
 exercise has positive and negative effects on immune system , susceptibility to minor illnesses .
regular moderate exercise is also associated with a reduced incidence of infection compared with a completely sedentary state . on the other hand , post exercise immune system dysfunction
is mostly pronounced when the exercise is continuous and prolonged ( > 1.5 h ) , of moderate to high intensity ( 5575% maximum o2 uptake ) , and performed without food intake ( 1 ) . 
 

one of the most important functions of immune system is to produce soluble or cellular factors that provide the immunity against inflammatory agents .
some investigators believe that omega-3 fatty acids have a major effect on decreasing inflammation ; on the other hand omega-6 fatty acids consumption can lead to tissue damage and susceptibility for inflammatory disease .
as omega-3 fatty acids are not synthesized in body , they must be supplied in food .
omega-3 fatty acids exert their biologic effects by changing components and fluidity of cellular membrane lipids bilayer or by directly providing the metabolic substrate .
the results of some studies show that increasing consumption of omega-3 fatty acids may increase the body resistance against oxidative stress ( 2 , 3 ) .
eicosapentaenoic acid ( epa ) is an unsaturated fatty acid that inhibits the arachidonic acid metabolism and subsequently causes decreased leukotriene 4 , prostaglandin e2 and inflammatory cytokines production .
although docosa - hexaenoicacid ( dha ) does not have a direct effect on producing eicosanoids , but it plays an essential anti - inflammatory role by modifying cellular translation factors ( 4 ) .
although it is believed that the inflammation is beneficent to the host defence system ; however , it also acts as an important factor in many chronic diseases such as cancer , cardiovascular disease and diabetes ( 5 ) .
it has been reported that exercise can increase the inflammatory and pre - inflammatory cytokines much like infection or tissue damage ( 6 ) . 
 

the cytokines , such as interlukin-17(il-17 ) act as an intercellular messenger molecules , when attached to the target cell , initiate their biologic effects .
several experimental and clinical studies show that il-17 family members are involved in specific inflammatory processes .
most published evidence supports a role for il-17 , as a promoter of granulopoiesis , neutrophil accumulation , and neutrophil activation in the lung , joint space , central nervous system , and intestinal tissue .
although il-17 is a t cell - derived , pro - inflammatory cytokine , but is suggested to be involved in the development of various inflammatory diseases ( 4 , 7 ) , il-6 secretion ( 8) and neutrophil activation ( 9 ) .
c - reactive protein ( crp ) is an acute phase protein which increases remarkably during infection , inflammation , and tissue damage ( 8) . it is mainly produced and secreted into circulation by liver in response to circulatory inflammatory mediators ( 10 ) .
crp level is the best indicator to determine tissue damage because crp increased immediately at early phase of tissue deterioration ( 11 ) .
recent studies reveal that crp not only is an inflammatory mediator , but it plays very important role in inflammation ( 12 ) .
the injured muscle also releases protein such as creatinekinase ( ck ) and myoglobin into blood circulation ( 11 , 13 ) . 
 

immediately after marathon race , the level of crp does not change , however , after the ultra - marathon race its level increases remarkably .
after the end of ultra - marathon race the level of ck increases by 35 times , but after marathon race its levels increases only by 4 times ( 13 ) .
it is also demonstrated that 12 weeks of intensive exercise increased il-17 levels , but in another group which had moderate exercise no change was observed ( 14 ) .
the effect of exercise along with fish oil supplement consumption on inflammatory mediators levels has not been investigated . in this study we investigated the effect of incremental aerobic exercise accompanied with fish oil supplement consumption on il-17 levels and some inflammatory markers . 


 fifty four adult healthy male mice ( age : 2 months old ) were used in this study ( table 1 ) .
this study protocol has been approved by shiraz medical sciences university ethics committee of experimental animals ( shiraz , iran ) . 
 
