Quercetin was purchased from Sigma-Aldrich (Q4951; Shanghai, China). Drug solutions were prepared by suspending the compound in 0.5% carboxymethyl cellulose sodium. Animals were gavaged daily with 0.1 ml solution of quercetin (60 mg/kg) or vehicle alone, which began 2 weeks prior to AAA induction and continued for 8 weeks. The dose regimen for quercetin was based on previous studies demonstrating beneficial effects of the drug in mouse models of aortic atherosclerosis (12).

A total of 60 male C57BL/6 wild-type mice (age, 6–7 weeks) were obtained from Vital River Laboratory Animal Technology (Beijing, China). All animals were treated and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Washington DC, 1996) and the experimental protocols were approved by the Animal Care and Use Committee (Nanjing University, Nanjing, China). The mice were randomly assigned to one of four groups (n=15 in each group): Vehicle treatment plus sham operation control (VC), vehicle treatment plus AAA (VA), quercetin treatment plus AAA (QA) and quercetin treatment plus sham operation control (QC). AAA was induced in the infrarenal abdominal aorta (age, 8 weeks) by periaortic application of CaCl₂, as previously described (16). NaCl (0.9%) was substituted for CaCl₂ in sham operation animals. Six weeks later, the mice were laparotomied and the aortic diameters (ADs) were measured; the abdominal incision was carried upwards as a thoracoabdominal incision, the animals were then sacrificed by left-heart injection of potassium chloride and the aortic tissues were collected. An aneurysm was defined as an increase in the AD of >50% of the original AD.

A computerized, non-invasive tail-cuff system with a four-channel mouse platform (BP-2000; Visitech Systems, Inc., Apex, NC, USA) was used to measure blood pressure and heart rate. To train mice, daily measurements were performed for five consecutive days prior to the actual recorded measurements. Hemodynamic parameters were measured one day pre- and 6 weeks post-AAA induction. The first 10 of 30 values recorded at each session were disregarded and the remaining 20 values were averaged and used for analysis, according to the manufacturer’s instructions.

Dihydroethidium (DHE) oxidative fluorescence dye was used to evaluate in situ production of ROS (17). DHE stock solution was prepared by dissolving DHE (D7008; Sigma-Aldrich) in dimethylsulfoxide at a concentration of 5 mM. The stock solution was stored in the dark and diluted in phosphate-buffered saline (PBS) to a final concentration of 5 μM immediately prior to use. The abdominal aorta was harvested and the aortic segment (10 mm) was embedded in Tissue-Tek OCT compound (Sakura Finetech Japan, Tokyo, Japan) and snap-frozen. DHE working solution (200 μl) was topically applied to the aortic sections and the slides were subsequently incubated at 37°C in the dark for 30 min. Excess DHE was rinsed off twice with PBS and the images were immediately captured with a fluorescent microscope (BX51; Olympus, Tokyo, Japan) at excitation and emission wavelengths of 520 and 610 nm, respectively.

A portion of the snap-frozen aortic tissue (n=5 per group) was crushed in a prechilled mortar and resuspended in PBS at a concentration of 50 mg/ml. The homogenate was centrifuged at 10,000 × g for 10 min at 4°C to collect the supernatant. The lipid peroxidation product, malondialdehyde (MDA), was assessed using the thiobarbituric acid reactive substances (TBARS) assay kit (A003; Jiancheng Bioengineering, Shanghai, China). Briefly, 100 μl supernatant was added to 100 μl sodium dodecyl sulfate (SDS) lysis solution and mixed thoroughly. Following the addition of 250 μl thiobarbituric acid (TBA) reagent, samples were incubated at 95°C for 1 h and centrifuged at 1,500 × g at room temperature for 15 min. The absorbance of each supernatant was measured at 532 nm using a spectrophotometer (BioPhotometer; Eppendorf, Hamburg, Germany). Values of TBARS are expressed as nmol equivalents of MDA per mg protein. Mn-SOD activity was measured using an assay kit (A001-2; Jiancheng Bioengineering) according to the manufacturer’s instructions. Assay conditions were 65 μmol phosphate buffer (pH 7.8), 1 μmol hydrochloric hydroxylamine, 0.75 μmol xanthine and 2.3×10⁻³ IU xanthine dismutase. The supernatant (50 μl) was incubated in the system for 40 min at 37°C and terminated with 2 ml 3.3 g/l p-aminobenzene sulfonic acid and 10 g/l naphthylamine. For inhibition of CuZn-SOD activity, the assay was conducted in the presence of 10 mm KCN following preincubation for 30 min. The supernatant was transferred to a microplate (Eppendorf) for determination of the absorbance at 550 nm and 1 unit SOD was defined as the quantity of enzyme required to produce 50% dismutation of superoxide radical. Mn-SOD activity was calculated by subtraction of CuZn-SOD activity from total SOD activity. The standard curves were created as described in the manufacturer’s instructions. Images were assessed by Image J 1.44 software (National Institute of Health, Bethesda, MD, USA).

