Eight to ten weeks old C57BL/6 mice (Charles River Laboratories International Inc., Wilmington, MA) were exposed to elastase and LPS for four consecutive weeks as described previously 15 . Animals were exposed by the intranasal route to 1.2 U of porcine pancreatic elastase (Elastin Products, Owensville, MO) on day one and 7 μg (approximately 70 endotoxin units) of LPS from E. coli O26:B6 (Sigma-Aldrich, St. Louis, MO) on day four of the week for four consecutive weeks. Control mice were exposed to PBS. Seven days after the last exposure to LPS, mice were orally gavaged with 300 μl of 50% propylene glycol (vehicle) or 0.2 mg of quercetin (10 mg/kg body weight; Sigma-Aldrich, St. Louis, MO) dissolved in 50% propylene glycol, once a day for 10 days. We chose this dose of quercetin based on previous studies in which quercetin at 10 mg/kg body weight reduced airways responsiveness and lung inflammation in an allergic mouse model of asthma 19 20 . In some experiments, mice were treated intraperitoneally with 100 μl PBS or PBS containing sirtinol (0.5 mg/kg body weight, Calbiochem, Gibbstown, NJ) along with quercetin or vehicle for 10 days. Mice were sacrificed 1 h after the last quercetin treatment. In some experiments, mice were examined 7 and 17 days after the last exposure to LPS without any treatment in order to examine the progression of emphysema after cessation of exposure to elastase/LPS. Unexposed mice treated with vehicle or quercetin (10 mg/kg body weight) were used as negative controls. All experiments described herein were approved by the Animal Care and Use Committee of the University of Michigan.

Mice Were Anesthetized By Intraperitoneal Injection Of Ketamine (2.5-5 Mg/100 G Body Weight) And A Steel Cannula Was Inserted Into The Trachea And Connected To A Miniature Computerized Flexivent Ventilator (Scireq, Montreal, Quebec, Canada). Sodium Pentabarbitol (2 Mg/100 G Body Weight) Was Also Given To Provide Further Sedation And Allow Stabilization On The Ventillator. To Determine Elastic Recoil, Lungs Were Gradually Inflated To 30 Cm H₂O And Pressure And Lung Volume Measured Continuously During Inflation And Deflation Of The Lungs. Static Elastance And Compliance Were Recorded By Inflating The Lungs To Full Capacity.

Lungs Were Perfused With 20 Mm Edta And Inflation-Fixed With 10% Buffered Formalin, And Embedded In Paraffin. Five Micron Thick Sagittal Sections Were Stained With Hematoxylin And Eosin (H & E) Or Periodic-Acid Schiff'(Pas) Reagent. Alveolar Chord Length Was Determined Using Sagittal Sections Obtained At 5 Mm Intervals Through The Length Of The Lungs, And Diameter Of The Airspaces Was Measured In Random Areas Using Nih Image J Analysis Software 15 .

Mice Were Euthanized And Lungs Were Lavaged With Pbs. Bal Fluid Was Centrifuged And The Supernatant Was Collected For Determination Of Mmp Levels. Total And Differential Cell Counts In Bal Fluid Were Determined As Described Previously 15 22 .

After Relevant Treatment, Mice Were Euthanized, Lungs Were Collected, Homogenized In Pbs Containing Complete Protease Inhibitors And Centrifuged (Roche, Indianapolis, In). Cytokine Protein Levels In The Lung Homogenate Supernatants Were Measured Either By Elisa (R & D Systems, Minneapolis, Mn) Or Multiplex Immunoassay (Biorad, Hercules, Ca) 15 22 .

