Effects of eccentric exercise on toll-like receptor 4 signaling pathway in peripheral blood...

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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

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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

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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).

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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).


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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).


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(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).


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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).


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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).


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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).


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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).


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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.

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