Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Effects of Eccentric Exercise on NF-JBActivation in Blood Mononuclear Cells
DAVID GARCIA-LOPEZ, MARIA J. CUEVAS, MAR ALMAR, ELENA LIMA, JOSE A. DE PAZ,and JAVIER GONZALEZ-GALLEGO
Institute of Biomedicine, University of Leon, Leon, SPAIN
ABSTRACT
GARCIA-LOPEZ, D., M. J. CUEVAS, M. ALMAR, E. LIMA, J. A. DE PAZ, and J. GONZALEZ-GALLEGO. Effects of Eccentric
Exercise on NF-JB Activation in Blood Mononuclear Cells. Med. Sci. Sports Exerc., Vol. 39, No. 4, pp. 653–664, 2007. Purpose:
Changes in nuclear factor JB (NF-JB) activation induced in peripheral blood mononuclear cells (PBMC) by acute eccentric exercise
and by submaximal eccentric training were investigated. Methods: Eleven subjects carried out two bouts of eccentric exercise separated
by 6 wk of training. Results: Soreness, vertical jump height, and plasma creatine kinase were significantly modified after the first bout.
NF-JB activation, p50 and p65, phospho-IJB> and phospho-IKK protein level, and Mn-SOD expression increased in PBMC, whereas
IJB> protein level was significantly reduced. Changes were significantly attenuated after the second exercise bout. An additional group of
nine subjects carried out the two bouts of exercise without training. Effects on NF-JB activation were similar after the second bout
compared with the first, despite a reduction in markers of muscle injury (repeated bout effect). Conclusion: Training significantly
attenuates the NF-JB–dependent pathway changes induced in PBMC by eccentric exercise, with no contribution from the repeated bout
effect. Key Words: EXERCISE, HUMANS, REACTIVE OXYGEN SPECIES, TRANSCRIPTION FACTOR
Eccentric exercise has been reported to damage
skeletal muscle in a fiber-specific manner, and it
is known that oxidative stress plays a role in the
progression of muscle fiber injury after the initial mechan-
ical insult (10). However, little is known about the effects
of eccentric exercise on several oxidative stress-sensitive
signaling pathways that play an important role in main-
taining cellular oxidant–antioxidant balance and that
mediate the expression of genes involved in the inflamma-
tory response. One of the most important signaling path-
ways that could be activated during eccentric exercise is
nuclear factor JB (NF-JB), which is present in the
cytoplasm in an inactive state, bound with the inhibitory
IJB subunit proteins (13). NF-JB is activated by a variety
of external stimulants, including reactive oxygen species
(ROS) and cytokines, via phosphorylation of IJB by IJBkinase (IKK) (21). When IJBs are phosphorylated, NF-JBis freed and migrates to the nucleus, where it binds to
specific JB-recognition elements in their promoter region
of target genes. In this way, NF-JB enhances deexpression
of genes encoding for cytokines, chemokines, growth
factors, cell-adhesion molecules, inflammatory enzymes,
antioxidant enzymes, or some acute-phase proteins (9).
Previous studies have indicated that an acute bout of
exercise increases NF-JB activation in rat muscle (13,15),
and it has been also shown that an incremental exercise test
until exhaustion (28), 5 min of intense exercise (26), or
short-term supramaximal anaerobic exercise (7) may
trigger NF-JB activation in human peripheral blood
leucocytes. Several studies have found increases in blood
markers of oxidative stress after eccentric exercise, both in
human and in animal models (16). Moreover, muscle fiber
injury during eccentric exercise may result in a local
inflammatory response associated with cytokines released
at the site of muscle injury, and there are data showing
that elevated plasma interleukins in response to exercise
may likely be a result of interleukins produced in the con-
tracting skeletal muscle (27). Thus, it is conceivable that
NF-JB activation in blood cells, which may represent a
population of cells on their way towards participation in
ongoing tissue surveillance and adaptations, could be a
consequence of stimuli arising from the damaged skeletal
muscle. Considering that multiple open biopsies can have
effects similar to those of eccentric cycling exercise in
human skeletal muscle, conclusions regarding eccentric
exercise–induced damage that are based solely on results
from research with open muscle biopsies should be
reconsidered (18), and the study of changes in blood cells
could provide useful information. In this line, Malm and
Address for correspondence: Javier Gonzalez-Gallego, Institute of
Biomedicine, University of Leon, 24071 Leon, Spain; E-mail: jgonga@
unileon.es.
Submitted for publication August 2006.
Accepted for publication November 2006.
0195-9131/07/3904-0653/0
MEDICINE & SCIENCE IN SPORTS & EXERCISE�
Copyright � 2007 by the American College of Sports Medicine
DOI: 10.1249/mss.0b013e31802f04f6
653
BASIC
SCIEN
CES
Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
coworkers (18) indicate that circulating monocytes may
have a governing function over muscular events. Thus, a
first objective of our study was to investigate whether a
bout of eccentric exercise would induce NF-JB activation
in peripheral blood mononuclear cells (PBMC). It was
hypothesized that muscle damage would be accompanied
by changes in the NF-JB–dependent pathway in blood
cells.
It has been demonstrated that exercise training induces
adaptations that provide additional protection during times
of intense physical stress and that attenuate the typical rise
in protein, lipid, and DNA oxidation after a single bout
of exercise (24). Recently, it has been shown that regu-
lar exercise attenuates age-associated oxidative stress and
NF-JB activation in rat liver (23); this might help provide
the molecular basis for the beneficial effects of training.
