as the longevity of humans increases , osteoarthritis ( oa ) has become a frequent disorder 
 among older adults .
oa is a disorder with a multifactorial origin that involves the 
 articular cartilage , synovium , subchondral bone , ligaments , and/or the neuromuscular 
 apparatus .
there are many risk 
 factors for the progression of knee oa , which include age , previous injury , increased body 
 mass index ( bmi ) , genetic factors , and high workload1 .
several studies have suggested that exercises such as joint - specific strength and general 
 aerobic conditioning are generally recommended for oa treatment2 .
also , several studies have shown that increased muscle 
 strength provides joint stability and reduces pain and disability3 , 4 .
the expected benefits of these 
 programs include reduction in pain , improved function , and altered joint load5 , 6 . fragments of structural proteins can be detected in serum as the reflection of cartilage 
 degeneration .
numerous studies have reported the discovery of putative biomarkers in blood 
 using a mass spectrometer7 , 8 .
there are three commonly used serum biomarkers associated 
 and/or correlated with oa and joint progression : antigenic keratin sulfate ( agks)9 , hyaluronan ( ha)10 , 11 , and cartilage 
 oligomeric matrix protein ( comp)12 . in this study ,
the serum levels of proteins in rats with oa were considered to reflect the 
 level of systemic inflammation .
therefore , we investigated the changes in serum protein 
 levels by proteomic analysis and confirmed one of the inflammatory proteins , c9 . in 
 addition , we further investigated the correlation between specific inflammatory protein 
 levels and treadmill exercise .
adult male sprague - dawley rats ( 810 weeks of age , weighing 250300  g , n = 30 ) were housed 
 at 22  c under a 12:12-h light 
louis , mo , usa cat # i2512 ) was used to induce 
 arthritis as previously reported13 , 14 .
rats were randomly divided into five groups : ( 1 ) no mia injection or exercise group 
 ( control group , n = 10 ) , ( 2 ) mia injection without treadmill running exercise group ( mia 
 injection group , n = 10 ) , ( 3 ) mia injection with low - intensity exercise group ( le , n = 10 ) , 
 ( 4 ) mia injection with middle - intensity exercise group ( me , n = 10 ) , and ( 5 ) mia injection 
 and with high - intensity exercise group ( he , n = 10 ) . the running protocol was performed 
 according to a modified version of ueda s method15 , 
 16 . all procedures followed daegu 
 university s guidelines and were approved by the institutional animal care and use committee 
 ( iacuc ) .
liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) analysis was carried out using an 
 ltq mass spectrometer ( thermo electron , san jose , ca , usa ) equipped with nsi sources ( thermo 
 electron , san jose , ca ) , as previously reported17 .
briefly , 12 ul of peptide sample from in - gel digestion was 
 injected and loaded onto a peptide trap cartridge ( agilent , palo alto , ca , usa ) .
trapped 
 peptides were eluted onto a 10-cm rp picofrit column packed in - house ( 5  mm , 300a pore size , 
 c18 ) and then separated by gradient elution .
the mobile phases consisted of h2o 
 ( a ) and acn ( b ) , and both contained 0.1% v / v formic acid .
the spray voltage was 1.9 kv , and the temperature of the ion transfer tube was 
 set at 195c .
the gradient started at 2% b and reached 60% b in 50  min , 80% b in the next 
 5  min , and 100% a in the final 15  min .
full ms spectra ( ms survey scan ) were recorded in the 
 ion trap cell , and data - dependent ms / ms spectra for the five most intense ions were 
 subsequently acquired by the ltq under the 35  ev of collision energy and an isolation with 
 of 2 da . to validate c9 protein ,
we used western blot analysis , which was performed as previously 
 reported17 . a western blot analysis 
 system ( amersham biosciences , little chalfont , buckinghamshire , uk ) was used to detect 
 immunoreactive proteins .
the thickness of the bands was photographically measured using 
 scion image ( scion , frederick , md , usa ) .
an image of a calibration grid slide was measured 
 using scion image , and the distance of 250  mm was calculated to be equivalent to 733 pixels . 

