Asia Pac Allergy.  2017 Jul;7(3):163-170. 10.5415/apallergy.2017.7.3.163.

Increased antioxidant activity after exposure of ozone in murine asthma model

• Kim YK
• Koo SM
• Kim K
• Uh ST
• Jang A
• Park CS

• KMID: 2396940
• DOI: http://doi.org/10.5415/apallergy.2017.7.3.163

Abstract

BACKGROUND
Ozone is well known as an important component of ambient air pollutants. Ozone can aggravate respiratory symptoms in patients with bronchial asthma, but, not in healthy person. We hypothesized asthma itself may show different response to ozone compared to nonasthma.
OBJECTIVE
This study was performed to evaluate the differences of response to ozone between normal and asthmatic mice model in terms of status of oxidant injury and antioxidant activity.
METHODS
Three parts per million of ozone was exposed to ovalbumin (OVA)-induced murine asthma model for 3 hours at 3, 7, 14, 21 days after completion of asthma model. Airway responsiveness to methacholine was measured after completion of asthma model. Bronchoalveolar lavage (BAL), protein extraction from lung for Western blot and immunohistochemistry of 4-hydroxy-2-nonenal (4-HNE), proliferating cell nuclear antigen (PCNA), NF-E2 related factor 2 (Nrf-2), and activity of glutathione were performed at before and each ozone exposure day.
RESULTS
Airway hyper-responsiveness and increased eosinophils in BAL fluid were observed in asthma model. In asthma model, the expression of 4-HNE already more increased at baseline (without ozone) compared to those in sham model. This increased expression is more enhanced at 3 days after ozone exposure. The expression of PCNA was significantly increased in OVA-model compared to those in sham model. The expression of Nrf-2 was observed at baseline, and 3 and 7 days after exposure ozone in asthma model, but not in sham model. The activity of glutathione increased significantly after exposure of ozone, but not in sham model.
CONCLUSION
Murine asthma model has enhanced oxygen toxicity and antioxidant activity response to ozone.

Keyword

Asthma; Oxidants; Antioxidants; Mice; Ozone

MeSH Terms

Air Pollutants
Animals
Antioxidants
Asthma*
Blotting, Western
Bronchoalveolar Lavage
Eosinophils
Glutathione
Humans
Immunohistochemistry
Lung
Methacholine Chloride
Mice
Ovalbumin
Oxidants
Oxygen
Ozone*
Proliferating Cell Nuclear Antigen
Respiratory Hypersensitivity
Air Pollutants
Antioxidants
Glutathione
Methacholine Chloride
Ovalbumin
Oxidants
Oxygen
Ozone
Proliferating Cell Nuclear Antigen

Figure

• 

  Fig. 1 Schematic diagram of the experimental protocol. Mice were sensitized on day 1 and 14 by intraperitoneal injection of ovalbumin (OVA) with aluminum sulfate. On day 21, 22, and 23 after the initial sensitization, the mice were challenged with an aerosol of 1% OVA using ultrasonic nebulizer. The mice housed in whole-body exposure chambers were exposed to ozone concentration of 2 ppm for 3 hours on day 24, 27, 31, 38, and 45 after initial OVA sensitization. ppm, parts per million; Penh, enhanced pause; BAL,bronchoalveolar lavage.

• 

  Fig. 2 Change of airway hyperresponsiveness to methacholine sitmulatoin in ovalbumin (OVA)-induced asthma and sham model. OVA-induced asthma model had a higher methacholine-induced enhanced pause (Penh) compared to the sham model at 25 and 50 mg/mL of methacholine concentration. *p < 0.01 vs. sham.

• 

  Fig. 3 (A) Total cell count and inflammatory cell counts in bronchoalveolar lavage fluid from the ovalbumin (OVA)-induced asthma (blue bar) and sham model (green bar). Total cell count significantly increased in OVA-induced asthma model compared to those in sham model during whole experimental period. (B, C) The numbers of macrophage and lymphocyte showed significantly increased in OVA-induced asthma model compared to those in sham model. (D) Eosinophils significantly increased at basal state, 3 and 7 days after ozone exposure in OVA-induced asthma model compared to those in sham model. (A) *p < 0.01 vs. 7, 14, and 21 days, #p < 0.01 vs. 14 and 21 days, ¶p < 0.01 vs. 21 days, $p < 0.05 vs. control. (B) *p < 0.01 vs. 14 and 21 days, $p < 0.05 vs. control. (C) *p < 0.01 vs. 21 days, #p < 0.05 vs. 14 and 21 days, $p < 0.01 vs. control. (D) *p < 0.01 vs. 7, 14, and 21 days, #p < 0.01 vs. 14 and 21 days, $p < 0.01 vs. control.

• 

  Fig. 4 Western blot analysis of 4-HNE (A), PCNA (B), and Nrf-2 (C) after exposure to ozone in ovalbumin (OVA)-induced asthma and sham-model. (A) In OVA-induced asthma model, the expression of 4-HNE already more increased at baseline compared to those in sham model. This increased expression is more enhanced at 3 days after ozone exposure. At 7 days after ozone exposure the expression decreased to baseline level and at 14 and 21 days after ozone exposure the expression is significantly decreased below the level of baseline. (B) In sham and OVA-induced asthma model, PCNA expressions were not different from baseline to 21 days. But the expression of PCNA was significantly increased in OVA-model compared to those in sham model. (C) In sham model, the expression of Nrf-2 was not observed at baseline and during experimental period. In contrast, in OVA-model, expression of Nrf-2 was observed at baseline, and 3 and 7 days after exposure ozone. OVA; ovalbumin, 4-HNE; 4-hydroxy-2-nonenal, PCNA; proliferative cellular nuclear antigen, Nrf-2; NF-E2-relatd factor 2.

• 

  Fig. 5 Glutathione activity of lung from ovalbumin (OVA)-induced asthma (blue bar) and sham-model (green bar). The activity of glutathione was more increased in OVA-induced asthma model compared to that in sham model. The exposure to ozone significantly increased glutathione activity in OVA-induced sham model. *p < 0.05 vs. sham model, #p < 0.05 vs. 0 day. CDNB, 1-Chloro-2,4-dinitrobenzene.

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