A Primary Study Of The Induction Of Formaldehyde On GSNO Reductase And The Mechanism

Asthma is a chronic respiratory disease characterized by airway hyperresponsiveness. In the recent 2 decades, the occurrence and death from asthma are continuing increasing all over the world. It has been generally accepted that asthma is mainly an inflammatory disease, and extensive studies have revealed the immunological basis for the occurrence of asthma and roles of inflammatory factors involved in asthma development. On the other hand, the role of endogenous airway relaxants in the pathogenesis of asthma has received less attention, and the relative importance of impaired airway relaxation versus active constriction is unknown. Recently, Que et al found that following allergen challenge, mice with genetic depletion of GSNO reductase (GSNOR) showed normal airway inflammatory reaction, but were protected from airway hyperresponsiveness, companioned with increase of S-nitrosothiols (SNOs), the substance of GSNOR. In contrast, wild type mice exhibiting airway hyperresponsivity have increased airway levels of the enzyme GSNOR and are depleted of lung SNOs. This work suggested that SNOs may serve to protect airway and this function is diminished in asthmatic airway because of the abnormally elevated GSNOR activity. As the regulating enzyme of SNOs, GSNOR may have a pivotal in asthma occurrence and may be a new target enzyme in asthma cure. However, the molecular regulation of GSNOR in airway is not clear.In recent years, the relationship between indoor airway pollutant formaldehyde and human health effect is extensively studied. Formaldehyde exposure is considered to be related with asthma risk, and animal studied revealed that formaldehyde exposure could alter airway smooth muscle activity. However, the molecular mechanism is poorly understood. Regarding the significance of GSNOR in protecting airway hyperresponsiveness, we hypothesized that formaldehyde exposure may up-regulate GSNOR activity, which may be an important mechanism in formaldehyde induced asthma. Meanwhile, since formaldehyde exposure is related with the elevation of oxidative stress in biological system, and asthma is a disease closely related with the increase of oxidative stress, we further hypothesized that formaldehyde induced GSNOR elevation in the airway is related with the increase of oxidative stress.We have conducted in vivo and in vitro studies to reveal the induction of GSNOR by formaldehyde exposure and the relationship with the increase of oxidative stress to testify our hypothesis, and results were as followings: 1 Up-regulation of GSNOR in mice airway by formaldehyde inhalationTo address whether gaseous formaldehyde could up-regulate GSNOR in mice airway, male Kun Ming mice were divided into 3 testing groups (n=6 each) placed in air chamber containing formaldehyde (FA) of 0, 1.0, 3.0 mg/m³ for inhale experiments, respectively. The inhaled groups were exposed to gaseous FA continuously for 72h, 3d. Immediately after exposure, GSNOR RT-PCR and enzyme activity assay were performed in lungs of mice. Results showed that GSNOR is expressed in lungs of mice, and GSNOR expression was significantly up regulated in lungs of 3.0 mg/m³ FA inhaled group (compared with 0 mg/m³ FA inhaled group, p<0.05). Consistent with RT-PCR results, enzyme activity assay showed that GSNOR activity increased in a dose-dependent manner, and the reductase activity increased significantly in lungs both of 1.0 mg/m³ FA inhaled group and 3.0 mg/m³ FA inhaled group (compared with 0 mg/m³ FA inhaled group, p<0.01). The results indicated that gaseous FA could activate GSNOR in lungs, which may be important mechanism in formaldehyde induced asthma occurrence.2. Up-regulation of GSNOR by Gaseous Formaldehyde via GSH pathwayIt has been suggested that asthma is a disease related with oxidative stress. Oxidative stress is significantly elevated in asthmatic airways, and reduced glutathione (GSH) is significantly decreased. Formaldehyde exposure could induce the increase of oxidative stress in organisms, to further investigate whether the up-regulation of GSNOR by gaseous formaldehyde is associated with the changes of GSH levels, GSNOR activity and GSH levels were measured in bronchoalveolar lavage of mice after the exposure to 0, 3.0 mg/m³ formaldehyde and mice that were intraperitoneally injected withα-lipoic acid. Results showed that GSNOR activity was significantly elevated in 3.0 mg/m³ formaldehyde inhaled mice, accompanied with the significantly decrease of GSH levels (compared with 0 mg/m³ formaldehyde inhaled mice); whileα-lipoic acid injection resulted in a significantly decrease of GSNOR activity and increase of GSH levels in formaldehyde inhaled mice. There was a negative relationship between GSNOR activity and GSH levels. These results suggested that regulation of GSNOR by gaseous formaldehyde is via GSH pathway. Formaldehyde might up-regulate GSNOR by inducing oxidative stress, which may provide a basis for the understanding the regulation of GSNOR in the airways. 3. Effects of injection of antioxidant on the transcription of GSNOR geneRecent studies showed that cellular response toward oxidative and nitrosative stress is mainly regulated at the transcriptional level. The sensing of oxidative and nitrosative stress is one of the basic cellular activities, and has important role in cell survival and cellular physiology. In order to test the hypothesis that formaldehyde and redox status in the airway could regulate GSNOR expression at the transcription level, effects of environmental formaldehyde exposure and antioxidantα-lipoic acid injection on GSNORexpression in mice lungs was investigated in the present study. Kunming purebred mice were used as experimental materials and the total RNA was extracted from the lungs immediately after exposed to formaldehyde and injection byα-lipoic acid. RT-PCR was applied to examine the transcriptional changes of GSNOR andβ-actin which was used as control gene. The results showed that compared with control groups, 3.0 mg/m³ formaldehyde exposure resulted in significant increase of GSNOR expression (p<0.01), and a-lipoic acid injection completely blocked the increase. These results suggested that inhaled gaseous formaldehyde could induce up-regulate GSNOR at transcription level, and the changes may be related to the altered redox status in lungs, which might provide a basis for the understanding of GSNOR expression in lungs.4. Effect of formaldehyde and H₂O₂ on GSNOR activity of Hela cellsTo explore the effects of formaldehyde on the activity of GSNO reductase (GSNOR) and the association of oxidative stress, Hela Cells were exposed to different concentrations of liquid formaldehyde and 100μmol/L H₂O₂ for 1h, and GSNOR activity was examined in the cellular lysates. The results showed that formaldehyde could up-regulate GSNOR in a dose-dependent manner, resulting in elevated GSNOR activity with the increase of formaldehyde concentrations, and the increase was statistically significant when the concentration of formaldehyde was up to 1250μmol/L (compared with 0μmol/L, p<0.05). Meanwhile, 100μmol/L H₂O₂ also had similar effect on GSNOR activity. These results suggested that formaldehyde could activate GSNOR in mammalian cells, and the activation might be associated with oxidative stress.