 at first 10 mice
were killed to determine the base line values and rest of them were divided randomly into four groups , control ( c , n=11 ) , supplement ( s , n=11 ) , training ( t , n=11 ) and supplement - training ( st , n=11 ) .
the mice were kept in separate cages ( each cage 6 mice ) , at ambient temperature of 23c with a 12-h/12-h light/ dark cycle .
the animals had free access to water and pellet chow containing of calcium and phosphorus . 
 

incremental aerobic exercise was performed using an animal treadmill system which allows 7 mice to run at the same time . in this study , we used a training protocol for treadmill running described by rico et al
the steep grade treadmill incline was used to stimulate high - intensity muscle activity in mice .
the mice started exercise trainings to be familiarized with treadmill running for a week . within the beginning of the familiarization period the speed , steep and running time of treadmill was 5 m/s , 0 degree and 10 minutes respectively . at end of the familiarization period the speed , steep and running time of treadmill
was increased to 7 m/s , 5 degree and 15 minutes respectively . in the principal protocol , running time was gradually increased from 15 min to 30 and 60 min per session for t , st groups .
treadmill speed increased up to 27 m/s with increments of 2 - 3 to reach a final grade inclination of 18. the chronological progression in the treadmill speed , grade inclination , and running time are shown in the table 2 .
control mice were kept in the cages at the same environmental conditions and inspected daily to control their health . 
 
 0.2 ml ( 0.06ml / g ) fish oil which contains epa and dha
was given to two groups of supplementation ( s ) and supplementation - training ( st ) daily for 8 weeks . 
 
 in order to determine the levels of variables , in each trial , 10 mice were killed for blood sampling ( for a total of 5 times including before starting the training protocol , once at each 2 weeks and at the end of the protocol ) .
serum il-17 levels were measured by elisa method using commercial elisa kits ( mouse il-17- kit - id labs inc , hungary ) and also crp was measured by ( crp - kit - neflometry , uk ) . in order to evaluate local inflammatory state of skeletal muscles , ck activity also was assessed by biochemical technique using uv - visible spectrophotometer ( shimadzu uv-1601 , japan ) . 
 

the values are presented as the mean  standard error of mean ( sem ) .
tukey 's hsd post hoc test was used in circumstances which there were significant differences between experimental groups , because of the equality of mice at each group . the 0.05 level of significance
the values are presented as the mean  standard error of mean ( sem ) .
tukey 's hsd post hoc test was used in circumstances which there were significant differences between experimental groups , because of the equality of mice at each group . the 0.05 level of significance

 there were no significant differences between the base line values in all groups ( table1 , 3 ) . at the end of the study , the results showed a significant difference in il-17 levels between groups ( c : 1210.52 , s : 771.29 , t : 1300.91 , st : 1302.25pg / ml ; figure 1a ) ( p<0.05 ) .
the result of post hoc test revealed significant differences between the control group with other groups , the training group with the supplement group and also the supplement group with the supplement - training group .
the maximum level of il-17 was seen in the fourth blood sampling of the training group ( 1380.65pg / ml ) , and the minimum level belonged to the fourth sampling of the supplement group ( 760.65pg / ml ; fig .
there were significant differences for the crp levels between the two groups of training and supplement - training ( t : 2.10.59 mg / l , st : 1.4  0.10 mg / l ) and also the other two groups ( s:0.250.56 mg / l , c : 1.00.64mg / l ) ( p<0.05 ) . 
 

comparison il-17(pg / ml ) , cpk(ul ) , crp(mg / l ) concentrations in 5 samplings between groups 
 
 the maximum level of crp belonged to the training and the supplement - training groups in the third sampling ( 2.50.6 ml / l and 2.40.26 mg / l respectively ) , and the minimum level belonged to the supplement group in fourth and fifth samplings ( 0.250.11 , 0250.57mg.l respectively)(fig .
we also observed that there were significant differences in ck levels between the supplement ( 583.11ul ) and other groups ( c : 123 4.3 , t : 2032.96 , st : 142 4.21 ul ; table 3 ) ( p<0.05 ) .
our results showed that there were significant differences between the supplement group and other groups and also between the training group and the supplement - training group .
1.c , maximum level of ck was seen in training group in fifth sampling ( 2032.96u / l ) , and the minimum level belonged to the fourth sampling of the supplement group ( 57.75 3.47u / l ; fig .

