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Cigarette smoke-induced cerebral cortical interleukin-6 elevation is not mediated through oxidative stress.

Lau WK, Mak JC, Chan KH, Law AC - Neurotox Res (2011)

Bottom Line: Our results demonstrated a significant increase in both IL-6 mRNA and protein levels.There were no significant changes in the cerebral cortical levels of MDA, AOPP, catalase activity, and the GSH/GSSG ratio.Nitrite level was significantly reduced, together with the decreased protein level of nNOS in the cerebral cortex, after 56 days of CS exposure.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Medicine, Department of Medicine, The University of Hong Kong, Hong Kong SAR, China.

ABSTRACT
The author group has previously established an in vivo subchronic cigarette smoke (CS) exposure rat model, in which the systemic oxidative burden as well as the modulation of local anti-oxidative enzymes in the lung has been demonstrated. Oxidative stress has been shown to induce pro-inflammatory cytokine release, including interleukin (IL)-6 in the airways. In this study, we aimed to investigate the changes in IL-6 production, as well as the oxidative/anti-oxidative responses in the cerebral cortex using the same in vivo model. IL-6 was determined by RT-PCR and western-blot analysis. Local oxidative and anti-oxidative responses were determined by measuring cerebral cortical malondialdehyde (MDA) and advanced oxidation protein product (AOPP) levels, superoxide dismutase (SOD) and catalase activities, and the reduced to oxidized glutathione (GSH/GSSG) ratio. Nitrite level was measured by fluorescent spectrophotometry. Our results demonstrated a significant increase in both IL-6 mRNA and protein levels. Reductions of SOD activity and manganese (Mn)SOD protein level were observed together with the increased level of superoxide measured by chemiluminescent signal, after 56 days of CS exposure. There were no significant changes in the cerebral cortical levels of MDA, AOPP, catalase activity, and the GSH/GSSG ratio. Nitrite level was significantly reduced, together with the decreased protein level of nNOS in the cerebral cortex, after 56 days of CS exposure. Our results suggest that exposure to CS induces IL-6 expression in the cerebral cortex, which is not mediated by the oxidative/anti-oxidative imbalance.

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Changes in nNOS, MnSOD protein levels, and superoxide levels after 56 days of CS exposure. a Protein levels of nNOS and MnSOD, with β-actin serving as an internal control. b, c Quantitative analysis of band intensity from the blots. Results are expressed as fold of control. d Superoxide level measured in cerebral cortex. Values represent the mean ± SEM. *p < 0.05, **p < 0.01, *** p < 0.001, when comparing between SA and 56 days of CS exposure groups (n ≥ 7)
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Fig4: Changes in nNOS, MnSOD protein levels, and superoxide levels after 56 days of CS exposure. a Protein levels of nNOS and MnSOD, with β-actin serving as an internal control. b, c Quantitative analysis of band intensity from the blots. Results are expressed as fold of control. d Superoxide level measured in cerebral cortex. Values represent the mean ± SEM. *p < 0.05, **p < 0.01, *** p < 0.001, when comparing between SA and 56 days of CS exposure groups (n ≥ 7)

Mentions: We further investigated the changes in different anti-oxidative markers in the cortex. We found that there was a significant reduction of total SOD activity (201.5 ± 82.7 mU/mg protein for control values) after CS exposure, in agreement with the earlier literature (Luchese et al. 2009), but not catalase activity (4.9 ± 0.6 U/mg protein for control values). Furthermore, no significant changes in GSH/GSSG ratio were observed after 56 days CS exposure (Fig. 3c–e). For further investigation of whether the decreased SOD activity would be due to reduction of its protein level, the protein level of manganese (Mn)SOD was determined after CS exposure. Our result demonstrated that CS reduced the protein level of MnSOD (Fig. 4a, c). SOD is the first line of anti-oxidative enzyme that is responsible for the defensive mechanism against ROS and other superoxide anion-free radicals (Zelko et al. 2002). The reduction of SOD activity might lead to the accumulation of superoxide anions in the brain. To confirm this, we examined the superoxide levels in the cerebral cortex after CS exposure by chemiluminescence assay. We found that the superoxide levels were significantly higher in the cortex (5.1 ± 1.9 RLU/μg protein versus 45.4 ± 6.0 RLU/μg protein) after CS exposure (Fig. 4d). The increased superoxide anions level can react with nitrogen species to form peroxynitrite which is a more potent oxidant involved in the nitration process of SOD. Post-translational modifications, including phosphorylation and nitration of SOD, have been associated with the reduced activity of SOD (Yamakura and Kawasaki 2010). The question as to whether these mechanisms are involved in the reduction of SOD activity in our model requires further investigation.Fig. 4


Cigarette smoke-induced cerebral cortical interleukin-6 elevation is not mediated through oxidative stress.

