trx) and redox factor-1 (Ref-1) (Hirota et al., 1997). Trx translocates from the cytosol into the nucleus in response to a variety of cellular stresses and regulates the DNA-binding protein activity, including Jun/Fos and NF-κB (Holmgren, 1989). In the nucleus, Trx forms physical interaction with Ref-1. The Trx/Ref-1 complex regulates transcriptional activity by altering the redox state of specific cysteine residues located in the basic DNA-binding region of these transcription factors (Hirota et al., 1997, 1999). Trx participates in redox reactions by the reversible oxidation of its active center, dithiol to disulfide and catalyzes dithiol-disulfide exchange reactions (Hu et al., 2002) involving many thiol-dependent cellular processes, including gene expression and signal transduction.
Ref-1 is a multifunctional protein that not only functions as a redox factor maintaining transcription factors in an active, reduced state but is also responsible for the repair of apurinic sites as a part of base excision repair (Heiss et al., 2001).
Flavonoids are of significant interest to the antioxidant-related research field because of their versatile and beneficial anti-inflammatory, cytostatic, anticarcinogenic, apoptotic, antiangiogenic, antioxidant, and estrogenic properties (Tinku et al., 2002). Hesperetin is derived from the hydrolysis of its aglycone, hesperidin (hesperetin 7-rhammnoglucoside) (Fig. 1) (Choi et al., 2004). The protective effect of hesperetin against the membrane lipid peroxidation involved in several physiological and pathological disorders, such as aging, inflammation, atherosclerosis, ischemia, and the toxicity of oxygen and chemical substances, has been largely studied (Tommasini et al., 2004).
For our current study, we chose to use the kidney because it mainly consists of endothelial and epithelial cells. Aging is associated with changes in blood vessel tone determined by the balance between vasoconstrictors and vasodilators. Endothelial cells are affected by this change because they line the blood vessels. We therefore chose to select endothelial cell type YPEN-1 cell line. Recent publications from our laboratory presented evidence that NF-κB activation increases during the aging process in kidney homogenate of rat and YPEN-1 cell (Chung et al., 2002; Kim et al., 2002; Go et al., 2005).
The objective of the present study was to investigate whether the antioxidative effect of hesperetin blocks age-related NF-κB activation in the NIK/IKK, ERK, p38, and JNK pathways and to show the potential of hesperetin to inhibit redox regulators Trx and Ref-1.
Effects of hesperetin on reactive species generation in young and aged rats
To assess the overall age-related oxidative status, total reactive species (RS) was measured with a DCFH-DA probe in kidney homogenates. The results showed that the significantly increased RS level with age was suppressed by the feeding of hesperetin and that the 20 mg kg−1 day−1 hesperetin-fed, old rat had lower levels of RS than the young control animals (Fig. 2), indicating the strong antioxidative effect of hesperetin in free radical scavenging. This antioxidative effect was stronger in the old animal than that observed in the young control. Therefore, hesperetin has an antioxidative effect regardless of the age of the rat. This finding was in agreement with our previous report that hesperetin inhibited intracellular RS generation (Kim et al., 2004).
Identification of NF-κB and its related gene expression by hesperetin
Hesperetin inhibited NF-κB transcriptional activity in YPEN-1, HEK 293T, and RAW 264.7 cells
To verify whether hesperetin modulates NF-κB transcriptional activity in YPEN-1, HEK 293T and RAW 264.7 cells, a luciferase reporter gene transfection assay was used. Activated macrophage induces NF-κB-related genes, COX-2 and iNOS (Yasuda et al., 2005). Cells were transiently transfected with a reporter plasmid carrying the luciferase gene. It has been reported that tert-butylhydroperoxide (t-BHP) induces NF-κB activation through IKK and MAP kinase (Lee et al., 2005). t-BHP activated NF-κB luciferase activity in YPEN-1 and its activity was attenuated by hesperetin in a dose-dependent manner (Fig. 3A). Also, hesperetin inhibited NF-κB activation by t-BHP in HEK 293T and RAW 264.7 cells (Fig. 3B,C). Hesperetin at 25 µm significantly decreased NF-κB activity.