 the results of the present study show that fish oil supplement consumption accompanied with incremental aerobic exercise significantly modulate inflammatory response by suppressing il-17 , crp and ck that are usually increased following intensive exercise ( 16 ) .
although preinflammatory cytokines production is necessary for immune defence , but over production of these cytokines may lead to inflammation and subsequently causing skeletal muscle damage , weakness and increased risk of infection ( 11 ) .
some researchers believe that the levels of il-17 may be a useful biochemical index to determine the produced acute inflammation in skeletal muscles ( 10 ) . 
 
 in some studies
, the effect of training intensity on il-17 as a major factor has been considered and the results showed that the levels of this cytokine increased in group that had strenuous training , but in another group no change was observed ( 14 ) . a similar pattern was also observed in our study with the increase in aerobic training ( increase in speed , steep and time of training ) ( fig.1.a ) .
in contrast to the result of this study , golzari et al ( 2010 ) found that applying compound exercise program ( including warming up , stretch trainings , aerobic trainings , strength trainings and relaxation trainings at the end of each session ) did not increase il-17 levels , and even in some cases , some decrease was observed which was attributed to the intensity of training ( 18 ) . in our study , the finding can be due to adaptation of animals to exercise .
on the other hand , levels of this cytokine in the supplement group are lower than other groups at the end of 8 week exercise .
these two conflicting result in both groups of training and supplement may be due to the supplement consumption .
however , the difference between two groups of supplement and training must be considered . despite supplement consumption , the level of this cytokine in the supplement - training group is higher than the supplement group .
the levels of il-17 decreased in the third sampling of the st group , but a significant increase was observed in fourth and fifth samplings that may be related to the amount of supplement consumption that was not enough to suppress inflammation induced by incremental exercise . 
 

distance of running must be considered as well . as the intensity and time of exercise increases , the level of this cytokine also increases . 
 

duzova et al ( 2009 ) have reported that strenuous exercises increase the il-17 production , but the moderate exercises do nt increase the il-17 production ( 14 ) .
possible involved mechanism could be releasing pre - inflammatory cytokines following strenuous exercise , which makes anti - inflammatory cytokines production such as il-2 , il-6 and il-10 .
it seems that sequence of production of pre - inflammatory and anti - inflammatory cytokines is the reason of production of il-17 by blood and skeletal muscle peripheral leucocytes ( 14 , 19 ) .
intensity and duration of exercise is an important factor that contributes in increasing il-17 production . in accordance to recent
result the finding showed that omega-3 supplement consumption can somehow prevent the increase of this cytokine
. 
 
 the normal level of crp is about 0.8 - 5 mg / l in adults , but it may be increased during infection or physical activity by 100 times , because the crp production depend on various agents such as cytokine production ( 13 ) , as it 's observed that omega-3 consumption for 8 weeks decreases crp levels(20 ) .
lakka et el ( 2005 ) reported that 6 gms epa consumption per day for 8 weeks decreases crp level from 11 mg / l to 8 mg / l ( probably due to il-6 decreasing ) ( 21 ) . in fig .
1.b , we can observe that crp levels are within normal range in all groups .
the level of crp in both supplement and control groups is lower than other groups and in the supplement group at least two samplings ( 4th and 5th blood samplings ) showed the lowest rate .
thus according to the results published in earlier studies , the duration of supplement consumption is an essential factor . in our study , the level of this protein was decreased after 8 weeks , but in some previous studies contrasting results has been observed ( 22 , 23 ) .
additionally , there is a non - significant relation between crp and dha levels ( 23 ) , and the fact that 4 grams of fish oil consumption per day does nt change crp levels in obese subjects ( 22 ) . 
 