Lau WK, Mak JC, Chan KH, Law AC - Neurotox Res (2011)

Changes in nNOS, MnSOD protein levels, and superoxide levels after 56 days of CS exposure. a Protein levels of nNOS and MnSOD, with β-actin serving as an internal control. b, c Quantitative analysis of band intensity from the blots. Results are expressed as fold of control. d Superoxide level measured in cerebral cortex. Values represent the mean ± SEM. *p < 0.05, **p < 0.01, *** p < 0.001, when comparing between SA and 56 days of CS exposure groups (n ≥ 7)
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Fig4: Changes in nNOS, MnSOD protein levels, and superoxide levels after 56 days of CS exposure. a Protein levels of nNOS and MnSOD, with β-actin serving as an internal control. b, c Quantitative analysis of band intensity from the blots. Results are expressed as fold of control. d Superoxide level measured in cerebral cortex. Values represent the mean ± SEM. *p < 0.05, **p < 0.01, *** p < 0.001, when comparing between SA and 56 days of CS exposure groups (n ≥ 7)
Mentions: We further investigated the changes in different anti-oxidative markers in the cortex. We found that there was a significant reduction of total SOD activity (201.5 ± 82.7 mU/mg protein for control values) after CS exposure, in agreement with the earlier literature (Luchese et al. 2009), but not catalase activity (4.9 ± 0.6 U/mg protein for control values). Furthermore, no significant changes in GSH/GSSG ratio were observed after 56 days CS exposure (Fig. 3c–e). For further investigation of whether the decreased SOD activity would be due to reduction of its protein level, the protein level of manganese (Mn)SOD was determined after CS exposure. Our result demonstrated that CS reduced the protein level of MnSOD (Fig. 4a, c). SOD is the first line of anti-oxidative enzyme that is responsible for the defensive mechanism against ROS and other superoxide anion-free radicals (Zelko et al. 2002). The reduction of SOD activity might lead to the accumulation of superoxide anions in the brain. To confirm this, we examined the superoxide levels in the cerebral cortex after CS exposure by chemiluminescence assay. We found that the superoxide levels were significantly higher in the cortex (5.1 ± 1.9 RLU/μg protein versus 45.4 ± 6.0 RLU/μg protein) after CS exposure (Fig. 4d). The increased superoxide anions level can react with nitrogen species to form peroxynitrite which is a more potent oxidant involved in the nitration process of SOD. Post-translational modifications, including phosphorylation and nitration of SOD, have been associated with the reduced activity of SOD (Yamakura and Kawasaki 2010). The question as to whether these mechanisms are involved in the reduction of SOD activity in our model requires further investigation.Fig. 4

Bottom Line: Our results demonstrated a significant increase in both IL-6 mRNA and protein levels.There were no significant changes in the cerebral cortical levels of MDA, AOPP, catalase activity, and the GSH/GSSG ratio.Nitrite level was significantly reduced, together with the decreased protein level of nNOS in the cerebral cortex, after 56 days of CS exposure.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Medicine, Department of Medicine, The University of Hong Kong, Hong Kong SAR, China.

ABSTRACT
The author group has previously established an in vivo subchronic cigarette smoke (CS) exposure rat model, in which the systemic oxidative burden as well as the modulation of local anti-oxidative enzymes in the lung has been demonstrated. Oxidative stress has been shown to induce pro-inflammatory cytokine release, including interleukin (IL)-6 in the airways. In this study, we aimed to investigate the changes in IL-6 production, as well as the oxidative/anti-oxidative responses in the cerebral cortex using the same in vivo model. IL-6 was determined by RT-PCR and western-blot analysis. Local oxidative and anti-oxidative responses were determined by measuring cerebral cortical malondialdehyde (MDA) and advanced oxidation protein product (AOPP) levels, superoxide dismutase (SOD) and catalase activities, and the reduced to oxidized glutathione (GSH/GSSG) ratio. Nitrite level was measured by fluorescent spectrophotometry. Our results demonstrated a significant increase in both IL-6 mRNA and protein levels. Reductions of SOD activity and manganese (Mn)SOD protein level were observed together with the increased level of superoxide measured by chemiluminescent signal, after 56 days of CS exposure. There were no significant changes in the cerebral cortical levels of MDA, AOPP, catalase activity, and the GSH/GSSG ratio. Nitrite level was significantly reduced, together with the decreased protein level of nNOS in the cerebral cortex, after 56 days of CS exposure. Our results suggest that exposure to CS induces IL-6 expression in the cerebral cortex, which is not mediated by the oxidative/anti-oxidative imbalance.

Show MeSH
Related in: MedlinePlus