Hesperetin suppressed NF-κB DNA-binding activity in young and aged rats kidney homogenate
To further investigate transfection results, electrophoretic mobility shift assay (EMSA) was carried out using nuclear proteins from young and aged rats. Results showed that increased NF-κB DNA-binding activity with age was decreased by the hesperetin treatment (Fig. 4, lanes 5–7). Also, hesperetin decreased NF-κB DNA-binding activity in young rats (Fig. 4, lanes 2–4). Furthermore, supershift assay using anti-p50 and anti-p65 antibodies appear to shift the NF-κB band (Fig. 4, lanes 8–9). The displacement pattern of NF-κB band as a result of incubation with specific antibodies thus supports the specificity of NF-κB binding to its consensus motif. Furthermore, binding specificity of the NF-κB complex was demonstrated using a 100-fold excess of an unlabeled oligonucleotide, which competed for binding (Fig. 4, lane 10). These results showed that hesperetin is a very powerful inhibitor of age-induced NF-κB activation.
Hesperetin decreased NF-κB-related gene expression in young and aged rats kidney homogenate
COX-2, iNOS, VCAM-1, and ICAM-1 genes are known to have an NF-κB binding site in their promoter regions and to be controlled by NF-κB regulation (Umezawa et al., 2002). Therefore, to elucidate the changes in NF-κB DNA-binding activity that correlate with NF-κB-dependent gene expression, gene expressions of COX-2, iNOS, VCAM-1, and ICAM-1 were examined. As shown in Fig. 5, COX-2, iNOS, VCAM-1, and ICAM-1 levels increased with age, but hesperetin lowered these levels. The 20 mg kg−1 day−1 hesperetin-fed old rats (HO20) showed lower levels of gene expression compared with the young control, indicating the strong antioxidant effects of hesperetin. This finding was in agreement with our previous report that hesperetin inhibited intracellular RS generation. These gene expressions also correlated well with NF-κB activity. Together, these results suggested that hesperetin modulates NF-κB activation.
Exploration of NF-κB cascade through NIK/IKK, ERK, p38, and JNK MAPK pathways
Hesperetin modulates NF-κB activation through NIK/IKK pathway
Inhibition of NIK/IKK and IκBα phosphorylation by hesperetin. To determine whether hesperetin modulates NF-κB activation through the NIK/IKK pathway, we examined the activation of NIK, IKK, and IκBα by detecting their phosphorylated forms using Western blot analysis. It has been shown that NF-κB activators induce the phosphorylation and degradation of IκB by activating the IKK complex, IKKα and IKKβ (Regnier et al., 1997; Karin, 1999). Both IKKα and IKKβ phosphorylate IκBα at both serines 32 and 36 in response to the proinflammatory cytokines TNF-α and IL-1β (Li et al., 2003).
NIK either directly or indirectly activates the IKKα–IKKβ complex, leading to IκB phosphorylation and degradation and NF-κB activation (Higuchi et al., 2002; Matata & Galinanes, 2002; Sakuma et al., 2003). Results showed that although the level of phospho-IκBα increased with age, rats fed hesperetin had lower levels than the control group. Furthermore, levels of phospho-NIK and IKK increased with age, but hesperetin also suppressed these levels (Fig. 6). Thus, these results indicated that hesperetin inhibited IκBα phosphorylation through the NIK/IKK pathway.
Suppression of translocation and activation of NF-κB through IκBα degradation by hesperetin. NF-κB is normally present in the cytoplasm in an inactive state and bound to a member of the IκB inhibitor protein family (Hatada et al., 2000). Following stimulation, IκBα is phosphorylated and degraded. Unbound NF-κB then translocates into the nucleus and transactivates various downstream genes (Poljokovic et al., 2003). In addition to this classical activation pathway, recent data suggest that the phosphorylation status of the NF-κB p65 subunit is also involved in NF-κB activation (Yeh et al., 2004). We found that increased degradation of the IκBα with aging was decreased by the hesperetin treatment (Fig. 6). Furthermore, as shown in Fig. 7, the nuclear translocation of NF-κB increased with age, and hesperetin prevented this change. Because hesperetin is a strong antioxidant, the 20 mg kg−1 day−1 hesperetin-fed young and old rats had lower levels of RS than of each control. The phosphorylation of p65 subunit affects neither IκBα degradation nor NF-κB nuclear translocation, but is required for the transcriptional activity of NF-κB, partly through the increasing interaction of NF-κB with other transcriptional cofactors. Also, the phosphorylation of the p65 subunit is mediated by IKK (Yasunari & Bharat, 2004; Yeh et al., 2004). Results showed that the increased phosphorylation of the p65 subunit with age was suppressed by hesperetin (Fig. 7). Based on these data, we believe that hesperetin inhibited the nuclear translocation of NF-κB through the NIK/IKK pathway.