An additional purpose of the present research was to
investigate whether effects on the NF-JB signaling path-
way induced by acute eccentric exercise are prevented by
submaximal eccentric training. It is well established that a
repeated bout of the same eccentric exercise within several
weeks results in significantly reduced symptoms of damage
(22). This phenomenon, referred to as the ‘‘repeated bout
effect,’’ has been demonstrated in numerous studies,
although the mechanism for this adaptation remains
unclear (20). Therefore, we also studied whether the
repeated bout effect could contribute to the changes
detected in NF-JB activation after a period of submaximal
eccentric training.
METHODS
Subjects and procedures. Moderately active male
students (N = 11) who regularly practiced physical activity
on a moderate to vigorous level (3 hIwkj1) took part in the
study. The subjects were not taking any medication known
to affect hormonal or metabolic responses to exercise.
Special care was taken to exclude subjects who were taking
any form of vitamin or antioxidant supplementation. The
subjects, who had not been involved in any resistance
training for the preceding 2 months, were directed to make
no major changes in their physical activity routines during
participation. Before data collection, subjects were
informed of the requirements and risks associated with
participation, and they provided written informed consent.
The study conformed to the code of ethics of the World
Medical Association (Declaration of Helsinki), and all
procedures were approved by the local ethics committee.
The study was completed in 9 wk, including pre- and
posttraining descriptive and baseline data collection, the
training period, and pre- and posttraining testing. Each
participant carried out two similar bouts of muscle-
damaging eccentric exercise separated by 8 wk (including
the 6-wk training period). During the performance of these
eccentric bouts (pre- and posttraining, respectively), blood
samples were collected for the damage and oxidative
stress–markers analysis. To investigate the rapid adaptation
phenomenon known as the repeated bout effect, a second
study (study 2) was conducted. Nine students followed an
exercise protocol similar to that used in study 1. Each
participant developed two bouts of eccentric exercise
separated by 8 wk (initial bout and repeated bout,
respectively) without training. Subjects were similar the
subjects from study 1with respect to age, body weight,
height, percentage of body fat, and training level.
Descriptive and baseline data collection.Descriptive and baseline data were collected during a
laboratory session carried out 1 wk before the first
eccentric bout and the second bout, respectively. Height
(cm) was measured with a Detecto stadiometer (model
D52, Webb City, MO), and weight (kg) was measured with
a Cobol electro scale with 20-g accuracy (model 20, Cobol,
Barcelona, Spain). Body fat percentage was estimated by
measuring skinfold thickness (six sites) using skinfold
calipers (Lafayette Instruments, Lafayette, IN) and the
equations developed by Yuhasz (30). Each skinfold site
was measured in triplicate by the same investigator and
was recorded to the nearest millimeter. Next, the
individuals carried out a standard 10-min warm-up and
then performed a vertical jump test in which the
countermovement jump (CMJ) modality was evaluated as
described Byrne and Eston (2). After the vertical jump test,
each subject performed a maximal voluntary isometric
contraction test (MVIC) in squat position; this value was
used to establish the load that would be used during the
eccentric exercise.
Eccentric-damaging protocol. One week later,
during a second evaluation session, all subjects performed
the eccentric bout, which consisted in 12 sets of 10
repetitions, using as eccentric movement the negative
phase of the barbell squat. The load was equivalent to the
60% of MVIC, and a 2-min rest period was permitted
between sets. For safety reasons, a multipower apparatus
was used (Salter, Barcelona, Spain). Thus, after each
repetition, in which the subject only had to lower the
imposed load from 180- of knee extension to 90-, fourassistants rose the load up. Subjects were asked to descend
the load with the most comfortable velocity for them,
registering the vertical displacements of the barbell through
an encoder system (Globus Real Power, Codogne, Italy). A
previous study has shown that letting subjects decide the
knee-flexion velocity allows them to pay more attention to
the squat technique; moreover, the results did not show
very different values between individuals. The only
recommendation given was that, in every moment, they
had to control the descent of the barbell, so that they could
stop it at the point equivalent to a knee flexion of 90-. Thesecond bout was similar to the first one in terms of load
and descent velocity.
Muscular soreness. The intensity of the soreness
perceived at quadriceps muscle level was assessed using a
10-cm visual analog scale (VAS) (6), the left and right
http://www.acsm-msse.org654 Official Journal of the American College of Sports Medicine
BASICSC
IENCES
Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
extremes of which refer to ‘‘no muscular soreness’’ and
‘‘maximum muscular soreness,’’ respectively. The VAS is
easily and quickly administered and has been used as a
reliable measurement for determining the intensity of
human pain. Thus, subjects were asked to place a vertical
line toward the amount of soreness they perceived, which
was quantified as the distance (with a precision of 0.1 cm)
between the left extreme of the line and the vertical line
carried out by the subject. All the subjects had been
previously familiarized with the VAS.
Submaximal eccentric training. Subjects performed
eccentric training three times weekly during 6 wk. The
same eccentric movement (eccentric phase of the barbell
squat) and the same multipower device used during the
eccentric damaging protocol were used during the training
sessions. The number of sets per session was between three
and five, with 10 repetitions per set. The load was
progressively increased from 40 to 50% of the MVIC.
The knee-flexion velocity was also monitored during
training sessions, used by the subjects as a feedback to
maintain a mean velocity similar to the one registered
during the initial eccentric damaging exercise.
Blood sample preparation. Venous blood samples
(15 mL) were taken, using EDTA as an anticoagulant.
