J Cancer Prev 2023; 28(1): 3-11
Published online March 30, 2023
© Korean Society of Cancer Prevention
1College of Pharmacy, Seoul National University, 2Cancer Research Institute, Seoul National University, 3Department of Physical Education, Sejong University College of Arts & Physical Education, Seoul, Korea
Correspondence to :
Young-Joon Surh, E-mail: email@example.com, https://orcid.org/0000-0001-8310-1795
Young-Soo Lee, E-mail: firstname.lastname@example.org, https://orcid.org/0000-0003-2275-2660
*These authors contributed equally to this work as co-correspondence authors.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
In the present study, we investigated the effects of exhaustive exercise and recovery on inflammatory, pro-apoptotic, and anti-oxidative responses in human peripheral blood mononuclear cells (PBMCs). Sixteen volunteers participated in a guided physical activity program in which they were subjected to progressive exercise on the treadmill until they were exhausted followed by an 1-hour recovery period. Isolated human PBMCs were collected before exercise, immediately after exercise, and after 1-hour recovery. Exhaustive exercise induced expression of heme oxygenase-1 and glutamate cysteine ligase catalytic subunit and activation of NF-κB and NF-E2 related factor 2 (Nrf2). Apoptosis, as measured by activity and cleavage of caspase-3 and its substrate PARP also significantly increased. However, induction of redox signaling and the pro-apoptotic response fully returned to the baseline level during the 1-hour recovery period. On the other hand, COX-2 expression was continuously elevated after exercise cessation throughout the 1-hour recovery period. Taking all these findings into account, we conclude that exhaustive exercise transiently induces Nrf2-mediated antioxidant gene expression and eliminates damaged cells through apoptosis as part of an adaptive cytoprotective response against oxidative and inflammatory stress.
Keywords: Adaptive stress response, Exhaustive exercise, Antioxidant response elements, NF-E2 related factor 2
Although moderate exercise provides many health benefits, intense/exhaustive exercise can cause oxidative stress due to massive production of reactive oxygen species (ROS) . A single bout of strenuous exercise induces oxidative stress in circulating human blood cells and in rodent splenocytes and thymocytes, leading to lipid peroxidation, DNA damage, mitochondrial perturbations, and protein oxidation [2-5]. ROS generation during exercise also contributes to inflammatory damage .
NF-κB is a master regulator of gene transcription in response to inflammation and oxidative stress in various cells. NF-κB promotes expression of genes encoding pro-inflammatory cytokines and enzymes, such as COX-2, a rate-limiting enzyme in the biosynthesis of prostaglandins and thromboxanes. Inappropriate overexpression of COX-2 has frequently been observed in various premalignant and malignant tissues .
Apoptosis, a selective, controlled, and genetically programmed cell death, is characterized by several distinct biochemical and molecular events . In mature animals, apoptosis balances cell division, maintaining the constancy of tissue mass. Removal of cells injured by genetic defects, aging, disease, or exposure to noxious agents is made possible by apoptosis . It has been reported that strenuous exercise can lead to apoptotic cell death [10-12]. A panel of caspases are recognized as an important mediator of apoptosis. Once caspases activated, caspases exert pro-apoptotic activity in part by cleaving PARP, a key enzyme essential for DNA repair and cell survival .
In response to oxidative stress, cells tend to initiate protective mechanisms by inducing expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1), a novel stress-responsive enzyme . Numerous studies have identified HO-1 as an important component of cytoprotective defense mechanisms against oxidative and inflammatory insults .
Reduced glutathione (GSH) is one of the major antioxidants synthesized in cells and plays a key role in maintaining redox homeostasis, scavenging peroxides, and detoxifying electrophilic xenobiotics. Glutamate cysteine ligase (GCL), which catalyzes the formation of γ-glutamylcysteine, is the rate-limiting enzyme for de novo synthesis of GSH. Catalytic subunit of GCL (GCLC) can be induced in response to various oxidative stressors. This inducible nature suggests that GCL is an essential component of the cellular adaptation machinery under oxidative stress .