The infrarenal abdominal aorta (n=5 per group) was dissected and fixed in 10% neutral-buffered formalin. Specimens were dehydrated through graded ethanols, embedded in paraffin and sliced into 4–6-μm sections. Immunohistochemical staining with a rabbit polyclonal anti-nitrotyrosine antibody (1:500; 06–284; Millipore, Temecula, CA, USA) was used as an indicator of peroxynitrite formation (18). Briefly, the slides were incubated in 3% hydrogen peroxide for 5 min to quench endogenous peroxidase activity and were then incubated with primary antibody overnight at 4°C. Subsequently, slides were washed with PBS and incubated (15 min; 37°C) with peroxidase-conjugated goat anti-rabbit IgG (AP132P; Millipore). Finally, the slides were incubated with diaminobenzidine and counterstained with hematoxylin.

RT-PCR was used to define the expression of glutathione peroxidase (GPx)-1, GPx-3, inducible nitric oxide synthase (iNOS) and p47phox NADPH oxidase mRNA. Total RNA was prepared with the TRIzol total RNA extraction kit (SK1321; Sangon Biotech, Shanghai, China). Primer sequences were as follows: Forward: 5′-ACC CCA AGT ACA TCA TTT GGT C-3′ and reverse: 5′-GCA GGG TTT CTA TGT CAG GTT C-3′ for GPx-1; forward: 5′-ATC TAC GAG TAT GGA GCC CTC A-3′ and reverse: 5′-GGC CCA AGT TCT TCT TGT AGT G-3′ for GPx-3; forward: 5′-CTT TGA CGC TCG GAA CTG TAG-3′ and reverse: 5′-AAC TCC AAG GTG GCA GCA T-3′ for iNOS; forward: 5′-CCC ATC ATC CTT CAG ACC TAT C-3′ and reverse: 5′-AAC CTC GCT TTG TCT TCA TCT G-3′ for p47 NADPH oxidase; and forward: 5′-AGG CCG GTG CTG AGT ATG TC-3′ and reverse: 5′-TGC CTG CTT CAC CAC CTT CT-3′ for GAPDH. Reverse transcription was performed with oligo-dT primers and the AMV First Strand cDNA Synthesis kit (SK2029; Sangon Biotech), according to the manufacturer’s instructions. The resultant cDNA was amplified by Taq DNA polymerase (SK2442; Sangon Biotech) in an Access RT-PCR System (Promega Corp., Madison, WI, USA). GAPDH mRNA was also amplified to serve as an internal control. The resultant PCR products were detected using an MSF-300G Scanner (Microtek Lab, Carson, CA, USA) and expressed as the ratio to GAPDH.

Total protein was extracted from the supernatants of tissue homogenate with T-PER tissue protein extraction reagent (Pierce Biotechnology Inc., Rockford, IL, USA) and stored at −80°C. Equal quantities (30 μg) of total protein were separated on 10% polyacrylamide gels and transferred to nitrocellulose membranes using a semidry transfer cell (#164-5052; Bio-Rad, Hercules, CA, USA) at 10 V for 40 min. The membranes were blocked for 60 min with 5% nonfat milk in Tris-buffered saline with Tween-20 (TBST) and subsequently washed. Primary antibodies for p47phox NADPH oxidase (sc-14015), c-Jun N-terminal kinase (JNK; sc-571) and phosphorylated JNK (sc-6254) (all Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added at a 1:500 dilution and incubated overnight at 4°C. Additionally, all blots were incubated with the anti-β-actin antibody (1:5,000; 4970; Cell Signaling Technology, Beverly, MA, USA) to confirm protein loading levels. Membranes were washed with TBST, incubated with horseradish peroxidase-conjugated species-appropriate secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature and developed using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA). Quantification of images was performed by scanning densitometry with Image J 1.44.

