Oxidative stress and Nrf2 expression in peripheral blood mononuclear cells derived from COPD patients: an observational longitudinal study

Abstract

Background: A persistent low inflammatory-oxidative status and the inadequacy of the antioxidant nuclear factor-E2-related factor 2 (Nrf2) have been implicated in chronic obstructive pulmonary disease (COPD) progression. Therefore this study was aimed to assess the association between lung function decline and oxidative-inflammatory markers and Nrf2 signaling pathway expression in peripheral blood mononuclear cells (PBMCs) over time.

Methods: 33 mild-moderate COPD outpatients (mean age 66.9 ± 6.9 years) were age-sex matched with 37 no-COPD subjects. A clinical evaluation, blood sampling tests and a spirometry were performed at baseline and after a mean follow-up of 49.7 ± 6.9 months.

Results: In COPD, compared to no-COPD, we found a faster lung function decline at follow-up. Although similar prevalence of smoking, hypertension, diabetes and dyslipidemia, systemic markers of inflammation (hs-CRP and white blood cells, WBCs) and oxidative stress (8-isoprostane) were significantly increased in COPD at follow-up, while the antioxidant glutathione (GSH) was significantly reduced. Moreover the expression of Nrf2 and of Nrf2-related genes heme oxygenase (HO)-1 and glutamate-cysteine ligase catalytic (GCLC) subunit in PBMCS were significantly down-regulated in COPD at follow-up, whereas no changes were observed in no-COPD. The percent variation (Δ) of FEV₁ detected after the follow-up in COPD patients was directly correlated with ΔNrf2 (r = 0.826 p < 0.001), ΔHO-1 (r = 0.820, p < 0.001) and ΔGCLC (r = 0.840, p < 0.001). Moreover ΔFEV₁ was also directly correlated with ΔGSH (r = 0.595, p < 0.01) and inversely correlated with Δ8-iso (r = - 0.587, p < 0.01) and with baseline smoking history (r = - 0.39, p < 0.03). No correlation was found between ΔFEV₁, ΔCRP and ΔWBCs. By means of hierarchical stepwise multiple linear regression, taking into account other baseline key factors related to FEV₁, ΔNrf2, ΔHO-1and ΔGCLC were found to be significant predictors of ΔFEV₁, explaining 89.5% of its variance.

Conclusions: Although our results must be confirmed in larger trial they suggest that the down-regulation of Nrf2/ARE gene expression in PBMCs may be one of the determinants of FEV₁ decline and of COPD progression. Therefore the future possibility to counteract Nrf2 decline in COPD patients may help in reducing the negative effects of the oxidative stress-induced progression of the disease.

Keywords: COPD progression; Nrf2/ARE gene expression; Oxidative stress; PBMCs.

Fig. 1

Detailed diagram of participant flow and group allocation

Fig. 2

Annual lung function decline in COPD and no-COPD groups after the period of follow-up. Data are expressed as mean ± SD and represent FEV₁ annual variations (in ml/year and %/ year) between baseline and follow-up values. *p < 0.001 vs no-COPD. Abbreviations: COPD, chronic obstructive pulmonary disease; FEV₁: Forced expiratory volume in 1st second

Fig. 3

Circulating markers of inflammation and oxidative stress in COPD and no-COPD groups at baseline and at follow-up. a High-sensitivity C-reactive protein (CRP) plasma concentrations. b White blood cells (WBCs) count. c 8-isoprostane (8-iso) plasma concentrations. d Glutathione (GSH) plasma concentrations. Data are expressed as mean ± SD. *p < 0.01 vs no-COPD baseline or follow-up. **p < 0.01 vs baseline

Fig. 4

Nrf2/ARE mRNA expression in PBMCs derived from both groups at baseline and at follow-up. mRNA was analyzed by quantitative real-time PCR; normalized gene expression levels are given as the ratio between the mean value for the target gene and β-actin in each sample. Data are expressed as mean ± SD; *p < 0.01 versus no-COPD. **p < 0.01 vs baseline. Abbreviations: GCLC, glutamate-cysteine ligase catalytic; HO-1, heme oxygenase-1; mRNA, messenger RNA; Nrf2, nuclear factor-E2-related factor 2; PBMCs, peripheral blood mononuclear cells; PCR, polymerase chain reaction. a Nrf2 mRNA expression. b HO-1 mRNA expression. c GCLC mRNA expression

Fig. 5

Correlations between FEV₁ percent predicted (% pred) and Nrf2 and HO-1 gene expression in both groups of subjects at baseline. a Correlation between FEV₁ and Nrf2 mRNA expression in PBMCs. b Correlation between FEV₁ and HO-1 mRNA expression in PBMCs. Abbreviations: HO-1, heme oxygenase-1; mRNA, messenger RNA; Nrf2, nuclear factor-E2-related factor 2; PBMCs, peripheral blood mononuclear cells

Fig. 6

Correlations between FEV₁ percent variation (Δ) and Δ Nrf2/ARE gene expression in COPD patients. a Correlation between FEV₁ and Nrf2 mRNA expression in PBMCs. b Correlation between FEV₁ and HO-1 mRNA expression in PBMCs. c Correlation between FEV₁ and GCLC mRNA expression in PBMCs. Abbreviations: GCLC, glutamate-cysteine ligase catalytic; HO-1, heme oxygenase-1; mRNA, messenger RNA; Nrf2, nuclear factor-E2-related factor 2; PBMCs, peripheral blood mononuclear cells