Expression of HO-1 and GCLC is known to be mediated by electrophilic response elements or antioxidant response elements (ARE), cis-acting enhancer sequences that mediate transcriptional activation of nuclear factor erythroid 2-related factor 2 (Nrf2) in response to oxidative stress and other noxious stimuli [17,18]. Nrf2 is a key mediator of physiological stress response that transcriptionally regulates antioxidant genes including HO-1, GCLC, NAD(P)H: quinone oxidoreductase 1 (NQO1), superoxide dismutase (SOD), catalase, GSH peroxidase, and GSH
Here, we report that exhaustive exercise induces transient expression of HO-1 and GCL via Nrf2-ARE signaling as an adaptive survival response against pro-inflammatory and pro-apoptotic insults in human peripheral blood mononuclear cells (PBMCs).
Rabbit polyclonal COX-2 and HO-1 antibodies were purchased from Lab Vision Co and Stressgen, respectively. Primary antibodies for caspase-3, cleaved caspase-3, PARP, and cleaved PARP were purchased from Cell Signaling Technology. Anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Zymed Laboratories. An oligonucleotide probe containing the NF-κB and Nrf2 consensus sequence were purchased from Promega. Enhanced chemiluminescence (ECL) detection kits and [γ32-32P] ATP were obtained from Amersham Pharmacia Biotech, and the bicinchoninic acid protein protein assay reagent was supplied by Pierce Biotechnology. All other chemicals were used in the purest form that were commercially available.
Sixteen healthy young (23.1 ± 1.1 years) male volunteers participated in this study, which was approved by the Seoul National University Institutional Review Board (SNU-IRB; approval number: 0709/001-001). Prospective participants were screened to exclude any individuals with chronic illness, acute infection, or current use of prescription drugs. The subjects, who had not been involved in any exercise training for the preceding one month, were directed to make no major changes in their physical activity routines during the study. All procedures were approved by the SNU-IRB. All participants were asked to provide written informed consents to participate in this study before collection of blood samples. Physiological characteristics of the subjects are presented in Table 1.
All subjects performed an incremental running test (Bruce protocol) to determine maximum oxygen uptake (VO2max) and maximum heart rate (HRmax) by the gas analyzer (Quinton) and the heart rate monitor (Polar Electro), respectively . All participants were subjected to progressive exercise on a treadmill until they were exhausted, followed by resting period. Subjects were instructed to avoid caffeinated beverages and exercise for at least 48 hours before the experiment. They were also fasted from 10:00 PM until the beginning of the exercise on the next day. At the time of the study, all subjects were transported to the laboratory, where they rested for at least 1 hour before the exercise test. After this rest period, each subject’s blood pressure was checked to screen for hypertension. And blood sample of resting venous (10 mL) was taken. Exercise was preceded by a 5-minute warm-up. After the warm-up, the subjects began the incremental running exercise on a treadmill and continued until they were exhausted. A second blood sample was taken immediately after discontinuing exercise and a third blood sample was collected after 1-hour recovery.