Hesperetin regulated NF-κB activation through the ERK, p38, and JNK pathways
Inhibition of NF-κB activation in ERK pathway by hesperetin. MAP kinases are involved in the expressional regulation of many genes that are induced by cytokines. ERK is one of the mitogen-activated protein kinases (MAPKs) and MAPK kinase kinase, an upstream kinase of ERK, contributes to cell differentiation, proliferation, and survival (Cho et al., 2003). Upon stimulation, Ras assumes its active GTP-bound form, and recruits the protein kinase Raf-1 (Miyazaki et al., 2000). Raf-1 stimulates MAP kinase kinases (MEK1 and MEK2), which in turn activate ERK1 (p44 MAPK) and ERK2 (p42 MAPK). A previous report showed that NF-κB activation by Raf is indicative of an alternative signaling pathway (Baumann et al., 2000) and that ERK phosphorylation is a requirement for the persistent activation of NF-κB. Another study (Castrillo et al., 2001) also reports that ERK kinase kinase activates NF-κB. Therefore, changes in the Raf-1/MEK1/2/ERK pathway by aging and hesperetin were investigated. As Fig. 8A shows, phosphorylated Raf-1, MEK1/2, and ERK increased with aging, suggesting that the activated Raf-1/MEK1/2/ERK pathway might contribute to age-related NF-κB activation. However, hesperetin suppressed the age-related increase of the Raf-1/MEK1/2/ERK pathway, which correlates with the age-related inhibition of NF-κB activation by hesperetin. Consequently, these results indicated that hesperetin modulates NF-κB activation via the ERK pathway.
Effect of hesperetin on NF-κB activation through p38 and JNK pathways. The p38 kinase family members are important effectors in the inflammatory response, and previous studies have reported the involvement of the p38 pathway in the activation of NF-κB-mediated transcription (Kim et al., 2002). The JNK pathway is also activated in response to stress and growth factors, and mediates signals that regulate apoptosis, cytokine production, and cell–cycle progression (Platanias, 2003). Therefore, we tested whether blocking the protein level of p38 with hesperetin could interfere with NF-κB activation. Both Rac, an upstream protein kinase, and phospho-p38 levels increased during aging. However, hesperetin suppressed the age-related increase of the Rac/p38 pathway, which correlates with an age-related down-regulation of NF-κB activity by hesperetin. Also, the phospho-JNK protein level was the same as p38 (Fig. 8B). Hesperetin exhibited the same pattern in young rats. Therefore, we found that hesperetin also modulated NF-κB activation through the p38 and JNK pathway.
Hesperetin modulated age-related changes of Trx and Ref-1
Several transcription factors, including AP-1, NF-κB, p53, and PEBP-2, and nuclear receptors like glucocorticoid receptors or estrogen receptors have been shown to be activated by Trx (Toru et al., 2000). Our results showed that hesperetin inhibits increased NF-κB activity and expression with age. Thus, when investigating the modification of Trx with age and hesperetin, we hypothesized that Trx exerts its effects during the aging process but that hesperetin could suppress these effects. As shown in Fig. 9, Trx in the nucleus increased with age, but in rats fed hesperetin, this age-related increase was blocked.
When Trx is imported into the nucleus, it forms a physical interaction with Ref-1, a nuclear protein with augmenting DNA-binding activity of AP-1. Ref-1 also enhances binding activity between NF-κB and DNA (Kiichi et al., 1999). Ref-1 protein levels in the nucleus of aged kidney were also assessed. The results showed that nuclear Ref-1 protein levels increased with age; however, hesperetin blunted this increase (Fig. 9). Based on these data, hesperetin is suggested to possibly contribute to the inactivation of redox-sensitive transcription factor NF-κB through the regulation of Trx and Ref-1.