one - way anova was used to evaluate the statistical significance of differences among three 
 or more groups .
all analyses were done with origin 8.0 , and p - values0.05 were considered 
 statistically significant .
blood samples were obtained from individuals with or without exercise followed by the serum 
 collection protocol . using 1-d sds - page ,
40 ug proteins of pooled serum from each of the 
 five groups were separated , and the gels were stained with coomassie brilliant blue staining 
 solution . to identify the proteins ,
gels were excised into five bands and subjected to 
 in - gel digestion , and then tryptic peptides were analyzed by lc - esi - ms / ms analysis .
the digested protein samples were run on an lc - esi - ms / ms .
proteins identified by lc - ms / ms analysis that increased in the mia injection 
 group by over 1.5-foldidentified proteins ( 17)accession numbermolecular weightnamialmhfold changegene_symbol = c9 c9 proteinipi0023142364 kda03000.gene_symbol=apoa4 apolipoprotein a - ivipi0032427244 kda012707.gene_symbol=serpinf2 serine ( or cysteine ) peptidase inhibitor , 
 clade f , member 2ipi0019969555 kda011690.gene_symbol=serpina4 kallistatinipi0020556848 kda010003.gene_symbol=serpina3k serine protease inhibitor a3  kipi0020059347 kda18643334393.557gene_symbol = ttr transthyretinipi0032438016 kda6191515283.255gene_symbol = loc299282 liver regeneration protein lrryanipi0039221668 kda21653334333.014gene_symbol = c4b complement c4ipi00213036192 kda10242721222.487gene_symbol = igh-6 igh-6 proteinipi0036888868 kda8212121242.453gene_symbol = hp isoform 2 of haptoglobinipi0038220242 kda8211923272.453gene_symbol = igg-2a loc367586 proteinipi0036134651 kda29667158712.299gene_symbol = afm afaminipi0020766869 kda71352541.810gene_symbol = loc678701 39 kda proteinipi0055759839 kda611149131.809gene_symbol = plg plasminogenipi0020678091 kda1118152691.709gene_symbol = itih3 inter - alpha - trypsin inhibitor heavy chain h3ipi0032698499 kda1322411181.644gene_symbol = f2 prothrombin ( fragment)ipi0018998170 kda580501.582gene_symbol = hpx hemopexinipi0019551651 kda25382755201.507fold - change = quantitative peptide hits of the mia group / quantitative peptide hits of 
 the control group shows the upregulated proteins identified by the lc - ms / ms analysis .
seventeen 
 proteins showed significant elevation in the samples from the mia injection group , with the 
 levels being 1.5-fold higher than those of the control group ( table 1 ) .
fold - change = quantitative peptide hits of the mia group / quantitative peptide hits of 
 the control group among the 17 upregulated proteins , 9 proteins increased in the mia injection group compared 
 with both the control group and exercise applied groups : complement component 9 , 
 apolipoprotein a - iv , serine peptidase inhibitor , kallistatin , serine protease inhibitor 
 a3  k , liver regeneration protein lrryan , complement c4 , interalpha - trypsin inhibitor heavy 
 chain h3 , and prothrombin ( table 1 ) . among the list of proteins upregulated in the mia injection group , c9
was first selected 
 for validation because it was at the top of the list ( table 1 ) . to validate the serum levels of c9 ,
the protein levels were analyzed by 
 western blot analysis in a total 50 serum samples , which comprised samples from the control 
 ( n=10 ) , mia injection ( n=10 ) , le ( n=10 ) , me ( n=10 ) and he groups ( n=10 ) .
( b ) the western blot data of the crude 
 sera of 10 individuals from each group were subjected to densitometry analysis .
the 
 density and area of the bands of the western blot were measured by using scion image , 
 as described in the materials and methods .