Human peripheral blood samples (10 mL) were obtained by venipuncture and transferred to an ethylene-diamine-tetraacetic acid (EDTA) anti-coagulant tube (Green Cross MS Corp.). Immediately after venipuncture, The PBMCs were separated as described previously . We carefully loaded 10-mL whole blood samples and centrifuged them for 10 min at 1,000
Isolated PBMCs were lysed in 200 μL of ice-cold hypotonic buffer A (10 mM HEPES [pH 7.8], 10 mM KCl, 2 mM MgCl2, 1 mM dithiothereitol [DTT], 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride [PMSF]). After 15-minute incubation on ice, the mixture was centrifuged for 2 minutes at 14,800
EMSA for measuring the DNA-binding activities of NF-κB and Nrf2 was performed using a DNA protein-binding detection kit (GIBCO BRL), according to the manufacturer’s protocol. Briefly, a synthetic double-stranded oligonucleotide containing the consensus sequence of NF-κB or Nrf2 was labeled by incubation with [γ-32P]ATP by T4 polynucleotide kinase and purified on a Nick column (Amersham Pharmacia Biotech). The binding reaction was carried out in 25 μL of a reaction mixture containing 5 μg of incubation buffer (10 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% glycerol, and 0.1 mg/mL sonicated salmon sperm DNA), 10 μg of nuclear extract, and 120,000 cpm of [γ-32P]ATP end-labeled oligonucleotide. After 50-minute incubation at room temperature, 2 μL of 0.1% bromophenol blue was added, and the samples were electrophoresed through 6% non-denaturing polyacrylamide gel at 150 V for 2 hours. Finally, the gel was dried and exposed to an X-ray film.
GSH levels were measured using a commercial kit (Bioxytech GSH-400; OxisResearch). The first step of the analysis leads to the formation of substitution products between a patented reagent and all of the mercaptans that are present in the sample. The second step specifically transforms the substitution product obtained with GSH into a chromophoric thione, which has maximal absorbance at 400 nm.
Isolated human PBMCs were washed with cold PBS and then lysed at 4°C in 500 μL of ice-cold lysis buffer (150 mM NaCl, 0.5% Triton-X 100, 50 mM Tris-HCl [pH 7.4], 20 mM ethylene glycol tetraacetic acid [EGTA], 1 mM DTT, 1 mM Na3VO4, and a protease inhibitor cocktail Tablet [Roche Molecular Biochemicals]) for 1 hour. The lysates were centrifuged at 14,800
Ac-DEVD-AMC (BD Bioscience) was used as a substrate for the caspase-3 assays based on the manufacturer’s instructions. A fluorescent assay kit was used to measure caspase-3 activity by monitoring the cleavage of a synthetic fluorescent substrate. In brief, cell lysates were prepared in the lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, and 1 mM PMSF) supplemented with protease inhibitor mixture (Roche), the lysates were centrifuged at 10,000
Values were expressed as the mean ± standard deviation of at least three independent experiments. Statistical significance was determined by repeated ANOVA as a post-hoc comparison.
GSH levels significantly declined after exhaustive exercise but were partially restored during the 1-hour recovery period (Fig. 1A). GSH restoration was accompanied by induction of GCLC expression after exhaustive exercise (Fig. 1B).
We also determined whether exhaustive exercise and 1 hour of recovery after exercise differentially modulated expression of COX-2 as an inflammatory response in human PBMCs. All subjects who participated in exhaustive exercise exhibited significantly elevated COX-2 expression during the 1-h recovery period (Fig. 2A). Because NF-κB is known to play a critical role in regulating induction of COX-2 expression, we also evaluated whether exhaustive exercise activated this transcription factor in nuclear extracts obtained from human PBMCs. As illustrated in Fig. 2B, NF-κB DNA-binding activity was significantly enhanced immediately after exhaustive exercise but declined during the 1-hour resting period.
A critical event in the activation of NF-κB is its dissociation from IκBα, which requires phosphorylation and subsequent degradation of this inhibitory subunit. To determine whether the enhancement by exhaustive exercise of NF-κB DNA binding was due to increased phosphorylation of IκBα, the cytoplasmic levels of phospho (p)-IκBα were measured by western blot analysis. Consistent with transient induction of NF-κB DNA binding, the cytoplasmic levels of p-IκBα were substantially elevated upon exhaustive exercise, but completely returned to the baseline level during the 1-hour recovery period (Fig. 2C).