Our data clearly show that hesperetin inhibited NF-κB activation through the NIK/IKK, ERK, p38, and JNK pathways. Because the phosphorylation of IκB is a key regulatory step that dictates NF-κB activation, the focus of our study was on the status of the essential enzyme IKK during aging and its modulation by hesperetin (Yang et al., 2001). First, we observed IKK to increase with age, but this increase was suppressed by hesperetin. Also, increased NIK levels with age were decreased by the hesperetin treatment. Second, MAPKs are important mediators involved in the intracellular network of proteins that transduce extracellular cues into intracellular responses. Previous studies report the involvement of the ERK, p38, and JNK pathways in the activation of NF-κB (Bian et al., 2001). In this study, hesperetin suppressed phospho-Raf-1, MEK1/2, and ERK levels. Similarly, Rac and phospho-p38 levels were decreased by hesperetin. Also, phospho-JNK level was diminished by the hesperetin treatment. Several studies exploring the regulation of NF-κB activity found that the nuclear DNA-binding activity of the transcription factor was strongly enhanced during aging in heart, liver, and brain tissue (Supakar et al., 1995; Helenius et al., 1996). Thus, increased NF-κB activity seems to be a widespread biological phenomenon in aged animals, as NF-κB is a critical transcription factor involved in the pathogenesis of many disorders, including inflammatory diseases such as rheumatoid arthritis, psoriasis, and even atherosclerosis (Helenius et al., 1996; Barnes, 1997).
Xagorari et al. (2001) reported that hesperetin was ineffective in blocking the release of IL-6 and NO. But our previous study revealed hesperetin effectively inhibited intracellular free radical activity (Kim et al., 2004). Our data further showed that hesperetin significantly decreased NF-κB and its related gene expressions such as COX-2, iNOS, VCAM-1, and ICAM-1. Moreover, other studies showed hesperetin reduced IL-1α which activated NF-κB and modulated inflammation (Liu & Chiou, 1996; Dekker et al., 2005). It is interesting to note that although hesperetin does not have a double bond at position C2–C3 of the C-ring, it still can modulate NF-κB and its related genes and cytokines.
It has been reported that t-BHP stimulates the redox status of the cell and has been extensively utilized to elucidate the nature of oxidative stress in cellular physiology and biochemistry (Elliott et al., 1989). Hydrogen peroxide (H2O2) and t-BHP in particular are metabolized by distinct enzymatic pathways, and are useful for investigating the complex transcriptional responses to oxidative stress in vitro (Weigel et al., 2002). H2O2 can induce DNA damage by generating hydroxyl radicals through Fenton's reaction in the presence of copper bound to DNA, while t-BHP generates alkoxyl and hydroxyl radicals through the same reactions (Banmeyer et al., 2004). Hydroxyl and alkoxyl radicals can generate a wide range of DNA damage including strand breaks, basic sites, and oxidized bases. The inactivation of t-BHP is predominantly accomplished by glutathione peroxidase (Aruoma et al., 1991; Termini, 2000). Both t-BHP and H2O2 generate ROS that are responsible for lipid peroxidation, DNA adduct formation and the induction of apoptosis (Haidara et al., 2002). Therefore, in our study, cells were treated with t-BHP to induce oxidative stress.
In the present study, hesperetin caused the down-regulation of renal NF-κB activity through NIK/IKK, ERK, p38, and JNK pathways. Since NF-κB is implicated in the exacerbation of inflammation by increasing the expression of proinflammatory genes, chronic activation of NF-κB throughout the aging process may be a major causative factor of inflammatory processes that lead to tissue injury, which might be due to the overgeneration and accumulation of ROS and RNS, from COX-2 and iNOS, respectively. It is important to note that because hesperetin suppressed NF-κB activation, it might serve well as an aging intervention agent.
The activation of redox-sensitive transcription factor NF-κB can be viewed as having two distinct steps with respect to cellular compartments. The first step is a cytoplasmic reaction (IκB degradation) following the nuclear translocation of proteins. The second step is the DNA-binding of the complex and transactivation into the nucleus.
Trx and Ref-1 are proteins involved in the second step. Both act as redox regulators of various transcription factors, such as NF-κB, AP-1, and HIF-1, thereby affecting the transcriptional-activating properties of these proteins. Trx forms a physical interaction with a Ref-1 in the nucleus, which activates redox-sensitive transcription factors in a redox-dependent manner. In the present study, it was revealed that age-related increases in the redox-sensitive transcription factor NF-κB consistently correlated with increases in nuclear Trx and Ref-1. In contrast, hesperetin was shown to suppress the NF-κB level and nuclear Trx and Ref-1 activity, suggesting that hesperetin modified the activities of the redox-sensitive transcription factor, NF-κB in a redox-dependent manner in both cytoplasm and nucleus.