Murine Alveolar Macrophages (Crl-2019, American Type Culture Collection, Manassas, Va) Were Cultured In Rpmi1640 Supplemented With 10% Fetal Bovine Serum, Penicillin (100 Units/Ml), Streptomycin (100 μG/Ml), 1% Glutamine And 0.01% β-Mercaptoethanol. To Determine The Effect Of Quercetin On Mmp Expression, Cells Were Seeded In 6 Well Plates And Grown For 24 H. Cells Were Then Exposed To Cell Culture Media Containing 1 Ng/Ml Lps For 8 Hours A Day For Three Days. In Between Exposures To Lps, Cells Were Maintained In Cell Culture Media Alone. Cells Were Then Shifted To Serum-Free Media Containing Quercetin Dihydrate Or Dmso, Incubated For 24 H, And Media And Cells Were Harvested. Cells Exposed To Media Alone Instead Of Lps Were Used As Negative Controls.

Mmp Activity Was Determined By Gelatin Zymography As Described. 23 . Briefly, Equal Volumes Of Bal Supernatant Or Conditioned Cell Culture Media Was Incubated With Non-Reducing Sample Buffer And Subjected To Electrophoresis On 8% Polyacrylamide Gels Impregnated With 0.1% Gelatin. Gels Were Washed With 1% Triton X-100, Developed In Tris Buffer Containing 10 Mm Cacl₂ And 5μM Zncl₂ And Stained With 0.5% Coomassie Blue.

Mice Were Sacrificed And Blood Was Collected By Cardiac Puncture In Tubes With Anticoagulant, Centrifuged And Plasma Was Collected. Levels Of Quercetin In Plasma Were Determined By Hplc As Described Previously 19 .

Chip Assays Were Performed With A Chip-It Kit (Active Motif, Carlsbad, Ca) Following The Manufacturer'S Instructions. Briefly, Cells Were Fixed, Lysed And Chromatin Was Subjected To Enzymatic Shearing. Chromatin Fragments Of 100-1000 Bp Were Immunoprecipitated With An Antibody To Acetylhistone H4 Antibody. Chip And Input Dna Were Purified And Subjected To Qpcr Using Primers Specific For The Nf-κB Binding Site In The Mmp9 And Mmp12 Promoters. Qpcr Conditions Were As Follows: 95°C For 15 Minutes; 95°C For 10 Seconds, 60°C For 30 Seconds, 72°C For 30 Seconds, Repeated For 50 Cycles, 72°C For 10 Minutes. The Primers Used For Qpcr Were Mmp9: Sense 5'-TTTAAACAGAAGAGGAAGGAT AGTGC-3' And Antisense 5'-CCTGATAGAGTCTTT GACTCAGCTTC-3'; Mmp12: Sense 5'-TTGCTGAATCATTTCATGGC-3' And Antisense 5'-AGTGCATAGGTATG TGAATGGG-3'.

Expression Of Mmp9, Mmp12, Sirt1, Inducible Nitric Oxide Synthase (Inos), Heme Oxygenase (Hmox)-1, And Muc5Ac Was Determined By Qpcr. All Pcr Reactions Were Performed In An Eppendorf Mastercycler (Westbury, Ny) And Gene Expression Was Quantified Using The Comparative Ct Method.

Nuclear Proteins Were Resolved By 7.5% Sds-Polyacrylamine Gel Electrophoresis, Proteins Transferred To Nitrocellulose Membrane And Probed With Antibody To Sirt1 And β-Actin (Santa Cruz Biotechnology, Santa Cruz, Ca). Specific Bands Were Quantified By Densitometry Using Nih Imagej And Expressed As A Ratio Of Sirt1/B-Actin Which Is Normalized To Untreated Control Mice.

The Amount Of Lipid Peroxidation Products In The Lungs Was Assayed As Thiobarburtic Acid Reacting Substances (Tbars) (Cell Biolabs, San Diego, Ca) Following Manufacturer'S Instructions.

Statistical Analysis Of Significance Was Calculated By One-Way Analysis Of Variance Followed By Tukey'S Post Hoc Test, Anova On Ranks With Dunn'S Post Hoc Analysis Or By Mann-Whitney Test As Appropriate. Results Represent Mean ± Sd Or Sem, Or Range Of Data With Median.