Blood samples were obtained by a catheter closed by stylet
(Vasoran and Mandrin, B. Braun) from the braquiocephalic
vein at rest, immediately after each eccentric bout, and at
2, 6, and 24 h after cessation of exercise. Immediately after
extraction, 3 mL of whole blood was centrifuged at 1500 gfor 10 min at 4-C, and plasma aliquots were stored at
j85-C until further determination of creatine kinase (CK),
thiobarbituric reactive species (TBARS), and protein
carbonyls. PBMCs were separated from the whole blood
by density-gradient centrifugation on Ficoll separating
solution (Biochrom AG). For each sample, two 15-mL
centrifuge tubes were used to layer 6 mL of blood onto
4 mL of Ficoll. The suspension was centrifuged for 30 min
at 275g and 20-C. The mononuclear cell layer was
removed with manual pipetteing, washed one time in
Hank`s solution, and centrifuged after the wash for
10 min at 20-C and 450g. Washed cells were resuspended
in 1 mL of PBS. Analyses were performed on frozen cells.
Assessment of CK activity, TBARS, and proteincarbonyls in plasma. CK activity was determined in
0.5 mL of plasma by a Cobas Integra 400 analyzer
(Hoffmann-La Roche, Basel, Switzerland). The con-
centration of thiobarbituric acid reactants was measured
according to a modified version of the high-pressure liquid
chromatography method of Richard et al. (25). A 100-KLsample was injected into the HPLC system, which was
equipped with a Prodigy analytical stainless steel column
(Phenomenex, 5 mm, 0.46 � 25 cm). Isocratic separation
was performed at a flow rate of 1.0 mLIminj1. The mobile
phase consisted of 50 mM phosphate buffer (pH 6.0):
methanol (58:42, v/v). The absorbance of each sample was
recorded at the column outlet at 532 nm. Carbonyl groups
were measured in plasma using the Zentech PC test (Protein
Carbonyl Enzyme Immunoassay Kit) from Zenith Tech-
nology (Dunedin, New Zealand), which uses derivatization
of protein carbonyls in samples and oxidized protein
standards with dinitrophenylhydrazine; this was followed
by ELISA with an anti-DNP antibody and standard ELISA
techniques for labeling and visualizing labeled molecules.
Absorbance was read at 450 nm on the Synergy HT
microplate reader (Bio-Tek, Winooski, VT). A standard
curve was plotted, and the carbonyl concentration of
samples was read off the curve.
TABLE 1. Subject characteristics.
Age (yr) Mass (kg) Height (cm) Body Fat (%)
Study 1First bout 22.0 T 0.7 79.9 T 2.0 181.0 T 1.3 9.6 T 0.4Second bout 79.6 T 2.4 9.4 T 0.4
Study 2First bout 23.6 T 0.8 76.2 T 1.9 177.4 T 1.4 9.1 T 0.4Second bout 75.9 T 2.0 8.9 T 0.3
Values are mean T SEM.
FIGURE 1—Quadriceps muscle soreness, countermovement jump
(CMJ) height, and plasma creatine kinase (CK) response to acute
eccentric exercise and submaximal eccentric training. N = 11 for each
time point. Values are means T SEM. * Significant change compared
with resting values (P G 0.05); # significant change compared with
pretraining values (P G 0.05).
ECCENTRIC EXERCISE AND NUCLEAR FACTOR JB Medicine & Science in Sports & Exercised 655
BASIC
SCIEN
CES
Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Nuclear extracts and electrophoretic mobilityshift assay. Binding activity of NF-JB was determined
in nuclear extracts of PBMC by means of electrophoretic
mobility shift assay (EMSA), as described Hofmann et al.
(12) Nuclear extracts of PBMC were harvested by the
method of Andrews and Faller (1). Oligonucleotides were
end labeled with [F-32P]ATP to a specific activity 9 5 �107 cpmIKgj1 DNA. Nuclear extract (26 Kg) was
incubated for 20 min at room temperature in binding
buffer in the presence of approximately 1 ng of labeled
oligonucleotide (~250 KCi; Amersham Redivue). For
competition studies, 3.5 pmol of unlabeled NF-JBoligonucleotide (competitor) or 3.5 pmol of labeled
NF-JB oligonucleotide mutate (noncompetitor) were
mixed 15 min before the incubation with the labeled
oligonucleotide. Protein–DNA complexes were separated
from the free DNA probe by electrophoresis through 6%
native polyacrylamide gels containing 10% amonium
persulfate and 0.5� Tris-borate-EDTA buffer. Gels were
dried under vacuum on Whatmann DE-81 paper and
exposed for 48–72 h to Amersham hyperfilms at j80-C.Western blot analysis. For Western blot analysis,
PBMC cells were homogenized with 150 KL of 0.25 mM
sucrose, 1 mM EDTA, 10 mM Tris, and a protease
inhibitor cocktail (21). Samples containing 50 Kg of
protein were separated by SDS-polyacrylamide gel
electrophoresis (9% acrylamide) and transferred to PVDF
membranes. Nonspecific binding was blocked by pre-
incubation of the PVDF membranes in PBS containing 5%
bovine serum albumin for 1 h. The membranes were then
incubated overnight at 4-C with appropriate antibodies.
Antibodies against IJB>, IKK>, were purchased from Cell
Signaling Technology, Inc., and antibodies against Mn-
SOD were purchased from Stressgen Biotechnologies.
Bound primary antibody was detected using a peroxidase-
conjugated secondary antibody (DAKO) by chemilumi-
nescence using the ECL kit (Amersham). The densities of
the specific bands were quantitated with an imaging
densitometer. The blots were stripped in 6.25 mM Tris,
pH 6.7, 2% SDS, and 100 mM mercaptoethanol at 50-C for
15 min and were probed again for anti–beta-actin anti-
bodies (Sigma) to verify equal protein loading in each lane.