In summary, the present study suggests that the active bioflavonoid component hesperetin suppressed NF-κB luciferase activity, NF-κB DNA-binding activity, and related gene expressions. Furthermore, hesperetin was shown to modulate NF-κB activation through the NIK/IKK, ERK, p38, and JNK pathways. Many of the currently accepted medical therapies for inflammatory bowel disease and rheumatoid arthritis inhibit NF-κB. Thus, NF-κB is a potential target for anti-inflammatory therapies. The identification of new and more selective inhibitors of NF-κB may lead to more effective treatments for inflammatory disorders. Because hesperetin modulated NF-κB and its gene expression, it therefore could be useful as an anti-inflammatory and anti-aging agent.
Hesperetin (3′, 5, 7-trihydroxy-4-methoxyflavanone) and t-BHP were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2,7-Di-chlorodihydrofluorescein diacetate (H2DCFDA) was from Molecular Probes (Eugene, OR, USA); polyvinylidene fluoride (PVDF) membrane (Immobilon-P) was obtained from Millipore Corp. (Billelica, MA, USA), and the chemiluminescence detection system was from Amersham Life Sciences, Inc. (Arlington Heights, IL, USA). Enhanced chemiluminescence (ECL) Western blotting detection reagents were from Amersham Life Science (Buckinghamshire, UK). Antibodies to p50, p65, IκBα, phospho-IκBα, phospho-NIK, phospho-IKKα/β, Raf-1, phospho-ERK1/2, Rac-1, phospho-p38, phospho-JNK, COX-2, iNOS, VCAM-1, ICAM-1, Ref-1, and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-p65 and anti-phospho-MEK1/2 were from Cell Signaling Technology, Inc. (Boston, MA, USA). The radionucleotide [γ-32P]-ATP was obtained from Amersham Life Science. All other chemicals were of the highest purity available from either Sigma Chemical Co. or Junsei Chemical Co. (Tokyo, Japan).
Cell line and culture conditions
YPEN-1 cells, rat endothelial cells, HEK 293T cells, human kidney epithelial cells, and RAW 264.7 cells, mouse macrophage, were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA). YPEN cells were cultured in Dulbecco's Modified Eagle Media (DMEM) (Nissui Co., Tokyo, Japan) supplemented with 5% heat-inactivated (56 °C for 30 min) fetal bovine serum (Gibco, Grand Island, NY, USA), 233.6 mg mL−1 glutamine, 100 µg mL−1 penicillin-streptomycin, and 0.25 µg mL−1 amphotericin B, and adjusted to pH 7.4–7.6 with NaHCO3 in an atmosphere of 5% CO2 and HEK 293T and RAW 264.7 cells were cultured in Dulbecco's Modified Eagle Media (DMEM) (Nissui Co.) supplemented with 10% heat-inactivated (56 °C for 30 min) fetal bovine serum (Gibco) containing 233.6 mg mL−1 glutamine, 100 µg mL−1 penicillin-streptomycin, and 0.25 µg mL−1 amphotericin B. The fresh medium was replaced after 1 day to remove non-adherent cells or cell debris.
Specific pathogen-free male Fischer 344 rats were obtained from Samtako (Osan, Korea) and fed a diet of the following composition: 21% soybean protein, 15% sucrose, 43.65% dextrin, 10% corn oil, 0.15%α-methionine, 0.2% choline chloride, 5% salt mix, 2% vitamin mix and 3% Solka-Floc. Hesperetin was mixed with powder and fed to the rats at 24 months of age at a dose of 10 or 20 mg kg−1 day−1. Rats at ages 6 and 24 months were divided into five rats per age group. After 10 days, the rats were killed.
Rats at 6 and 24 months of age were sacrificed by decapitation and the kidneys were quickly removed and rinsed in iced-cold buffer [100 mm Tris, 1 mm EDTA, 0.2 mm phenylmethyl-sulfonylfluoride (PMSF), 1 µm pepstatin, 2 µm leupeptin, 80 mg L−1 trypsin inhibitor, 20 mmβ-glycerophosphate, 20 mm NaF, 2 mm sodium orthovanadate (pH 7.4)]. The tissue was immediately frozen in liquid nitrogen and stored at −80 °C.
RS generation assay
Reactive species (RS) generation was measured as previously described (Thomas et al., 1992). A working solution of DCFDA (2.5 µm) was added to homogenate for 250 µL of final volume and then changes in fluorescence intensity were measured for 30 min on the microplate fluorescence (GENious, TECAN, Austria) with excitation and emission wavelengths set at 485 and 535 nm, respectively.