As observed previously 15 , mice at seven days after the four-week exposure to elastase/LPS showed an upward and leftward shift compared to control mice, demonstrative of reduced elastic recoil (Figure 1A). Because mice receiving quercetin or vehicle for 10 days were studied a total of 17 days after the last exposure to elastase/LPS, we also measured the lung function of untreated mice at this time point. The volume-pressure curve was shifted further to the left, indicating further progression of emphysema after cessation of exposure to elastase/LPS. This shift may be due to persistence of oxidative stress and MMP activity even after cessation of exposure to elastase/LPS. This situation is analogous to the further progression of emphysema in COPD patients even after cessation of smoking 24 .

Next, we examined the lung function of mice treated with quercetin (0.2 mg) or vehicle for 10 days starting one week after the four-week course of elastase/LPS-treatment. Compared to vehicle, mice receiving quercetin showed a rightward and downward shift in their volume-pressure curve (Figure 1B). Shifts in the pressure-volume loops were accompanied by appropriate changes in elastance and compliance (Figures 1C and 1D). Finally, compared to vehicle, quercetin treatment was associated with a reduction in alveolar chord length (Figure 1E). However, quercetin treatment did not completely reverse the emphysematous changes caused by elastase/LPS. Quercetin treatment did not affect any of these measurements in the lungs of mice exposed to PBS. Together, these data suggest that quercetin treatment prevented further progression of emphysema after elastase/LPS treatment rather than stimulating the regeneration of degraded alveoli.

To determine the mechanism by which quercetin prevents progression of emphysema in elastase/LPS-treated mice, we examined the effects of quercetin on indices of lung oxidative stress and inflammation. Elastase/LPS-exposed mice were treated with 0.2 mg of quercetin for 10 days and lung levels of TBARS, iNOS mRNA and Hmox-1 mRNA determined. Compared to unexposed mice either treated with vehicle or quercetin, elastase/LPS-exposed mice treated with vehicle showed significantly increased levels of TBARS and iNos, and decreased levels of Hmox-1 mRNA. The ratio of iNos/Hmox-1 was increased (Figures 2A to 2D). In contrast, elastase/LPS exposed mice treated with 0.2 mg of quercetin for 10 days showed significantly reduced TBARS, increased Hmox-1 mRNA and decreased iNos/Hmox-1 compared to vehicle treated controls. These results indicate that exposure of mice to elastase/LPS increases oxidative stress, and that treatment with quercetin reverses this effect.

Lung cytokine levels, histology, total and differential cells counts in the BAL, and expression of the mucin gene Muc5AC were determined to test whether quercetin treatment reduces inflammation in elastase/LPS exposed mice. As previously noted, elastase/LPS-exposed mice showed increased protein expression of the chemokines KC/CXCL-1, MIP-2/CXCL-2 and MCP-1/CCL2 and pro-inflammatory cytokines IL-1β, IL-12p40 and MIP-1β (Figure 3). Compared to vehicle, quercetin treatment significantly decreased the levels of all chemokines and pro-inflammatory cytokines examined. PBS-exposed mice treated with quercetin showed similar levels of all cytokines measured compared to mice treated with vehicle (data not shown).

Evaluation of H & E-stained lung sections showed wide-spread lung inflammation and emphysema in elastase/LPS exposed mice as observed previously (Figure 4A). Quercetin-treated elastase/LPS exposed mice showed an overall reduction in lung inflammation compared to vehicle treated mice (Figure 4C). Immunostaining of lung sections with anti-MUC5AC antibody showed intense signals in the airway epithelium of elastase/LPS-exposed mice treated with vehicle but not mice treated with quercetin (Figures 4B and 4D). Consistent with the histologic changes, elastase/LPS-exposed mice showed increased total cell counts, macrophages and neutrophils compared to PBS-exposed mice, and each of these variables was significantly reduced by quercetin (Figure 4E). We also observed decreased mRNA expression of Muc5AC in quercetin-treated, elastase/LPS-exposed mice compared to vehicle-treated mice (Figure 4F).