Statistical analysis. Data were expressed as mean Tstandard error of means (SEM). The results for NFJB, IJB,IKK, and Mn-SOD are presented as percentages from
resting values. All data were analyzed using an analysis of
variance (ANOVA) with repeated measures for time
(baseline, post, and 2, 6, and 24 h after) and training
(pre- and posttraining). Bonferroni post hoc analysis was
used where appropriate. A value of P G 0.05 was regarded
FIGURE 2—Nuclear factor JB activation in PBMC in response to acute eccentric exercise and submaximal eccentric training. Left panel,Representative EMSA. N = 11 for each time point. Right panel, Results expressed as percentages of resting values (means T SEM). * Significant
change compared with resting values (P G 0.05); # significant change compared with pretraining values (P G 0.05).
http://www.acsm-msse.org656 Official Journal of the American College of Sports Medicine
BASICSC
IENCES
Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
as significant. SPSS version 13.0 statistical software
(Chicago, IL) was used.
RESULTS
Age, height, mass, and body fat percentage did not differ
significantly between groups in study 1 and study 2.
Physical characteristics before eccentric bout 1 and
eccentric bout 2 values did not show significant differences
within each group (Table 1).
Eccentric training and muscle damage markers.Figure 1 shows the evolution over time of soreness before
and after the training period in study 1. Muscle soreness
induced by the first eccentric exercise (pretraining)
increased significantly over time (P G 0.01). The pain
perceived at 24 h was significantly higher than that
experienced just after finalizing the exercise or 6 h later.
A significant effect of training was detected (P G 0.01),
with the soreness response after the training period
significantly lower than the corresponding pretraining
values at each temporal point. The vertical jump height
also experienced a significant variation in response to the
first eccentric exercise (pretraining) (P G 0.01), with a
reduction in performance, already significant immediately
after the exercise (j25%), persisting for 6 h (j29%) and
24 h (j33%) (Fig. 1). After the 6-wk training period, the
vertical jump height showed a significant change
immediately after the eccentric bout (P G 0.01), although
the decrease relative to the baseline was lower than that
before the training program. Thus, a training effect was
observed (P G 0.01), with significant differences in
response to submaximal eccentric exercise between pre-
and posttraining values. Figure 1 also displays CK activity
values in response to the 6-wk training period. Pretraining
CK changed significantly over time (P G 0.01), reaching
the highest value at 24 h (+670%). A significant effect of
training was observed (P G 0.05), showing this significant
differences for this muscular protein between pretraining
and posttraining, at 6 and 24 h after the eccentric bout.
Eccentric training and the NF-JB–dependentpathway. A single bout of eccentric exercise caused a
significant increase in NF-JB binding activity to NF-JBconsensus sequence (P G 0.01) that was significant at 2, 6,
and 24 h after exercise compared with baseline values.
After the training program, no significant change was
detected after the eccentric bout. There was a significant
training effect (P G 0.01), with significant differences
between pre- and posttraining values at 2, 6, and 24 h
(Fig. 2). Data shown in Figure 3 indicate that p50 content
in PBMC increased gradually after a single bout of exercise
FIGURE 3—Western blot analysis of p50 and p65 in PBMC in response to acute eccentric exercise and submaximal eccentric training. Left panel,Representative Western blot photographs. N = 11 for each time point. Right panel, Results expressed as percentages of resting values (means TSEM). * Significant change compared with resting values (P G 0.05); # significant change compared with pretraining values (P G 0.05).
ECCENTRIC EXERCISE AND NUCLEAR FACTOR JB Medicine & Science in Sports & Exercised 657
BASIC
SCIEN
CES
Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
(P G 0.01), returning to resting level at 24 h after exercise.
A second bout of eccentric exercise after training did
not alter p50 protein. Thus, a training effect was observed
(P G 0.01), with significant differences in responses
to submaximal eccentric exercise between pre- and
posttraining values. Figure 3 also shows that p65 protein
in nuclear extracts increased progressively, reaching a
maximal level at 6 h (P G 0.01). Post–training program
values were lower, but they remained elevated after the
second eccentric bout at 2, 6, and 24 h after exercise
(P G 0.01). Again, a training effect was detected (P G 0.01),
with significant differences at 2 and 6 h. A significant
decrease in IJB> protein levels was observed in response
to eccentric exercise before the training program (P G 0.01)
(Fig. 4). This decrease was more pronounced at 2, 6, and
24 h after exercise (j41, j37, and j44%, respectively).
IJB> protein levels after the 6-wk training program did not
significantly change throughout time, and a training effect
was detected (P G 0.05), with significant differences at 2
and 6 h. A marked increase of cytosolic phospho-IJB>protein level was clearly demonstrated immediately after
exercise and was maintained at 2 and 6 h. (P G 0.01). After
the training program, values of phospho-IJB> did not elicit
any change (Fig. 4). Thus, a training main effect (P G 0.01)
was also observed, with significant differences in the
response to the 6-wk training program immediately after
the second bout and at 2 and 6 h. As shown in Figure 5, the
first session of eccentric exercise produced a significant
increase in phospho-IKK> protein level (P G 0.05). Values
increased immediately after the exercise bout and at 2 h
(+146 and +161%, respectively). A training main effect
(P G 0.01) was detected, with significant differences in the
response between pre- and posttraining values immediately
after exercise and also at 2 h. Mn-SOD expression (Fig. 6)
changed significantly in response to the first eccentric bout
(P G 0.01), with significant increases at 2, 6, and 24 h
(+129, +151, and +143%, respectively). After the training
program, values decreased after the second eccentric bout.
A significant training effect was present (P G 0.01), with
significant differences between pre- and posttraining
values at 2, 6, and 24 h.
