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Inhibition of aldose reductase prevents experimental allergic airway inflammation in mice.

Yadav UC, Ramana KV, Aguilera-Aguirre L, Boldogh I, Boulares HA, Srivastava SK - PLoS ONE (2009)

Bottom Line: Our results indicate that inhibition of AR prevents RWE -induced apoptotic cell death as measured by annexin-v staining, increase in the activation of NF-kappaB and expression of inflammatory markers such as inducible nitric oxide synthase (iNOS), cycloxygenase (COX)-2, Prostaglandin (PG) E(2), IL-6 and IL-8.Our results indicate that inhibition of AR prevents airway inflammation and production of inflammatory cytokines, accumulation of eosinophils in airways and sub-epithelial regions, mucin production in the bronchoalveolar lavage fluid and airway hyperresponsiveness in mice.These results suggest that airway inflammation due to allergic response to RWE, which subsequently activates oxidative stress-induced expression of inflammatory cytokines via NF-kappaB-dependent mechanism, could be prevented by AR inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Immunology, University of Texas Medical Branch, Galveston, Texas, United States.

ABSTRACT

Background: The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress-induced inflammation by affecting the NF-kappaB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be anti-inflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, ragweed pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma.

Methods and findings: Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on ragweed pollen extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE -induced apoptotic cell death as measured by annexin-v staining, increase in the activation of NF-kappaB and expression of inflammatory markers such as inducible nitric oxide synthase (iNOS), cycloxygenase (COX)-2, Prostaglandin (PG) E(2), IL-6 and IL-8. Further, BALB/c mice were sensitized with endotoxin-free RWE in the absence and presence of AR inhibitor and followed by evaluation of perivascular and peribronchial inflammation, mucin production, eosinophils infiltration and airway hyperresponsiveness. Our results indicate that inhibition of AR prevents airway inflammation and production of inflammatory cytokines, accumulation of eosinophils in airways and sub-epithelial regions, mucin production in the bronchoalveolar lavage fluid and airway hyperresponsiveness in mice.

Conclusions: These results suggest that airway inflammation due to allergic response to RWE, which subsequently activates oxidative stress-induced expression of inflammatory cytokines via NF-kappaB-dependent mechanism, could be prevented by AR inhibitors. Therefore, inhibition of AR could have clinical implications, especially for the treatment of airway inflammation, a major cause of asthma pathogenesis.

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Inhibition of AR prevents RWE-induced expression and activation of inflammatory, apoptotic and cell cycle proteins and redox-sensitive transcription factors NF-κB and AP-1 in SAEC.(I) Approximately 2×105 SAEC were seeded in 6-well plates and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (150 µg/ml) for 24 h and cell lysate was prepared. Immunoblotting was performed using antibodies against (A) COX-2, iNOS, (B) Bcl-XL, Bax, and (C) Cyclin D1, E2F2 to determine the expression of various proteins. GAPDH was used as loading control. Representative blots are shown (n = 3), numbers below the blots represent fold changes. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, ragweed pollen extract. (II) (A) For EMSA, approximately 2×106 SAEC were seeded in T-150 cm2 flasks and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (50 µg/ml) for 3 h. Nuclear extract was prepared and EMSA was performed to assess the DNA binding activity of NF-κB and AP-1. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, ragweed pollen extract. (B) Approximately, 1×105 SAEC were plated in 24-well plate and growth-arrested by preincubatin in serum-free basal medium with AR inhibitor or carrier for 24 h followed by transfection with NF-κB-pSEAP vector or control (pTAL) vector. After 6 h, transfected cells were incubated with RWE (50 µg/ml) for 48 h. Medium was collected, cleared by centrifugation and NF-κB-dependent reporter SEAP activity was measured by chemiluminescence's method essentially as described by the manufacturer. Bars represent Mean±SD (n = 4). #p<0.001 Vs. Control; *p<0.01 Vs RWE. zop, zopolrestat; RWE, ragweed pollen extract.
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pone-0006535-g004: Inhibition of AR prevents RWE-induced expression and activation of inflammatory, apoptotic and cell cycle proteins and redox-sensitive transcription factors NF-κB and AP-1 in SAEC.(I) Approximately 2×105 SAEC were seeded in 6-well plates and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (150 µg/ml) for 24 h and cell lysate was prepared. Immunoblotting was performed using antibodies against (A) COX-2, iNOS, (B) Bcl-XL, Bax, and (C) Cyclin D1, E2F2 to determine the expression of various proteins. GAPDH was used as loading control. Representative blots are shown (n = 3), numbers below the blots represent fold changes. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, ragweed pollen extract. (II) (A) For EMSA, approximately 2×106 SAEC were seeded in T-150 cm2 flasks and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (50 µg/ml) for 3 h. Nuclear extract was prepared and EMSA was performed to assess the DNA binding activity of NF-κB and AP-1. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, ragweed pollen extract. (B) Approximately, 1×105 SAEC were plated in 24-well plate and growth-arrested by preincubatin in serum-free basal medium with AR inhibitor or carrier for 24 h followed by transfection with NF-κB-pSEAP vector or control (pTAL) vector. After 6 h, transfected cells were incubated with RWE (50 µg/ml) for 48 h. Medium was collected, cleared by centrifugation and NF-κB-dependent reporter SEAP activity was measured by chemiluminescence's method essentially as described by the manufacturer. Bars represent Mean±SD (n = 4). #p<0.001 Vs. Control; *p<0.01 Vs RWE. zop, zopolrestat; RWE, ragweed pollen extract.