kim et al ( 2009 ) investigated the effect of distance in a marathon ( 42.195 km ) and ultra - marathon ( 200 km ) on this protein levels , and they found that after marathon race , the crp level does n't change , but after a day its level increased 3.4 times , and after 4 days it was recovered to its basal level .
they also found that after ultra - marathon race , the crp level was increased by 40 times , and remained nearly at noticed range up to 6 days after race ( 13 ) . 
 

there was an increase in the supplement - training group similar to the training group in first three samplings , but in the second four weeks , the level of crp gradually decreased .
it can be suggested that fish oil supplement consumption to some extent can downregulate exercise induced inflammatory response , consequently decreasing crp secretion . 
 

evidence showed that intensive exercise may lead to muscular damage at microscopic level , and finally resulting in inflammation .
as noticed , when exercise causes muscle damage , the activity of ck increases in blood .
creatine kinase is usually considered as reliable biomarker in determining muscle damage ( 24).the effect of two types of exercise ( eccentric and concentric ) on some anti - inflammatory indexes has shown a significant relation exists between il-6 increase and muscle damage measured by ck levels . in another study it was distinguished that carrying out 3 times 90 minutes - exercise training along with carbohydrate supplement cause a significant decrease at second and third times , but no change was observed at first time ( 25 ) . in another study ,
the results showed that there is a direct relation between the intensity and duration of exercise and cpk levels ( 26 ) . in the present study the lowest level of cpk in different phases
comparing cpk levels in two supplement - training and training groups distinguishes that the cpk increase in the supplement- training group has been decreased by supplement consumption whereas in the training group the cpk level has not been decreased .
the results in the training group shows that if increasing cpk is considered as an index for showing inflammation , it can be said that increased cpk levels reflects the micro - tears of skeletal muscle damage in mice .
a recent study revealed that the time period of supplementation is a critical cause for changes in second and third blood samplings .
( 27)the extent and range of injury in different types of exercise modality is dependent on various factors such as duration , intensity , the type of exercise , gender and the level of subject s physical fitness ( 13 , 26 , 27 ) . 


 fish oil supplement consumption downregulates incremental aerobic training induced il-17 plasma levels increase . on the other hand ,
moreover , production of these il-6 and il-17 lead to crp secretion which increases ck secretion . as shown in the present study
, a direct and significant correlation was observed between the changes of crp and ck ( 18 ) .
the results of present study should be confirmed by forthcoming studies that investigate the role of fish oil supplement on il-17 and its role in acute inflammatory process of skeletal muscle that takes place with exercise . 

 
 background : exercise has positive and negative effects on immune system . 
 herein , we would like to investigate the effects of incremental aerobic training and fish oil supplementation on the plasma levels of crp , cpk and il-17 in trained mice . 
 one of the major roles of immune system is to produce soluble or cellular components that provide the immunity against inflammatory agent . 
 the purpose of this study is to investigate distinct and combine effects of incremental aerobic training and fish oil supplement on plasma levels of il-17 , cpk and crp in trained male mice . 

 
 methods : totally , 54 healthy male mice ( 2 months old , weight= 341 grams ) were selected . at first 10 mice 
 were killed to determine base line values , the rest of them were randomly divided into four groups , control group ( c , n=11 ) , supplement group ( s , n=11 ) , training group ( t , n=11 ) and supplement - training group ( st , n=11).the supplement and supplement - training groups were fed with 0.2cc / day fish oil for 8 weeks . 
 training and supplement - training groups underwent exercise for 5 sessions per week for a period of 8 weeks on animal treadmill . 
 spss 16.0 software and multivariate analysis of variance were used for statistical analysis of data 

 results : exercise and fish oil supplement lead to a decrease in crp levels and subsequently causing a reduction in plasma levels of il-17 and ck in mice ( p<0.05 ) . 

 
 conclusion : combination of exercise and fish oil can reduce regulate inflammatory response caused by incremental exercise . 
 

Introduction


Methods


Statistical Analysis


Results


Discussion


Conclusion