Eccentric training and oxidative stress markers.Figure 7 reports plasma TBARS and plasma protein
carbonyls. The results of the two-factor ANOVA revealed
no significant time effects (P = 0.141 and 0.460 for the pre-
and posttraining bouts, respectively). However, a main
training effect was observed (P G 0.01). The post hoc test
demonstrated that posttraining TBARS decreased
significantly at 2 and 6 h after cessation of exercise.
Similar data were observed for protein carbonyls (Fig. 7).
FIGURE 4—Western blot analysis of IJB> and phospho-IJB> in PBMC in response to acute eccentric exercise and submaximal eccentric training.
Left panel, Representative Western blot photographs. N = 11 for each time point. Right panel, Results expressed as percentages of resting values
(means T SEM). * Significant change compared with resting values (P G 0.05);#significant change compared with pretraining values (P G 0.05).
http://www.acsm-msse.org658 Official Journal of the American College of Sports Medicine
BASICSC
IENCES
Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Only a training main effect was measured (P G 0.01), with
significant differences immediately after eccentric exercise
when comparing pre- and posttraining conditions. Repeated bout effect. A second study was carried
out to identify the repeated bout effect and its potential
contribution to the changes detected in study 1. Figure 8
displays the evolution of soreness over time, after both the
initial and the repeated bout. As expected, muscle soreness
induced by the initial eccentric exercise increased
significantly over time (P G 0.01). Again, the pain
perceived at 24 h was significantly higher than that
experienced just after finalizing the exercise or 6 h later.
The second bout also induced a significant muscle soreness
(P G 0.05), and a significant repeated bout effect was
observed (P G 0.01), with a significant difference at 24 h in
soreness responses to the first and second eccentric bouts.
The vertical jump height changed significantly in response
to the initial eccentric exercise (P G 0.01), with reductions
in performance already significant immediately after
exercise (j29%) that were maintained at 6 h (j28%)
and 24 h (j32%) (Fig. 8). After the repeated bout, vertical
jump height also showed a significant change over time
(P G 0.01), although the decrease relative to the baseline
was lower than that shown in the initial bout (j22, j19,
and j20%, immediately, 6 h, and 24 h after the eccentric
exercise, respectively). Thus, a repeated bout main effect
(P G 0.05) was also observed, with significant differences
in responses to the first and the second bouts at 6 and 24 h.
As in study 1, CK activity also changed significantly over
time after the initial bout of eccentric exercise (P G 0.01),
FIGURE 6—Western blot analysis of Mn-SOD in PBMC in response to
acute eccentric exercise and submaximal eccentric training.Upper panel,Representative Western blot photographs. N = 11 for each time point.
Lower panel, Results expressed as percentages of resting values (means TSEM). * Significant change compared with resting values (P G 0.05);# significant change compared with pretraining values (P G 0.05).
FIGURE 7—PlasmaTBARS and plasma protein carbonyl concentration
in response to acute eccentric exercise and submaximal eccentric
training. N = 11 for each time point. Values are means T SEM.# Significant change compared with pretraining values (P G 0.05).
FIGURE 5—Western blot analysis of phospho-IKK> in PBMC in
response to acute eccentric exercise and submaximal eccentric train-
ing. Upper panel, Representative Western blot photographs. N = 11 for
each time point. Right panel, Results expressed as percentages of
resting values (means T SEM). * Significant change compared with
resting values (P G 0.05); # significant change compared with
pretraining values (P G 0.05).
ECCENTRIC EXERCISE AND NUCLEAR FACTOR JB Medicine & Science in Sports & Exercised 659
BASIC
SCIEN
CES
Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
with a maximum at 24 h. After the second bout, there was a
significant time effect (P G 0.05), with increases at 6 and
24 h. Responses to the first and second bouts were
significantly different at 6 and 24 h, indicating the presence
of a repeated bout main effect (P G 0.01) (Fig. 8). Figure 9
shows the comparison between study 1 and study 2 for the
percent change from the first to the second bout in markers
of muscle damage. Results indicate that changes in
soreness, vertical jump height, and CK activity were
attenuated to a higher significant degree by the program
of submaximal eccentric training.
NF-JB activation in PBMC in response to both eccentric
bouts is shown in Figure 10. The initial bout caused a
progressive increase in the activation of the transcription
factor NF-JB (P G 0.01); this increase was already
significant immediately after the exercise (+129%) and
persisted for 24 h (+138%). A significant difference over
time was also detected after the repeated bout (P G 0.05).
The increase was significant immediately after exercise
(+138%), at 2 h (+146%), and at 6 h (+142%), returning to
basal values at 24 h. No repeated bout effect was detected
(P = 0.50). Mn-SOD expression (Fig. 11) changed sig-
nificantly in response to the initial bout (P G 0.05), with
significant increases at 2 and 6 h (+135 and +145%,
respectively). A significant difference over time was found
after the repeated bout (P G 0.01). The increase was sig-
nificant at 2 h (+146%), 6 h (+178%), and 24 h (+159 = %).
No repeated bout effect was present (P = 0.21).
DISCUSSION
One of the characteristics of the eccentric muscle actions
is the capacity to induce muscular damage inherent to the
unaccustomed active lengthening. Symptoms associated
with exercise-induced muscle damage include soreness,
changes in range of motion, loss of strength, and release of
muscle proteins, such as CK, into the circulation (20). In
our study, the severe muscular soreness symptoms and
impairment in explosive force after the first eccentric test
are in agreement with data from the literature concerning
exercise-induced muscle damage (16). Its partial prevention
FIGURE 8—Quadriceps muscle soreness, countermovement jump
(CMJ) height, and plasma creatine kinase (CK) response to a repeated
bout of eccentric exercise. N = 9 for each time point. Values are means TSEM. * Significant change compared with resting values (P G 0.05);#significant change compared with initial bout values (P G 0.05).
