Roles Of PPARγ And PGC-1α Act Cooperatively To Regulate Nrf2 And γ-GCS In Chronic Obstructive Pulmonary Disease

【Objective】To evaluate the expression patterns and their relationship of PPARγ,PGC-1α,Nrf2 andγ-GCS-HS in lungs of rats and patients with COPD,to investigate effects of PPARγand its agonist RGZ,PGC-1αon gene expression of Nrf2 andγ-GCS-HS, and its role in the pathophysiological course of chronic obstructive pulmonary disease,and provide a new theoretical basis for the prevention and treatment of COPD【Methods】The study was composed of two parts: animal experiment and clinical trial. (1)Thirty six adult male Sprague- Dawley(SD) rats were randomly divided into control group,COPD model and RGZ-treated group with twelve rats in each group. The rat COPD model was established by intratracheal instillation of lipopolysaccharide twice and exposed to cigarette smoke daily. The lung function measurements were carried out , and the pathological changes were observed. mRNAs expression of PPARγ,PGC-1α,Nrf2,γ-GCS-HS in lung tissue of rats was measured by in site hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR) . Proteins expression of PPARγ,PGC-1α,Nrf2,γ-GCS-HS were observed by immunohistochemistry(IH) and western blot.(2) Clinical peripheral lung tissue samples were obtained from forty patients undergoing resection for lung cancer, and all patients were were measured lung function prior to they were operated.Those patients were divided into four groups: controls, Mild COPD group,Moderate COPD group,Severe COPD group. COPD classification based on the Chinese Medical Association established standards in 2007. mRNA expression of PPARγ,PGC-1α,Nrf2,γ-GCS-HS in clinical lung tissue was measured by in site hybridization (ISH) . Protein expression of PPARγ,PGC-1α,Nrf2,γ-GCS-HS in clinical lung tissue were observed by immunohistochemistry(IH). 【Results】1. Animal experiments:(1)The results of rats pulmonary function showed: forced expiratory volume in first 0. 3 second(FEV0.3),percentage of forced expiratory volume in first 0. 3 second to forced vital capacity (FEV0.3/FVC%) and peak expiratory flow (PEF) were all obviously decreased in COPD group compared with control group (P<0.01) and which were all significantly improved in RGZ-treated group compared with COPD model group (all P < 0.01).Lung pathological changes in COPD model group conformed morphological character of COPD,however, pathological changes of rats lung tissue in RGZ-treated group markedly reduced compared with COPD model group.(2)In COPD model group, ROS levels was markedly raised compared to controls(P<0.01), whereas in RGZ-treated group, ROS levels was remarkably reduced compared to COPD model group(P<0.01).γ-GCS-HS activity assay showed that, in COPD model group and RGZ-treated group,γ-GCS-HS activity was all observably increased compared to controls(all P<0.01), and which was further increased in RGZ-treated group compared to COPD model group(P<0.01).(3)In situ hybridization showed that the position of PPARγ,PGC-1α,Nrf2 andγ-GCS-HS mRNAs expression are basically consistent in the lung tissue of three group rats, which were mainly located in alveolar epithelial cells,bronchial epithelial cells,bronchial smooth muscle cells and part of the vascular smooth muscle cells. proteins expression of PPARγ,PGC-1α,Nrf2 was mainly found in the nucleus of alveolar epithelial cells and bronchial epithelial cells of rats in each group, while, staining ofγ-GCS-HS proteins was found not only in the nucleus but also in the cytoplasm of alveolar epithelial cells and bronchial epithelial cells of rats in each group(4)PPARγ,PGC-1αandγ-GCS-HS mRNAs in rat lung tissues were significantly higher in COPD model group than those in controls(P<0.01), and those were further elevated in RGZ-treated group than those in COPD model group(P<0.05). mRNA expression of Nrf2 were positive in COPD model group and control group,and there were no significant difference between the two groups(P>0.05),while mRNA expression of Nrf2 were strongly positive in RGZ-treated group,and there were significant differences compared with COPD model group and control group(P<0.01).(5)Proteins expression of PPARγ,PGC-1αandγ-GCS-HS in rat lung tissues were significantly higher in COPD model group and RGZ-treated group than those in controls(P<0.01), and those were manifestly increased in RGZ-treated group compared with COPD model group(P<0.05).(6)Linear correlation analysis showed that proteins expression of PPARγ,PGC-1αwere positively correlated with Nrf2 mRNA and protein(all P<0.01), proteins expression of PGC-1αwas positively correlated with ROS contents(P<0.01), and expression of PPARγ,Nrf2 were no significantly correlated with ROS contents(P>0.05). proteins expression of PPARγ,PGC-1α,Nrf2 were positively correlated withγ-GCS-HS activity,mRNA and protein (all P<0.01).2. Clinical experiments:(1)mRNAs expression of PPARγ,PGC-1α,γ-GCS-HS were strongly positive in lungs of patients with Mild COPD, and which were markedly increased compared with normal control subjects(all P<0.01), and showed a progressive decrease in lungs of patients with Moderate and Severe COPD,and were obviously decreased compared with normal control subjects and patients with Mild COPD(all P<0.01). mRNA expression of Nrf2 Showed weakly positive in lungs of each group patients with COPD and normal control subjects,and there are no significant differences between groups(P>0.05).(2)Proteins expression of PPARγ,PGC-1α,Nrf2,γ-GCS-HS were strongly positive in lungs of patients with Mild COPD, and which were markedly increased compared with normal control subjects(all P<0.01), and showed a progressive decrease in lungs of patients with Moderate and Severe COPD, and which were obviously decreased compared with normal control subjects and patients with Mild COPD(all P<0.01).(3)Linear correlation analysis showed proteins expression of PPARγ,PGC-1α,Nrf2,γ-GCS-HS were positively correlated with FEV1(%)(all P<0.01),were no significantly correlated with FEV1/FVC(%)(P>0.05). Proteins expression of PPARγ,PGC-1αwere positively correlated with Nrf2 protein,γ-GCS-HS mRNA and protein(all P<0.01), and were no significantly correlated with Nrf2 mRNA(P>0.05).【Conclusions】1. Antioxidant enzymeγ-GCS may be compensatory up-regulate to against oxidative stress in the early stage of COPD, but which maybe a progressive decompensation with increasing COPD severity.2. Activation of the PPARγand PGC-1αpathway may reduce the extent of COPD oxidant/antioxidant imbalance , and they both may protect against COPD progression by up-regulatingγ-GCS enzyme activity and gene expression as well as relieving ROS levels.3. In the pathogenesis of COPD, oxidative stess may mainly induce expression of Nrf2 protein,however,activation of the PPARγand PGC-1αpathway may up-regulatingγ-GCS to increase GSH synthesis against oxidative stress by influencing levels of Nrf2 mRNA and protein.