One of the well-defined hallmarks of apoptosis is cleavage of PARP by pro-apoptotic signaling. Thus, we examined whether exhaustive exercise induced cleavage of PARP in human PBMCs. As illustrated in Fig. 3A, PARP cleavage was evident upon exhaustive exercise. However, initial induction was almost completely abolished after 1 hour of recovery. In another experiment, we determined whether exhaustive exercise induced cleavage and catalytic activity of caspase-3, a key enzyme involved in cleavage of PARP, in human PBMCs. Cleavage (Fig. 3B) and activity (Fig. 3C) of caspase-3 were both significantly elevated after exhaustive exercise. This activation of caspase-3 through proteolytic cleavage returned nearly to the baseline level during the 1-hour recovery period.
The onset of pro-inflammatory response was accompanied by transient induction of an antioxidant response. As shown in Fig. 4A, the initial increase in HO-1 expression was followed by a decline during the 1-hour recovery period. Nrf2 is known to play a critical role in regulating the induction of HO-1 expression. In parallel with transient HO-1 induction, the Nrf2-ARE binding activity was significantly enhanced after exhaustive exercise and returned to the baseline level during 1 hour of recovery (Fig. 4B).
There is substantial evidence for the effectiveness of periodic exercise in primary and secondary prevention of various disorders including cancer and premature death . However, exhaustive or strenuous exercise can cause injuries and chronic fatigue, leading to overtraining syndrome, which occurs partly due to oxidative stress via production of ROS [3,24,25]. Davies et al.  reported increased production of ROS in the muscle tissue of rats subjected to strenuous exercise. A study by Gomez-Cabrera et al.  revealed the protective role of xanthine oxidase against oxidative damage that occurs as a consequence of vigorous exercise in rats and humans. The present study clearly demonstrates that exhaustive exercise increases oxidative stress, as revealed by diminished GSH in erythrocytes. Oxidative stress is essentially an imbalance between pro- and antioxidants. Increased oxidative stress can be harmful to all cellular macromolecules, such as lipids, proteins, and DNA . In contrast, transiently elevated ROS often mediate the activation of distinct intracellular signaling pathways which regulate transcription of a number of genes encoding antioxidant proteins, DNA-repair proteins, stress-regulated chaperones, and anti-apoptotic proteins .
NF-κB has a pleiotropic function in regulation of many genes involved in cellular immunity and inflammatory responses . In particular, NF-κB promotes expression of COX-2, a rate-limiting enzyme in the biosynthesis of prostaglandins that mediate inflammatory processes. We noted that exhaustive exercise did induce expression of COX-2 as well as NF-κB activation, as an inflammatory response in human PBMCs. Although some studies have reported a reduction in NF-κB DNA binding activity after exercise [31,32], we found that NF-κB binding activity was markedly elevated following exhaustive exercise. This finding is also in good agreement with the results of other studies demonstrating that exercise is a powerful activator of NF-κB in blood, brain, kidney, and muscle cells [33-35].
We also observed that NF-κB activation, initially induced by exhaustive exercise, almost completely returned to the baseline level during 1-hour of recovery, while COX-2 expression continuously increased during the recovery period. This sustained increase in COX-2 expression even during the recovery period may reflect the interval between transactivation of NF-κB and expression of the
It has been reported that strenuous/vigorous exercise can cause apoptotic cell death [10-12]. Caspases are recognized as important mediators of apoptosis. Several previous studies have shown that exercise causes DNA damage in leukocytes [37,38]. Using single-cell gel electrophoresis, a sensitive measurement of DNA strand damage and alkali-labile damage, appearance of leukocytes with damaged DNA after exhaustive exercise has been demonstrated . Our results reveal that exhaustive exercise does induce apoptosis in human PBMCs, as evidenced by elevation of caspase-3 activity and proteolytic cleavage of caspase-3 and PARP. Although exhaustive exercise significantly induced the aforementioned hallmarks of apoptosis, there was an almost complete decline in these markers during the 1-hour recovery period. This decline in apoptosis during the resting period may be attributable to induction of antioxidant or other defense/repair mechanisms as an adaptive survival response to the oxidative and inflammatory stresses caused by exhaustive exercise.