FIGURE 9—Comparison between study 1 (eccentric training) and
study 2 (repeated bout effect) for changes in soreness, vertical jump
height, and plasma creatine kinase (CK). Histograms show the
percent change from the first to the second bout of eccentric exercise
in study 1 and study 2. * Significant change compared with study 1
(P G 0.05).
http://www.acsm-msse.org660 Official Journal of the American College of Sports Medicine
BASICSC
IENCES
Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
by 6 wk of submaximal eccentric training indicates that
regular training attenuates the magnitude of muscle
damage induced by acute eccentric exercise. Because CK
in the circulation reflects its release from muscle fibers and
its clearance from the circulation, interpretation of this
parameter should be made with caution. Increases in
plasma CK levels detected in the present research
presumably indicate persistent damage to the muscles,
resulting in leakage of proteins into the blood (3); our data
also show that the increases in CK activity were not seen
after the training period.
Recent reports suggest that acute exercise activates
NF-JB signaling in different tissues. Under normal phys-
iological conditions NF-JB forms a complex with its
inhibitors, the IJBs, and is maintained in the cytosol in
this inactive state. The activation pathway of NF-JB leads
to the translocation of NF-JB to the nucleus from the
cytoplasm on dissociation from IJB, which is phosphory-
lated by IKK (9). The phosphorylated IJB undergoes
ubiquitination and then degradation by the 26S proteasome
complex. Thus, the inhibition of IJB and the phosphor-
ylation of IKK are essential steps in the NF-JB–activationprocess (15). Increased NF-JB activation in rat muscle in
response to an acute bout of exercise has been reported
(13), as has local activation of IKK in muscle (11), re-
sulting in decreases in the cytosolic content of IJB> (4).
Vider et al. (27) provided the first evidence that performing
an incremental exercise test until exhaustion could trigger
NF-JB activation in human peripheral blood lymphocytes,
and we have recently shown that short-term supramaximal
anaerobic exercise gives rise in PBMC to an activation of
NF-JB, accompanied by a degradation of IJB (7). These
data are consistent with the current study, revealing that
after an acute bout of eccentric exercise, increased expres-
sion of phospho-IJB> and proteolysis of IJB> are coinci-
dent with activation of NF-JB and nuclear translocation of
p50/p65 subunits in mononuclear cells. This scenario is also
supported by IKK having been activated (i.e., phosphory-
lated) in the PBMC. The antibody used in the current study
could detect both IKK> (85 kDa) and IKKA (87 kDa) when
the kinase was activated by phosphorylation at Ser-180 or
Ser-181, respectively. In our experiments, the major band
detected was identified as the 85-kDa IKK>. These effects
on the NF-JB signaling pathway were prevented by a period
of submaximal eccentric training, which resulted in no
activation of NF-JB and an absence of significant changes
FIGURE 10—Nuclear factor JB activation in PBMC in response to a repeated bout of eccentric exercise. Left panel, Representative EMSA. N = 9
for each time point. Right panel, Results expressed as percentages of resting values (means T SEM). * Significant change compared with resting
values (P G 0.05).
ECCENTRIC EXERCISE AND NUCLEAR FACTOR JB Medicine & Science in Sports & Exercised 661
BASIC
SCIEN
CES
Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
of IJB>, phospho-IJB>, and phospho-IKK> contents after
a second bout of acute exercise.
Translocation of NF-JB to the nucleus results in binding
to the JB domain of gene promoters, resulting in changes
in the activity of different enzymes, including Mn-SOD
(15). Thus, we decided to study Mn-SOD expression as a
potential marker of the activation of the NF-JB–dependentpathway. As one of the major antioxidant enzymes, SOD
plays an important role in catalyzing the dismutation of
superoxide radicals to hydrogen peroxide, thereby prevent-
ing the dangerous Haber–Weiss reaction, which generates
hydroxyl radicals (8). NF-JB is present in the promoter of
the Mn-SOD gene, and although oxidative stress has been
shown to activate its binding, little is known about the
effects of exercise-induced NF-JB activation on SOD gene
expression. The sole exception is the observation of
activation of muscle Mn-SOD gene expression after acute
bouts of exercise (13,15). In the present study, a single
bout of eccentric exercise was accompanied by increases in
SOD protein content in PBMC, but after the training
program, neither NF-JB activation nor Mn-SOD expres-
sion were increased. Although it is not possible to conclude
from our study whether there is a causal relationship
between these events, SOD data suggest that a bout of
eccentric exercise induces changes in NF-JB–mediated
gene expression and that submaximal eccentric training
may prevent effects on the NF-JB signaling cascades.
Because it has been well documented that a repeated
bout of the same eccentric exercise within several weeks
results in significantly less damage, the potential contribu-
tion of the repeated bout effect to the changes detected in
PBMC NF-JB activation after the proposed period of
eccentric training cannot be overlooked, and this should be
explored. Data obtained in study 2 demonstrate that damage
induced by the second bout was significantly less than the
damage induced by the initial one, with a significant
reduction in soreness, increased CK activity, and explosive
force loss, confirming similar results from the literature
(20,22). However, despite this protective effect, significant
NF-JB activation and overexpression of Mn-SOD were
detected in mononuclear cells. These findings indicate that
the repeated bout effect does not contribute to the
protection against acute exercise-induced changes in the
NF-JB signaling cascade observed in subjects who per-
formed a period of eccentric training at submaximal levels.
Our results are in agreement with those of Willoughby et al.
