Nrf2 expression is increased in peripheral blood mononuclear cells derived from mild–moderate ex-smoker COPD patients with persistent oxidative stress

Figures & data

Table 1 GOLD stage, lung function, and medical history of non-COPD and COPD subjects

	Non-COPD N=30	COPD N=30	P-value
Age (years)	67.8±6.8	69.3±6.6	NS
Sex (M/F)	24/6	23/7	NS
Past smoking exposure (pack-years)	27.8±36.6	34.2±26	NS
Duration of smoking (years)	23.8±11.2	27.5±12.4	NS
GOLD 1 n (%)	0	16 (53.3%)
GOLD 2 n (%)	0	14 (46.7%)
FEV1 (L)	2.98±0.7	1.95±0.7	0.001
FEV1 (% of predicted)	105.0±16.8	75.06±20.2	0.001
FEV1/FVC	75.3±4.5	58.0±8.4	0.001
SpO2 (%)	97.5±1.4	95.3±2.6	0.01
Hypertension (%)	70	76.7	NS
Type 2 diabetes mellitus (%)	20	23.3	NS
Dyslipidemia (%)	83.3	73.3	NS
Antihypertensive drug use (%)	41.4	66.6	0.01
Oral antidiabetic drug use (%)	13.3	13.3	NS
Statin use (%)	20	40	0.01

Note: Data are expressed as mean ± SD unless otherwise stated.

Abbreviations: COPD, chronic obstructive pulmonary disease; F, female; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; GOLD, Global initiative for chronic Obstructive Lung Disease; M, male; NS, not significant; SD, standard deviation; SpO₂, percutaneous oxygen saturation.

Table 2 Clinical and laboratory data of non-COPD and COPD subjects

Clinical and laboratory data	Non-COPD N=30	COPD N=30	P-value
Waist circumference (cm)	96.1±11.8	101.3±12.2	NS
BMI (kg/m^2)	27.6±4.8	28.3±4.3	NS
SBP (mmHg)	140.6±20.7	130.2±16.9	0.01
DBP (mmHg)	85.1±9.5	80.3±10.6	NS
Total cholesterol (mmol/L)	5.48±0.97	4.87±1.02	0.01
LDL cholesterol (mmol/L)	3.56±0.95	2.89±0.78	0.01
HDL cholesterol (mmol/L)	1.47±0.42	1.34±0.32	NS
Triglycerides (mmol/L)	1.27±0.67	1.48±0.7	NS
Plasma glucose (mmol/L)	5.64±1.45	5.79±1.12	NS
hsCRP (mg/L)	1.07±0.51	2.72±1.32	0.01
White blood count (mm^3)	6,160±1,500	7,199±1,940	0.02

Note: Data are expressed as mean ± SD.

Abbreviations: BMI, body mass index; COPD, chronic obstructive pulmonary disease; DBP, diastolic blood pressure; HDL, high-density lipoprotein; hsCRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein; NS, not significant; SBP, systolic blood pressure; SD, standard deviation.

Figure 1 Nrf2 and HO-1 mRNA and protein expression in PBMC of non-COPD and COPD subjects.

Notes: (A) mRNA expression of Nrf2 and HO-1. 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<0.001 versus non-COPD. (B) Representative Western blot analysis for the indicated proteins and average quantification obtained by densitometric analysis of all the samples derived from non-COPD and COPD subjects. Row 1, target protein; row 2, β-actin; row 3, lamin B. Data are expressed as mean ± SD; *P<0.001 versus non-COPD.

Abbreviations: COPD, chronic obstructive pulmonary disease; HO-1, heme oxygenase-1; mRNA, messenger RNA; Nrf2, nuclear factor-E2-related factor 2; PBMC, peripheral blood mononuclear cells; PCR, polymerase chain reaction; SD, standard deviation.

Figure 2 UPR mRNA and protein expression in PBMC of non-COPD and COPD subjects.

