Increased antioxidant activity after exposure of ozone in murine asthma model

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.

The activation of NLRP3-inflammsome by stimulation of diesel exhaust particles in lung tissues from emphysema model and RAW 264.7 cell line

BACKGROUND/AIMS: Diesel exhaust particles (DEPs) lead to elevation of reactive oxygen species, which can activate the nucleotide-binding oligomerization domain-like receptor (NLR) family members containing the pyrin domain 3 (NLRP3)-inf lammasome. In this study, we elucidated whether NLRP3 -inf lammasome is activated by DEPs and whether antioxidants (N-acetylcysteine [NAC]) could inhibit such activation. METHODS: RAW 264.7 cells and ex vivo lung tissues explants obtained from elastase-induced emphysema animal models were stimulated with cigarette smoking extract (CSE), DEPs, and lipopolysaccharide, and levels of interleukin-1β (IL-1β), caspase-1 and nucleotide-binding oligomerization domain-like receptor (NLR) family members containing the pyrin domain (NLRP3)-inflammasome were assessed by Western blotting and immunohistochemistry. RESULTS: NAC and caspase-1 inhibitor suppressed CSE- and DEP-induced secretion of IL-1β in RAW 264.7 cells. The expression levels of the NLRP3-inflammasome and caspase-1 were upregulated in RAW 264.7 cells by stimulation with CSE and DEPs and were inhibited by NAC. CSE and DEPs increased the secretion of IL-1β in lung tissues from both the normal and elastase-induced emphysema groups. The secretion of IL-1β by CSE and DEPs was increased in the elastin-induced emphysema group more than that in the normal group (CSE: 309 ± 19 pg/mL vs. 151 ± 13 pg/mL, respectively, p < 0.05; DEP: 350 ± 24 pg/mL vs. 281 ± 15 pg/mL, respectively, p < 0.05). NAC inhibited CSE- and DEP-induced IL-1β secretion in both the normal and elastase-induced emphysema groups. NLRP3-inflammasome expression as determined by immunohistochemistry was increased by CSE and DEPs in both the normal and elastin-induced emphysema groups, and was suppressed by NAC. CONCLUSIONS: The NLRP3-inf lammasome is activated by DEPs in ex vivo tissue explants from elastase-induced emphysema animal model, and this activation is inhibited by NAC.

The Role of c-Jun N-terminal Kinase in the Radiation-Induced Lung Fibrosis / 결핵

BACKGROUND: The undrlying pathogenesis of radiation-induced lung fibrosis (RTLF) has not been very well defined. However, the role of TGF-β in the generation of RTLE has been a major focus because there is an increase in the expression of both the TGE-β stimulated lung fibrosis includes the activation of many mediators such as Smad and c-Jun N-terminal kinase (JNK) through TAK1. It is we hypothesized that JNK activation may play a pivotal role in RTLF pathogensis through increased transcription of the fibrogenic cytokines. The present study evaluates JNK activity in alveolar macrophages after irradiation and the relationship between JNK activity and the amount of collagen in the lung tissues. METHODS: C57BL/6 mice(20-25 gr. males) received cholorotetracycline(2g/L) in their drinking water 1 week prior to irradiation and continuously there after. The mice were irradiated once with 1400 cGy of 60COγ-ray over the whole chest. The cellular composition of the whole lung bronchoalveolar lavage fluids(BALF), elastin expression in the lung tissues, the level of hydroxyproline in lung tissues, and an in vitro JNK assay was measured before irradiation and one, four, and eight weeks after irradiation (RT). RESULTS: The volumes of BALF retrieved from instilled 4ml of saline with 2% heparin were 3.7-3.8ml for each group. The cell numbers were similar before(4.1×10(4)±0.5±10(4)/ml) and 1 week(3.1×10(4)±0.5±10(4)/ml) after RT. At four and eight weeks after RT, the cell number reached to 14.0×10(4)±1.5±10(4)/ml and 10.0×10(4)±1.3±10(4)/ml, respectively. There we no changes in the lymphocytes and neutrophils population obseved in the BALF after RT. The H-E stain of the lung tissues did not show any structural and fibrotic change in the lung tissues at 4 and 8 weeks after RT. In addition, the amount of elastin and collagen were not different on Verhoeff staining of the lung tissues before RT to eight weeks after RT. The hydroxyproine content was measured with the left lung dissected from the left main bronchus. The lung were homogenized and hydrolyzed with 6 N HCI for 12 hours at 110℃ then measured as previously described. The content of hydroxyproline, standardized with a lung protein concentration, reached a peak 4 weeks after RT. and thereafter showed a plateau. An In vitro JNK assay using c-Jun(1-79)-GST sepharose beads were performed with the alveolar macrophages obtained from the BAL. JNK activity was not detected prior to RT, However, the JNk activity increased from one week after RT and reached a peak four weeks after RT. CONCLUSION: JNK may be involved in the pathogensis because the JNK activity showed similar pattern observed with the hydroxyproine content. However, it is necessary to clarify that the JNK increases the transcription of fibrogenic cytokines through the transcription factor.

The Activity of c-Jun N-terminal Kinase (JNKb) in Patients with UIP / 결핵

BACKGROUND: TNF-alpha is related to the generation of lung fibrosis in patients with UIP. The precise mechanism leading to lung fibrosis by TNF-alpha is unknown. However, the activation of a transcription factor like AP-1(down stream of c-jun N-terminal kinase, JNK) by TNF-alpha may be related to the induction of fibrogenic cytokines like PDGF or IGF-I. Furthermore, JNK was reported to be activated in the radiation-in-duced lung fibrosis model. This study examined JNK activity in patients with UIP. METHODS: The expression of phosphorous JNK(p-JNK), macrophage/moncoyte specific markers, CD68, and cytokeratin was evaluated by immunohistochemical (IHC) staining of lung tissues from patients with UIP and lung cancer. An in vitro kinase assay was performed with alveolar macrophages obtained by a bronchollung cancer. An in vitro kinase assay was performed with alvolar macrophages obrtained by a bronchol avleolar lavage from patients with UIP and healthy persons as the control. RESULTS: The IHC stain showed that p-JNK is expressed in the almost all of the alveolar macrophages and smooth muscle cells in patients with UIP. In case of the normal areas of the lung from patients with lung cancer, the alveolar macrophages showed little p-JNK expression. Interestingly, increased JNK activity was not found in the in vitro kinase assay of the alveolar macrophages obtained from both patients with UIP and healthy persons as the control. Furthermore, 10 ng/ml of TNF-alpha failed to increase the JNK activity of the alveolar macrophages in both patients with UIP and healthy people. CONCLUSION: The JNK was activated constitutionally in patients with UIP. However, the role of JNK in the pathogenesis of lung fibrosis needs to be clarified.