Increased MMP levels are thought to play a role in the development and/or progression of emphysema in COPD patients 25 26 27 . Consistant with this, lungs of elastase/LPS exposed mice showed increased mRNA and activity levels of MMP9 and MMP12 compared to vehicle treated PBS-exposed mice (Figure 5A-C). Lung MMP levels did not change in quercetin treated PBS-exposed mice (data not shown). Quercetin treatment significantly decreased the mRNA and activity levels of both MMP9 and MMP12 in elastase/LPS-exposed mice.

MMP9 transcription is negatively regulated by a histone deacetylase, SIRT1 (2). We examined whether reductions in Mmp9 and Mmp12 mRNA levels were associated with increases in SIRT1 expression in quercetin-treated, elastase/LPS-exposed mice. Vehicle-treated elastase/LPS-exposed mice showed an 83.2% reduction in mRNA expression of Sirt1 compared to mice unexposed to elastase/LPS (Figure 5D). Similarly, we observed 53% reduction in protein levels of Sirt1 in the lungs of vehicle-treated, elastase/LPS-exposed mice (Figure 5E and 5F). Quercetin treatment of elastase/LPS exposed mice increased both Sirt1 mRNA and protein levels. These results suggest that quercetin may suppress MMP9 and MMP12 expression by increasing Sirt1 levels.

To determine the contribution of Sirt1 expression to the observed effects of quercetin on lung phenotype, elastase/LPS exposed mice were treated with polyethylene glycol or quercetin along with sirtinol, an inhibitor of Sirt1 activity 28 . As observed earlier, compared to vehicle-treated elastase/LPS-exposed mice, lungs of quercetin-treated mice showed reduced mRNA expression of MMP9 and MMP12 (Figure 6A and 6B) and improved elastic recoil as indicated by leftward and downward shift in pressure-volume loops, decreased static compliance and increased static elastance (Figure 6C and 6E). In contrast, mice treated with quercetin along with sirtinol showed a similar level of MMP mRNA expression as vehicle treated elastase/LPS mice. Sirtinol treatment also blocked the improvements of elastic recoil, static compliance and static elastance induced by quercetin. These results are consistent with the notion that quercetin exerts its effects by increasing Sirt1 levels, which negatively regulate MMP expression.

Chromatographic analysis of plasma obtained from mice treated with 0.2 mg quercetin for 10 days revealed a major peak which corresponded to quercetin aglycone and other minor peaks as we observed previously 19 . Quantification of the major peak indicated a mean plasma quercetin level of 0.131 ± 0.038 μM in mice treated with quercetin. This level was significantly higher than the quercetin level observed in vehicle-treated mice (0.004 ± 0.0019 μM).

Sirt1, a histone deacetylase, negatively regulates MMP9 transcription by deacetylating histone H4 in the promoter region NF-κB binding site 7 . To determine whether quercetin increases H4 deacetylation at this site, we employed an in vitro cell culture system. Murine alveolar macrophages exposed to low levels of LPS for three days showed increased Mmp9 and Mmp12 mRNA levels, with a concomitant decrease in Sirt1 expression (Figure 7A-C). We also observed increased MMP9 activity (Figure 7D) and decreased SIRT1 protein expression (Figure 7E and 7F) in LPS-treated alveolar macrophages. Consistent with these in vivo results, in vitro quercetin treatment of alveolar macrophages significantly decreased LPS-induced mRNA expression of Mmp9 and Mmp12 as well as MMP9 activity, while increasing mRNA and protein expression of Sirt1. Next, we examined whether quercetin increases histone deacetylation of the MMP9 and MMP12 promoter NF-κB binding sites by ChIP assay. Histone H4 acetylation at the MMP9 and MMP12 promoter NF-κB binding sites was increased in LPS-exposed alveolar macrophages compared to media-treated controls, and this was completely inhibited by treatment with quercetin (Figures 7G and 7H). These results suggest that quercetin inhibits H4-acetylation of MMP promoters by increasing Sirt-1 expression, thereby regulating MMP expression at the transcriptional level.