Using the terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labeling assay, Mars et al.  demonstrated that apoptosis occurred in 63% of lymphocytes immediately after high-intensity exercise. At 24 hours after exercise, 86.2% of the cells still exhibited an apoptotic profile of DNA distribution . According to the results of our present study, however, initial induction of caspase-3 activity and cleavage of PAPR were almost completely abolished during the 1-hour recovery period. This discrepancy between the two results may be due to the different exercise protocols and cell types examined. The mechanisms responsible for exercise-related apoptosis of human blood cells remain poorly understood. Alterations in the cellular redox status may influence apoptotic signaling. In numerous cells, ROS are involved as both initiators and mediators of apoptosis . Treatment with antioxidants has been shown to attenuate exercise-induced DNA damage, indicative of a pro-apoptotic function of ROS . In contrast to intensive exercise, moderate exercise attenuates lymphocyte apoptosis induced by oxidative stress, possibly by potentiating intracellular anti-oxidative capabilities . Thus, excessive production of ROS during intensive exercise is considered responsible for a series of biochemical and physiological changes that account for adaptive response to oxidative stress [24-26].
The human body maintains distinct antioxidant defense systems against oxidative stress. Nrf2 up-regulates a battery of antioxidant enzymes and other cytoprotective proteins . One of the principal stress-responsive cytoprotective proteins whose gene transcription is regulated by Nrf2 is HO-1 . HO-1 has potent anti-inflammatory, anti-oxidative, and anti-apoptotic functions [14,43,44]. HO-1-mediated cytoprotection has been shown to be critical for tissues that are vulnerable to oxidative stress . Notably, an acute bout of exercise has been found to increase expression of HO-1 in human lymphocytes . Although Nrf2 and HO-1 are well known to have cytoprotective functions, there is a paucity of data on the induction of Nrf2 activation and HO-1 expression by exercise. Consistent with previous studies on exercise-induced expression of HO-1 in human lymphocytes and leukocytes [45-47], our study documents exhaustive exercise-related upregulation of HO-1 expression in conjunction with Nrf2 activation in human PBMCs.
HO-1 is an important stress-response enzyme induced by oxidative and inflammatory stimuli. Previous studies have shown that a competitive half-marathon , treadmill test until exhaustion , and prolonged treadmill running , but not a short run or eccentric exercise , increase HO-1 expression. Although our exercise trial was not a long-duration type, there was pronounced induction of HO-1 expression and Nrf2-ARE binding activity immediately after exercise. High-intensity exercise may disrupt the fragile balance between oxidative stress and anti-oxidative defense, and as a result, the mitochondrial respiratory chain and other metabolic pathways generate ROS [49-51]. This, in turn, provokes adaptive cellular antioxidant responses to resolve oxidative and inflammatory stresses [24,25].
In addition to HO-1, exhaustive exercise induced induction of other antioxidant enzymes, such as NQO1, SOD, and GST (Figure S1), which also constitute an important part of the cellular defense against pro-oxidative/pro-inflammatory insults. Therefore, moderate exercise may offset inflammation via ROS generation, likely by timely induction of antioxidant gene expression through activation of Nrf2-ARE signaling.
In conclusion, exhaustive exercise triggers oxidative and inflammatory responses and induces expression of cellular antioxidant enzymes, including HO-1 in human PBMCs. In addition, exhaustive exercise-induced apoptosis in human PBMCs appears to facilitate elimination of cells that cannot survive oxidative and inflammatory injuries caused by vigorous exercise. Further studies will be necessary more precisely assess the effects of exhaustive exercise on human health and disease.
Supplementary materials can be found via https://doi.org/10.15430/JCP.2023.28.1.3.
This work was supported by the grant (NRF-2010-G00134) funded by the National Research Foundation of Korea.
No potential conflicts of interest were disclosed.
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