Notes: (A) mRNA expression of BiP, ATF6, PERK, IRE1, and CHOP. 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. (B) Representative Western blot analysis for the indicated proteins (BiP, ATF6, PERK, IRE1, and CHOP) and the average quantification obtained by densitometric analysis of all the samples derived from non-COPD and COPD subjects. Row 1, target protein; row 2, β-actin. Data are expressed as mean ± SD.
Abbreviations: ATF6, activating transcription factor 6; BiP, binding immunoglobulin protein; CHOP, CCAAT/enhancer-binding protein homologous protein; COPD, chronic obstructive pulmonary disease; IRE1, inositol-requiring kinase 1; mRNA, messenger RNA; PBMC, peripheral blood mononuclear cells; PCR, polymerase chain reaction; PERK, protein kinase-like ER kinase; SD, standard deviation; UPR, unfolded protein response.

Figure 3 MDA and GSH plasma concentrations in non-COPD and COPD subjects.

Notes: Data are expressed as mean ± SD; *P<0.01 versus non-COPD.
Abbreviations: COPD, chronic obstructive pulmonary disease; GSH, glutathione; MDA, malondialdehyde; SD, standard deviation.

Figure 4 oxPAPC (PGPC and POVPC) concentrations in PBMC of non-COPD and COPD subjects.

Notes: Data represent the mean ± SD of measurements performed in triplicate in all subjects; *P<0.01 versus non-COPD.

Abbreviations: COPD, chronic obstructive pulmonary disease; oxPAPC, oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine; PBMC, peripheral blood mononuclear cells; PGPC, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine; POVPC, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine; SD, standard deviation.

Figure 5 p47phox mRNA and protein expression in PBMC of non-COPD and COPD and correlations between p47phox mRNA expression and PGPC and POVPC concentrations in PBMC of all subjects.

Notes: (A) mRNA expression of p47phox. 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<0.001 versus non-COPD. (B) Representative Western blot analysis for p47phox protein and the average quantification obtained by densitometric analysis of all the samples derived from non-COPD and COPD subjects. Data are expressed as mean ± SD; *P<0.001 versus non-COPD. (C) Correlation between p47phox mRNA expression and PGPC concentrations in PBMC of all non-COPD and COPD subjects. (D) Correlation between p47phox mRNA expression and POVPC concentrations in PBMC of all non-COPD and COPD subjects.

Abbreviations: COPD, chronic obstructive pulmonary disease; mRNA, messenger RNA; PAPC, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine; PBMC, peripheral blood mononuclear cells; PGPC, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine; POVPC, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine; SD, standard deviation.

Figure 6 Correlation between p47phox and Nrf2 mRNA expression in PBMC of all non-COPD and COPD subjects.

Abbreviations: COPD, chronic obstructive pulmonary disease; mRNA, messenger RNA; Nrf2, nuclear factor-E2-related factor 2; PBMC, peripheral blood mononuclear cells.

Figure 7 Correlations between lung function (expressed as FEV₁ % predicted) and circulating markers of oxidative stress and antioxidant mRNA expression in all non-COPD and COPD subjects.

Notes: (A) Correlation between FEV₁ and MDA plasma concentration in all non-COPD and COPD subjects. (B) Correlation between FEV₁ and GSH plasma concentration in all non-COPD and COPD subjects. (C) Correlation between FEV₁ and Nrf2 mRNA expression in PBMC of all non-COPD and COPD subjects. (D) Correlation between FEV₁ and HO-1 mRNA expression in PBMC of all non-COPD and COPD subjects.
Abbreviations: COPD, chronic obstructive pulmonary disease; FEV₁, forced expiratory volume in 1 second; GSH, glutathione; HO-1, heme oxygenase-1; MDA, malondialdehyde; mRNA, messenger RNA; Nrf2, nuclear factor-E2-related factor 2; PBMC, peripheral blood mononuclear cells; pred, predicted.