Mentions: Since biosynthesis of PGE2 and NO from their precursors is catalyzed by COX-2 and iNOS enzymes respectively, we next examined the effect of AR inhibition on RWE-induced COX-2 and iNOS expression in SAEC by immunoblotting. As shown in Fig. 4(I) A, treatment of SAEC with RWE significantly (∼3-folds) increased COX-2 and iNOS protein expression and pre-treatment of SAEC with AR inhibitor, zopolrestat, significantly (>90%) prevented the increase. This indicates that RWE-induced COX-2 and iNOS overexpression is mediated by AR, which is obligatory for RWE-induced PGE2 and NO production that exacerbate allergic inflammation.


Inhibition of aldose reductase prevents experimental allergic airway inflammation in mice.

Yadav UC, Ramana KV, Aguilera-Aguirre L, Boldogh I, Boulares HA, Srivastava SK - PLoS ONE (2009)

Inhibition of AR prevents RWE-induced expression and activation of inflammatory, apoptotic and cell cycle proteins and redox-sensitive transcription factors NF-κB and AP-1 in SAEC.(I) Approximately 2×105 SAEC were seeded in 6-well plates and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (150 µg/ml) for 24 h and cell lysate was prepared. Immunoblotting was performed using antibodies against (A) COX-2, iNOS, (B) Bcl-XL, Bax, and (C) Cyclin D1, E2F2 to determine the expression of various proteins. GAPDH was used as loading control. Representative blots are shown (n = 3), numbers below the blots represent fold changes. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, ragweed pollen extract. (II) (A) For EMSA, approximately 2×106 SAEC were seeded in T-150 cm2 flasks and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (50 µg/ml) for 3 h. Nuclear extract was prepared and EMSA was performed to assess the DNA binding activity of NF-κB and AP-1. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, ragweed pollen extract. (B) Approximately, 1×105 SAEC were plated in 24-well plate and growth-arrested by preincubatin in serum-free basal medium with AR inhibitor or carrier for 24 h followed by transfection with NF-κB-pSEAP vector or control (pTAL) vector. After 6 h, transfected cells were incubated with RWE (50 µg/ml) for 48 h. Medium was collected, cleared by centrifugation and NF-κB-dependent reporter SEAP activity was measured by chemiluminescence's method essentially as described by the manufacturer. Bars represent Mean±SD (n = 4). #p<0.001 Vs. Control; *p<0.01 Vs RWE. zop, zopolrestat; RWE, ragweed pollen extract.
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pone-0006535-g004: Inhibition of AR prevents RWE-induced expression and activation of inflammatory, apoptotic and cell cycle proteins and redox-sensitive transcription factors NF-κB and AP-1 in SAEC.(I) Approximately 2×105 SAEC were seeded in 6-well plates and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (150 µg/ml) for 24 h and cell lysate was prepared. Immunoblotting was performed using antibodies against (A) COX-2, iNOS, (B) Bcl-XL, Bax, and (C) Cyclin D1, E2F2 to determine the expression of various proteins. GAPDH was used as loading control. Representative blots are shown (n = 3), numbers below the blots represent fold changes. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, ragweed pollen extract. (II) (A) For EMSA, approximately 2×106 SAEC were seeded in T-150 cm2 flasks and incubated until 80% confluency. The cells were starved in serum-free basal medium with or without zopolrestat for 24 h. The cells were treated with RWE (50 µg/ml) for 3 h. Nuclear extract was prepared and EMSA was performed to assess the DNA binding activity of NF-κB and AP-1. Lanes: 1, control; 2, RWE; 3, control+zop; 4, RWE+zop. zop, zopolrestat; RWE, ragweed pollen extract. (B) Approximately, 1×105 SAEC were plated in 24-well plate and growth-arrested by preincubatin in serum-free basal medium with AR inhibitor or carrier for 24 h followed by transfection with NF-κB-pSEAP vector or control (pTAL) vector. After 6 h, transfected cells were incubated with RWE (50 µg/ml) for 48 h. Medium was collected, cleared by centrifugation and NF-κB-dependent reporter SEAP activity was measured by chemiluminescence's method essentially as described by the manufacturer. Bars represent Mean±SD (n = 4). #p<0.001 Vs. Control; *p<0.01 Vs RWE. zop, zopolrestat; RWE, ragweed pollen extract.