NF-κB activity assay by transfection and luciferase reporter
NF-κB activity was examined using a luciferase plasmid DNA, pTAL-NF-κB that contains a specific binding sequence for NF-κB (BD Biosciences Clontech, CA, USA). Transfection was carried out using FuGENE 6 Reagent (Roche, Indianapolis, IN, USA). Briefly, 1.5 × 104 cells per well were seeded in 48-well plates. When cultured cells reached about 50% confluence, cells were treated with 0.1 µg DNA/0.5 µL FuGENE 6 complexes in a total volume of normal media (5% serum contained) with 500 µL for 42 h. Subsequently, hesperetin of various concentrations was treated after the plate was changed with serum-free media and incubated for 1 h and then 20 µm of t-BHP was treated and incubated for 6 h. Cells were washed with PBS and added by Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA) to the plate. Luciferase activity was measured by a luminometer (GENious, TECAN).
Raw luciferase activities were normalized by protein concentration per well.
Preparation of nuclear extract
All solutions, tubes, and centrifuges were maintained at 0–4 °C. For each nuclear extract preparation, three male Fischer rat kidneys were used. The preparation of rat kidney nuclear extracts was based on previous methods (Kerr, 1995). The nuclear extract was frozen at −80 °C in aliquots until EMSA was done.
EMSA and supershift assay for NF-κB
The EMSA method was used to characterize the binding activities of NF-κB in nuclear extracts (Kerr, 1995). Protein-DNA-binding mixture containing 15–20 µg of nuclear protein extract were incubated for 20 min at 4 °C in binding medium containing 5% glycerol, 1 mm MgCl2, 50 mm NaCl, 0.5 mm EDTA, 2 mm DTT, 1% NP-40, 10 mm Tris (pH 7.5), and 1 µg of poly (dI-dC)·poly (dI-dC). Radiolabeled transcription factor consensus oligonucleotide [γ-32P]-ATP (Amersham) was added, and the complex mixture was incubated for an additional 20 min at room temperature. Also, supershift experiments were conducted using specific antibodies (rabbit antip50 and antip65, Santa Cruz Biotechnology). Reactions were identical to gel shift reaction conditions except 1 µg of antibody was added to the binding reaction mixtures before the addition of labeled probe and the reaction mixtures were incubated for 40 min at room temperature. DNA-binding complexes were resolved by 7% native poly acrylamide gel electrophoresis with 0.5× TBE (0.045 m Tris-borate/0.001 m EDTA) with 5 mm Tris/38 mm glycine running buffer for 90 min at 200 V. The gel was dried, and complexes were detected by autoradiography. The identity of the complexes was established with excess unlabeled oligonucleotide.
Western blot analysis
The prepared samples in gel buffer, pH 6.8 (12.5 mm tris[hydroxymethyl] aminomethane, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2-mercaptoethanol and 0.2% bromophenol blue) in a ratio of 1 : 1 were boiled for 5 min. Total protein equivalents for each sample were separated on 8–17% SDS-polyacrylamide minigel at 100 V and transferred to a PVDF membrane at 100 V for 90 min in a wet transfer system (Bio-Rad, Hercules, CA, USA). The membrane was immediately placed into a blocking solution (5% w/v skim milk powder in TBS-Tween buffer containing 10 mm Tris, 100 mm NaCl, and 0.1 mm Tween-20, pH 7.4) at room temperature for 1 h. The membrane was washed in TBS-Tween buffer for 30 min and then incubated with a primary antibody (1% w/v skim milk, diluted 1 : 500 in TBS-Tween buffer) at room temperature for 3 h. After three 10-min washings in TBS-Tween buffer, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody from sheep (1% w/v skim milk, diluted 1 : 10 000 in TBS-Tween buffer) at room temperature for 2 h. Then, after three 10-min washings in TBS-Tween buffer, antibody labeling was detected using ECL and exposed to radiographic film. Pre-stained blue protein markers (Bio-Rad) were used for molecular-weight determination.
Data are expressed as mean ± standard deviation (SD) of three or five experiments. Statistical analysis was confirmed by Student's t-test. Values of P < 0.05 were considered to be statistically significant.
This work was supported by a grant from the KOSEF (R01-2000-000-00120-0). We are grateful to the ‘Aging Tissue Bank’ (R21-2005-000-10008-0) for supplying aged tissue. We express our thanks to Dr Byung Pal Yu for his critical evaluation and comments on this manuscript. This work was supported by the Brain Korea 21 Project in 2006.