Physical Exercise Reduces Cytotoxicity and Up-Regulates Nrf2 and UPR Expression in Circulating Cells of Peripheral Artery Disease Patients: An Hypoxic Adaptation?

Abstract

Aim: Ischemia-reperfusion (I-R) produces reactive oxygen species (ROS) that damage cells and favour cytotoxicity and apoptosis in peripheral artery disease (PAD) patients. Since brief episodes of I-R (ischemic conditioning) protect cells against ischemic harms, we evaluated whether a short-course of supervised treadmill training, characterized by repeated episodes of I-R, makes peripheral blood mononuclear cells (PBMCs) from PAD patients with intermittent claudication more resistant to I-R injuries by reducing oxidative stress and by inducing an adaptative response of unfolded protein response (UPR) and nuclear factor-E2-related factor (Nrf2) pathway expression.

Methods: 24 PAD patients underwent 21 sessions of treadmill training and a treadmill test as indicator of acute response to I-R.

Results: Maximal and pain free walking distance improved (p＜0.01), whereas LDH leakage and apoptosis of PBMCs decreased (p＜0.01); plasma malondialdehyde and ROS generation in PBMCs declined, while plasma glutathione augmented (p＜0.01). Moreover we demonstrated an up-regulation of UPR and Nrf2 expression in PBMCs (p＜0.01). To understand whether treadmill training may act as a trigger of ischemic conditioning, we examined the effect of repeated episodes of I-R on adaptative response in PBMCs derived from the patients. We showed an up-regulation of UPR and Nrf2 gene expression (p＜0.01), while oxidative stress and cytotoxicity, after an initial increase, declined (p＜0.01). This positive effect on cytotoxicity was reduced after inhibition of UPR and Nrf2 pathways.

Conclusions: Treadmill training in PAD patients through UPR and Nrf2 up-regulation may trigger hypoxic adaptation similar to conditioning, thus modifying cell survival.

Keywords: Nrf2; Oxidative stress; PAD; UPR.

Fig. 1.

Effect of supervised physical training on cytotoxicity in PBMCs derived from PAD patients, and correlation between the changes (delta) of maximal walking distance (MWD) with those of LDH leakage. (a) LDH leakage and apoptosis of PBMCs, (b) correlation between the changes of MWD with those of LDH leakage. Data are expressed as mean ± SD; *p < 0.01 vs start.

Fig. 2.

Effect of supervised physical training on reactive oxygen species (ROS) generation in PBMCs derived from PAD patients and on plasma malondialdehyde (MDA) and glutathione (GSH), and correlations between the changes (delta) of maximal walking distance (MWD) with those of MDA and GSH. (a) Reactive oxygen species (ROS) generation in PBMCs, (b) plasma MDA and GSH concentrations, c) correlation between changes of MWD and those of MDA, d) correlation between changes of MWD and those of GSH. Data are expressed as mean ± SD; *p < 0.01 vs start.

Fig. 3.

Effect of supervised physical training on acute response to ischemia-reperfusion (treadmill test) on (a) reactive oxygen species (ROS) generation in PBMCs, (b) malondialdehyde (MDA), and (c) glutathione (GSH) plasma concentrations, at the start and at the end of the supervised physical training. *p < 0.01 vs start; ^¶p < 0.01 vs T0.

Fig. 4.

Effect of supervised physical training on UPR and Nrf2 pathway gene expression in PBMCs derived from PAD patients. (a) mRNA expression of PERK and IRE1, (b) mRNA expression of Nrf2, HO-1, and GCLC, (c) nuclear ATF4, XBP1, and Nrf2 concentrations. 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 vs start, ^¶p < 0.01 vs T0.

Fig. 5.

Correlations between changes (delta) in PERK with those of LDH leakage and changes in Nrf2 and reactive oxygen species (ROS) in PBMCs. a) Correlation between changes in PERK mRNA with those of LDH leakage (%). b) Correlation between changes in Nrf2 mRNA with those of ROS.

Fig. 6.

Effect of multiple (5) cycles of ischemia-reperfusion on PERK, IRE1, and Nrf2 gene expression in PBMCs derived from PAD patients. (a) mRNA expression of PERK, IRE1, and Nrf2; (b) nuclear ATF4, XBP1, and Nrf2 concentrations. 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 vs control.

Fig. 7.

Oxidative stress and cytotoxicity in PBMCs derived from PAD patients submitted to multiple (5) cycles of ischemia-reperfusion. (a) reactive oxygen species formation (ROS), (b) malondialdehyde (MDA) and glutathione (GSH) concentrations in culture media, (c) LDH leakage and apoptosis. Data are expressed as mean ± SD; *p < 0.01 vs control.

Fig. 8.

Requirement of PERK, IRE1, and Nrf2 for PBMC survival after multiple (5) cycles of I-R. (a) LDH leakage, (b) apoptosis. Data are expressed as mean ± SD; *p < 0.01 vs cycle 1. ^¶p < 0.01 vs multiple I-R; ^§p < 0.05 vs multiple I-R; GSKc39 = GSK compound 39, trigo = trigonelline.