Mentions: Since biosynthesis of PGE2 and NO from their precursors is catalyzed by COX-2 and iNOS enzymes respectively, we next examined the effect of AR inhibition on RWE-induced COX-2 and iNOS expression in SAEC by immunoblotting. As shown in Fig. 4(I) A, treatment of SAEC with RWE significantly (∼3-folds) increased COX-2 and iNOS protein expression and pre-treatment of SAEC with AR inhibitor, zopolrestat, significantly (>90%) prevented the increase. This indicates that RWE-induced COX-2 and iNOS overexpression is mediated by AR, which is obligatory for RWE-induced PGE2 and NO production that exacerbate allergic inflammation.

Bottom Line: Our results indicate that inhibition of AR prevents RWE -induced apoptotic cell death as measured by annexin-v staining, increase in the activation of NF-kappaB and expression of inflammatory markers such as inducible nitric oxide synthase (iNOS), cycloxygenase (COX)-2, Prostaglandin (PG) E(2), IL-6 and IL-8.Our results indicate that inhibition of AR prevents airway inflammation and production of inflammatory cytokines, accumulation of eosinophils in airways and sub-epithelial regions, mucin production in the bronchoalveolar lavage fluid and airway hyperresponsiveness in mice.These results suggest that airway inflammation due to allergic response to RWE, which subsequently activates oxidative stress-induced expression of inflammatory cytokines via NF-kappaB-dependent mechanism, could be prevented by AR inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Immunology, University of Texas Medical Branch, Galveston, Texas, United States.

ABSTRACT

Background: The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress-induced inflammation by affecting the NF-kappaB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be anti-inflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, ragweed pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma.

Methods and findings: Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on ragweed pollen extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE -induced apoptotic cell death as measured by annexin-v staining, increase in the activation of NF-kappaB and expression of inflammatory markers such as inducible nitric oxide synthase (iNOS), cycloxygenase (COX)-2, Prostaglandin (PG) E(2), IL-6 and IL-8. Further, BALB/c mice were sensitized with endotoxin-free RWE in the absence and presence of AR inhibitor and followed by evaluation of perivascular and peribronchial inflammation, mucin production, eosinophils infiltration and airway hyperresponsiveness. Our results indicate that inhibition of AR prevents airway inflammation and production of inflammatory cytokines, accumulation of eosinophils in airways and sub-epithelial regions, mucin production in the bronchoalveolar lavage fluid and airway hyperresponsiveness in mice.

Conclusions: These results suggest that airway inflammation due to allergic response to RWE, which subsequently activates oxidative stress-induced expression of inflammatory cytokines via NF-kappaB-dependent mechanism, could be prevented by AR inhibitors. Therefore, inhibition of AR could have clinical implications, especially for the treatment of airway inflammation, a major cause of asthma pathogenesis.

Show MeSH
Related in: MedlinePlus