Nuclear protein lysates were harvested using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Inc.) according to the manufacturer’s instructions. Activator protein (AP)-1 DNA-binding activities were analyzed using Gel Shift Assay systems (Promega Corporation, Madison, WI, USA), according to the instructions previously described (16). Briefly, the AP-1 consensus oligonucleotide probe (5′-CGC TTG ATG AGT CAG CCG GAA-3′) was end-labeled with [γ-³²P]-ATP (Furui Biotech, Beijing, China). The extracted nuclear proteins (10 μg) were incubated for 20 min at 37°C with the ³²P-labeled oligonucleotide (0.30 pmol) in a binding buffer. Reaction products were then separated in a 4% polyacrylamide gel, followed by autoradiography. The reactive bands were quantified as described in western blot analysis.

Protein extracts (10 μg) were mixed with SDS buffer and separated by electrophoresis on 10% SDS-polyacrylamide gels containing 1.0% gelatin. Following electrophoresis, the gels were renatured in renaturing buffer (LC2670; Invitrogen Life Technologies, Carlsbad, CA, USA) and incubated with developing buffer (LC2671; Invitrogen Life Technologies) for 30 min at room temperature. Subsequently, the gel was incubated in fresh developing buffer overnight at 37°C. The gel was stained with 0.5% Coomassie blue R-250 for 30 min and destained with destaining solution containing 10% acetic acid and 40% methanol. The relative molecular weight of each band was determined using protein standards (Pierce Biotechnology, Inc.). Areas of protease activity appeared as unstained bands against a blue background. Images were assessed by Image J 1.44.

All values are expressed as the mean ± standard deviation. Statistical analyses were performed with SPSS for Windows version 17.0 (SPSS, Inc., Chicago, IL, USA). Within-group comparisons of hemodynamic parameters at various intervals were performed using paired Student’s t tests. Between-group comparisons were performed using the Fisher’s exact test or analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

No significant difference was found in AD at the time of surgery among the groups (data not shown). Six weeks later, the VA mice showed a marked increase in AD following CaCl₂ treatment with 10/15 (66.7%) developing aneurysms. Only 3/15 (20%) of the aortas became aneurysmal in QA mice; this difference in aneurysm incidence was considered to be highly significant (P<0.05). No aneurysm formation was observed in the VC or QC mice. Quercetin treatment had no effect on mean arterial pressure or heart rate when measured pre- and post-surgery (Table I).

To evaluate the effect of quercetin on ROS generation, aortic sections were exposed to DHE, which is transformed to the highly fluorescent molecule, oxyethidium, in the presence of superoxide (17). As shown in Fig. 1A, ROS production was extremely low in aortas from VC and QC mice. At 6 weeks post-AAA induction in VA mice, oxyethidium fluorescence was higher, being significantly enhanced throughout the vascular wall (Fig. 1A and B). However, it was attenuated in QA mice, indicating decreased ROS production due to quercetin treatment.

As increased production of ROS may lead to further peroxynitrite accumulation, which induces protein damage by formation of nitrotyrosine (18), immunohistochemistry was performed with a polyclonal antibody against nitrotyrosine in aortic cross sections. Staining appeared only weakly in the aorta of VC and QC animals, however, VA mice revealed marked brown nitrotyrosine staining in the aortic wall. By contrast, a decreased immunoreactivity was observed in QA mice (Fig. 1A and C). Similar observations were noted for lipid peroxidation production (MDA) levels (TBARS) in aortic tissues. The TBARS concentration in QA mice was found to be significantly lower than that in the VA mice (Table II).

Increased levels of Mn-SOD activity were observed at AAA regions of the VA mice, while quercetin significantly decreased its activity in QA mice (Table II). In addition, quercetin caused a relative decrease in mRNA expression of GPx-1 and GPx-3, which are involved in antioxidative status (Fig. 2).

Expression of iNOS and the NADPH oxidase subunit, p47phox, was also examined. Compared with the VA group, QA mice showed relative decreases in iNOS and p47phox mRNA levels (Fig. 2) and this result was confirmed by western blot analysis of p47phox (Fig. 3A and B).

Since quercetin reduced oxidative stress, the specific contribution of quercetin to the regulation of AP-1 activation in experimental AAA was examined. Levels of AP-1 DNA binding activity in QA mice, determined by EMSA, were significantly inhibited by quercetin when compared with controls in the VA group (Fig. 4A and B). Western blotting showed that phosphorylated-JNK was significantly upregulated in VA mice and downregulated following treatment with quercetin. In addition, QA animals had significantly less total JNK than VA controls (Fig. 3A and B).

Gelatin zymography revealed that MMP-2 and -9 activities were elevated in VA mice, however, quercetin treatment resulted in a marked decrease in MMP-2 and -9 activities (Fig. 5).