( a ) representative 
 western blot of crude sera from each group . ( b )
the western blot data of the crude 
 sera of 10 individuals from each group were subjected to densitometry analysis . the 
 density and area of the bands of the western blot
were measured by using scion image , 
 as described in the materials and methods . in the results ,
c9 significantly decreased in the me group compared with the mia injection 
 group ; however , the c9 level in the le / he groups did not show significant changes ( fig .
oa is a common chronic degenerative disease characterized by the loss of articular 
 cartilage components due to many reasons . according to studies of oa biology
the objective of this study was to determine the change in serum protein levels in a rat 
 osteoarthritis model with or without exercise .
therefore , we investigated the proteome 
 profiles in serum samples using lc - ms / ms .
the proteome profile results showed that many 
 serum protein levels were changed ; some proteins were increased and others were 
 decreased . among the upregulated proteins , 9 proteins showed increases compared with the control group 
 and decreases in the exercise intervention group ( table 
 1 ) . among these 9 proteins , c9 is well known to be a terminal member in cell 
 killing through the action of the membrane attack complex , which consists of c5b , c6 , c7 , 
 c8 , and c918 .
we focused on the change in 
 c9 expression in the inflammation environment caused by mia injection .
the results showed that c9 expression in the mia injection 
 and 3 exercise groups changed significantly compare with the control group .
only the me 
 group showed a significantly reduced increase in c9 expression after mia injection . also , 
 the c9 level of the me group was significantly lower than those of the le / he groups . 
 however , the le and he groups did not show significant decreases in the c9 level due to 
 induction by mia ( fig .
c9 is one of the proteins in the complement system , which is a well - known 
 inflammatory - related pathway . during late complement pathway processes , which involve c5-c9
, 
 the inflammation sites recruit c5a by chemotaxis , and this leads to the formation of 
 terminal complement complex ( tcc)19 .
oa 
 is a systemic inflammatory disease ; therefore , the complement system may be an important 
 effector system in various arthritides . based on our results ,
it is possible that the c9 
 protein level was increased by the systemic response to the induced oa .
further , the 
 increased level of c9 decreased due to exercise , which indicates improvement of the oa 
 condition .
in several previous studies , oa cartilage degeneration was examined based on certain 
 protein levels in the serum . in a previous study ,
. therefore , investigation of whole serum proteins in rats with oa would provide 
 supportive information for such biomarkers . in this study
therefore , due to the masking 
 effects of several highly abundant proteins , such as albumin and iggs , proteins present at 
 low levels that might be potential novel biomarker candidates , could not be detected .
it 
 appears to be necessary to discover more proteins with different serum preparation methods 
 and to confirm them in human patients with oa .
also , many other different experimental 
 methods could be used in the study of correlation between exercise and oa .
one recent study 
 suggested that the glycosamionoglycan content in cartilage increases after exercise21 , and another showed that increased muscle 
 strength provides joint stability and reduces pain and disability4 .
here , we showed that treadmill exercise could decrease the inflammatory response protein c9 
 in serum of an oa rat model , which demonstrates that appropriate intensity exercise might be 
 beneficial for patients with oa .
   [ purpose ] to investigate the correlation between the effect treadmill exercise and 
 change in serum proteins in rats with osteoarthritis , a study of proteins was carried out 
 using a mass spectrometer . 
 [ subjects and methods ] rats were randomly divided into five 
 groups . 
 after 4 weeks of treadmill training , serum from each rat was analyzed by liquid 
 chromatography - electrospray ionization tandem mass spectrometry . 
 complementary component 9 
 ( c9 ) was discovered to be downregulated in the serum of the exercise groups , and this was 
 validated by western blot . [ results ] seventeen proteins 
 were discovered to be elevated in 
 the monosodium iodoacetate injection osteoarthritis group samples by more than 1.5 fold 
 compared with the control group . 
 one of the proteins upregulated , c9 protein , was 
 validated , and it was found to decrease in the middle - intensity exercise group . 
 
 [ conclusion ] we showed that the serum level of c9 , an inflammatory - related protein , 
 decreased after treadmill exercise . 
 therefore , treadmill exercise with an appropriate 
 intensity might be recommended for oa patients . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION