| [Objective] To investigate the expressions of NF-E2-related factor 2(Nrf2),BTB and CNC homology 1 (Bach1),Keap1(Kelch-like ECH associating protein 1),PERK(PKR-like endoplasmic reticulum kinase) andγ-glutamylcysteine synthetase(γ-GCS) in pulmonary tissues of rats with chronic obstructive pulmonary disease,to elucidate the possible important role ofγ-GCS and Nrf2,Bach1,Keap1,P-PERK in pathogenesis of COPD, and provide theoretical basis and new remedy method for COPD.[Methods] The study was composed of two parts: animal experiment and clinical trial.Part1: Sixteen male SD rats were randomly divided into COPD model and control group. The rat COPD model was established by intratracheal instillation of lipopolysaccharide (LPS200μg/200μL) twice and exposed to cigarette smoke daily. The lung function measurements FEV0.3 and FEV0.3/FVC% were carried out .The pathological changes were also observed. The levels ofγ-GCS,Nrf2,Bach1 and Keap1mRNA expression in lung tissue was measured by in site hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR) . The protein expression ofγ-GCS and Nrf2,Bach1,Keap1,P-PERK were observed by immunohistochemistry(IH) and western blot.Part2: Lung tissues from eighteen patients undergoing resection for lung disease were drawn,for study,and the expression of Nrf2,Bach1,γ-GCS protein by IH and mRNA by ISH.[Results]1. FEV0.3 and FEV0.3/FVC% were decreasing significantly in COPD group compared with control group( all P<0.05) , implied airflow obstruction, The pathological findings were consistent with chronic bronchitis and obstructive emphysema.2. The ISH showed that the levels ofγ-GCS and Nrf2mRNA expression in the lung tissue of COPD group were significantly higher than that in the control group(P <0.05),but the levels of Bach1 mRNA in COPD group and control group were stable. RT-PCR analysis showed that the expression ofγ-GCS mRNA was higher in lung of rats in COPD group than in control group (P <0.05).3. As to immunohistochemical (IH), the levels of Nrf2,P-PERK andγ-GCS protein in COPD group were significantly higher than those in control group (P<0.01); while the expression of Bach1 and Keap1 protein in COPD group had no significant difference compared with the control group (P>0.05).4. Nuclear/cytosol fractionation and western blot assay demonstrated that Nrf2 protein was mainly expressed in cytoplasm in control group. But Nrf2 protein was mainly expressed in nucleus in COPD group. Bach1 protein of cytoplasm was mainly detected in COPD group. Keap1 protein were mainly detected in cytoplasm in both groups. Furthermore, the protein expressions of Nrf2 and P-PERK were significantly higher in COPD group as compared to control group (P<0.01); the protein expressions of Bach1 and Keap1 in both groups were stable (P>0.05).5.ISH analysis showed that the expressions ofγ-GCS and Nrf2 mRNA were higher in lung of patients in COPD group than those in control group (P<0.05),there was no difference of Bach1 protein when COPD group was compared to control group(P>0.05). IH assay demonstrated that the expressions ofγ-GCS and Nrf2 protein in COPD group were strongly increased as compared to control group (P<0.01), the expression of Bach1 protein in two groups was stable.5. By linear correlation analysis, we found there was positive correlation between Nrf2 expressed in nucleus andγ-GCS.P-PERK protein was positively correlated with Nrf2 andγ-GCS.But there were negative correlation between Bach1 protein/Nrf2 protein in nucler and FEV0.3,FEV0.3/FVC%,γ-GCSmRNA,γ-GCS protein.[Conclusions]1. The level of Bach1 and Nrf2 protein in nucleus up-regulate or down-regulate the expression ofγ-GCS in COPD.2. The Keap1 protein mainly expressed in cytoplasm and may influence the stability of Nrf2 protein.3. PERK may play an important role in the mechanism of COPD oxidative stress vis activating of Nrf2 regulation ofγ-GCS. |
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