(29), who demonstrated that the mRNA and protein levels
of plasma interleukin-6, a well-known inducer of NF-JBactivation (15), were significantly elevated in response to
two eccentric exercise bouts performed 3 wk apart, with no
differences between the first and second bouts of eccentric
exercise. Nevertheless, it should be considered that effects
relative to NF-JB activation in blood cells also have been
reported after high-intensity cycling, which involves no
eccentric loading (7). Thus, it is possible that the molecular
effects detected in the present study were driven by
exercise stress and not specifically by the contractile mode;
this could explain why the repeated bout effect had an effect
on muscle damage but no impact on blood cell NF-JBsignaling. In fact, increases in NF-JB activation in blood
cells have been observed after a supramaximal cycling
sprint, reaching a peak value 1 h after the effort (7). How-
ever, given that the maximal increase in the present study
was detected 6 h after the eccentric protocol, we suspect
that the influence of effort-induced acute stress over
changes observed in NF-JB activation after an unaccus-
tomed eccentric bout may be minimal.
Despite the advances in recent years, the bases of
exercise-related NF-JB activation are elusive. It is possible
that the effect is triggered by a combination of multiple
factors, including intracellular ROS, which could act as a
second messenger, modulating the kinase activities of NF-JBsignaling pathways, IKK complex activation, and/or DNA
binding and transactivation activity of the NF-JB dimmers
(14). At this point, it should be noted that there are
conflicting results in the literature concerning oxidative
stress induced by eccentric exercise. Maughan et al. (19)
found that TBARS were significantly elevated after down-
hill running, and elevated lipid peroxidation has been
reported after eccentric exercise as measured by lipid
hydroperoxides (5). However, no change in MDA has been
found in blood or muscle in subjects after eccentric actions
with the knee extensors or the elbow flexors (4,17). The
lipid peroxidation data from the present study corroborate
such findings, showing no changes in TBARS levels after
an acute bout of eccentric exercise, either in pretraining or
posttraining conditions. Regarding protein carbonyls, it has
FIGURE 11—Western blot analysis of Mn-SOD in PBMC in response
to a repeated bout of eccentric exercise. Upper panel, RepresentativeWestern blot photographs. N = 9 for each time point. Lower panel,Results expressed as percentages of resting values (means T SEM).
* Significant change compared with resting values (P G 0.05).
http://www.acsm-msse.org662 Official Journal of the American College of Sports Medicine
BASICSC
IENCES
Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
been reported that high-force eccentric exercise can
increase blood protein oxidation (16), but our data point
to a nonsignificant increase immediately after and at 2 h
after the first eccentric bout. Indeed, the lack of signifi-
cance in the changes of plasma protein carbonyl concen-
tration might be caused by the activation of a mechanism
that removes the oxidatively modified proteins from the
circulation or, alternatively, activation of an antioxidant
mechanism that removes the ROS (3). A significant
training effect was observed both for TBARS and protein
carbonyl concentration, indicating that chronic training can
induce adaptations that attenuate oxidative stress. In fact,
several studies in humans have shown that regular exercise
reduces oxidative stress after acute exercise (24). In any
case, the present results do not clarify whether ROS
coming from the muscle could be directly involved in
NF-JB activation, because ROS formation may increase
without measurable signs of lipid peroxidation, and
potential differences may exist between tissues. A direct
measurement of ROS generation is needed to clarify this
point. Moreover, redox-sensitive signaling pathways often
use other activators, such as cytokines released in inflam-
mation process, to transfer signals from the cytosol to the
nucleus (27).
In summary, a single bout of acute eccentric exercise
induces muscular injury and changes in NF-JB activation
in PBMC. These alterations are significantly attenuated
as a result of submaximal eccentric training, indicating
that, although the molecular basis underlying the signal-
transduction pathway is largely unknown, NF-JB may be a
key transcription factor in the training-induced adaptation
process. Our data also indicate that the repeated bout effect
reduces symptoms of muscular damage but has no impact
on NF-JB activation in mononuclear cells.
REFERENCES
1. ANDREWS, N. C., and D. V. FALLER. A rapid micropreparation
technique for extraction of DNA-binding proteins from limit-
ing numbers of mammalian cells. Nucleic Acids Res. 11:2499,1991.
2. BYRNE, C., and R. ESTON. The effect of exercise-induced muscle
damage on isometric and dynamic knee extensor strength and
vertical jump performance. J. Sports Sci. 20:417–425, 2002.3. CHEVION, S., D. S. MORAN, Y. HELED, et al. Plasma antioxidant
status and cell injury after severe physical exercise. Biochemistry100:5119–5123, 2003.
4. CHILD, R., S. BROWN, S. DAY, A. DONNELLY, H. ROPER, and
J. SAXTON. Changes in indices of antioxidant status, lipid
peroxidation and inflammation in human skeletal muscle after
eccentric muscle actions. Clin. Sci. 96:105–1159, 1999.5. CHILDS, A., C. JACOBS, T. KAMINSKI, B. HALLIWELL, and
C. LEEWENBURGH. Supplementation with vitamin C and N-acetyl-
cysteine increases oxidative stress in humans after an acute
muscle injury induced by eccentric exercise. Free Radic. Biol.Med. 6:745–753, 2001.
6. CLARKSON, P. M., K. NOSAKA, and B. BRAUN. Muscle function
after exercise-induced muscle damage and rapid adaptation. Med.Sci. Sports Exerc. 24:512–520, 1992.
7. CUEVAS, M. J., M. ALMAR, J. C. GARCIA-GONZALEZ, et al. Changes
in oxidative stress markers and NF-kappaB activation induced by
sprint exercise. Free Radic. Res. 39:431–439, 2005.8. FRIDOVICH, I. Superoxide radical and superoxide dismutases.
Annu. Rev. Biochem. 64:97–112, 1995.9. GHOSH, S., M. J. MAY, and E. B. KOPP. NF-kappa B and Rel
proteins: evolutionarily conserved mediators of immune
responses. Annu. Rev. Immunol. 16:225–260, 1998.10. GOLDFARB, A. H., R. J. BLOOMER, and M. J. MCKENZIE. Combined
antioxidant treatment effects on blood oxidative stress after
eccentric exercise. Med. Sci. Sports Exerc. 37:234–239, 2005.11. HO, R. C., M. F. HIRSHMAN, Y. LI, et al. Regulation of IJB kinase
and NF-JB in contracting adult rat skeletal muscle. Am. J.Physiol. Cell Physiol. 289:C794–C801, 2005.
12. HOFMANN, M. A., S. SCHIEKOFER, M. KANITZ, et al. Insufficient
glycemic control increases nuclear factor-kappa B binding
activity in peripheral blood mononuclear cells isolated from
patients with type 1 diabetes. Diabetes Care 21:1310–1316,
1998.
13. HOLLANDER, J., R. FIEBIG, M. GORE, T. OOKAWARA, H. OHNO, and
L. L. JI. Superoxide dismutase gene expression is activated by a
single bout of exercise in rat skeletal muscle. Pflugers Arch. Eur.J. Physiol. 442:426–434, 2001.
14. JANSSEN-HEININGER, Y. M., M. E. POYNTER, and P. A. BAEUERLE.
Recent advances towards understanding redox mechanisms in the
activation of nuclear factor kappaB. Free Radic. Biol. Med.28:1317–1327, 2000.
15. JI, L. L., M. C. GOMEZ-CABRERA, N. STEINHAFEL, and J. VINA.
Acute exercise activates nuclear factor (NF)-kappaB signal-
ling pathway in rat skeletal muscle. FASEB J. 18:1499–1506,2004.
16. LEE, J., A. H. GOLDFARB, M. H. RESCINO, S. HEGDE, S. PATRICK,
and K. APPERSON. Eccentric exercise effect on blood oxidative-
stress markers and delayed onset of muscle soreness. Med. Sci.Sports Exerc. 34:443–448, 2002.
17. LENN, J., T. UHL, C. MATTACOLA, et al. The effects of fish oil and
isoflavones on delayed onset of muscle soreness. Med. Sci. SportsExerc. 34:1605–1613, 2002.
18. MALM, C., P. NYBERG, M. ENGSTROM, et al. Immunological
changes in human skeletal muscle and blood after eccentric
exercise and multiple biopsies. J. Physiol. 529:243–262, 2000.19. MAUGHAN, R. J., A. DONNELLY, M. GLEESON, P. WHITTING, and
K. WALKER. Delayed-onset muscle damage and lipid perox-
idation in man after a downhill run. Muscle Nerve 12:332,
1998.
20. MCHUGH, M. P. Recent advances in the understanding of the
repeated bout effect: the protective effect against muscle damage
from a single bout of eccentric exercise. Scand. J. Med. Sci.Sports 13:88–97, 2003.
21. PRITTS, T. A., E. S. HUNGNESS, D. D. HERSHKO, et al. Proteasome
inhibitors induce heat shock response and increase IL-6 expres-
sion in human intestinal epithelial cells. Am. J. Physiol. Regul.Integr. Comp. Physiol. 282:R1016–R1026, 2002.
22. PROSKE, U., and D. L. MORGAN. Muscle damage from eccentric
exercise: mechanism, mechanical signs, adaptation and clinical
applications. J. Physiol. 537:333–345, 2001.23. RADAK, Z., H. CHUNG, H. NAITO, et al. Age-associated increases in
oxidative stress and nuclear transcription factor (B activation are
attenuated in rat liver by regular exercise. FASEB J. 18:749–750,2004.
ECCENTRIC EXERCISE AND NUCLEAR FACTOR JB Medicine & Science in Sports & Exercised 663
BASIC
SCIEN
CES
Copyright @ 2007 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
24. RADAK, Z., A. W. TAYLOR, H. OHNO, and S. GOTO. Adaptation to
exercise-induced oxidative stress: from muscle to brain. Exerc.Immunol. Rev. 7:90–107, 2001.
25. RICHARD, M. J., P. GUIRAUD, J. MEO, and A. FAVIER. High-
performance liquid chromatographic separation of malondialdehyde-
thiobarbituric acid adduct in biological materials (plasma and
human cells) using a commercially available reagent. J. Chromatogr.577:9–19, 1992.
26. RICHLING, V. A., J. M. G. AREVALO, J. A. ZACK, and S. W. CODE.
Stress-induced enhancement of NF-JB-binding in the peripheral
blood leukocyte pool: effects of lymphocyte redistribution. BrainBehav. Immun. 18:231–237, 2004.
27. STEENSBERG, A., G. VAN HALL, T. OSADA, M. SACCHETTI, B. SALTIN,
and B. PEDERSEN. Production of interleukin-6 in contracting
human skeletal muscles can account for the exercise-induced
increases in plasma interleukin-6. J. Physiol. 529:237–242, 2000.28. VIDER, J., D. E. LAAKSONEN, A. KILK, et al. Physical exercise
induces activation of NF-JB in human peripheral blood lympho-
cytes. Antioxid. Redox Signal. 3:1131–1137, 2001.29. WILLOUGHBY, D. S., B. MCFARLIN, and C. BOIS. Interleukin-6
expression after repeated bouts of eccentric exercise. Int. J.Sports Med. 24:15–21, 2003.
30. YUHASZ, M. S. In: Physical Fitness Manual, Canada: Universityof Western Ontario, pp. 114–116, 1974.
http://www.acsm-msse.org664 Official Journal of the American College of Sports Medicine
BASICSC
IENCES
Top Related