doxorubicin has been used extensively for chemotherapy of cancer by the mechanism through which it systemically inhibits cell proliferation in vivo .
one of the most devastating symptoms associated with mortality of cancer patients during the chemotherapy is development of sarcopenia.1 in humans , survival time in patients who were developed sarcopenia was significantly shorter than in patients who were not developed sarcopenia.2 doxorubicin has been reported to cause muscle weakness and increase proinflammatory tumour necrosis factoralpha ( tnfalpha ) level.3 evidence from tumourfree animal study suggests that the sarcopenic effect of doxorubicin is not associated with tumour growth.1 
  muscle size is maintained by a dynamic balance between muscle loss and regeneration .
a restoration mechanism to encounter increased entropy of recruited muscle during daily physical challenge is essential , which is currently found to associate with macrophagemediated inflammation.4 , 5 , 6 macrophage functions to recognize unhealthy cells , activate phagocytosis , regenerate tissue , and finally resolve inflammation.4 , 7 distribution of m1 ( cd68 ) and m2 ( cd163 ) macrophages in inflamed muscle tissue regulates the development of skeletal muscle after damage.8 , 9 m1 macrophage is responsible for phagocytic events during early phase of inflammation , and m2 macrophage presents during regenerative phase of inflammation.4 
  q10 is currently considered as an adjuvant option in hope to minimize unwanted side effect during doxorubicinbased anticancer therapy,10 according to previous results on decreased toxicity of multiple organs during doxorubicin treatment.11 , 12 how doxorubicin may influence macrophage distribution during recovery process of eccentric exercisechallenged skeletal muscle and whether q10 supplementation can modulate this influence have not yet been examined .
exercise containing eccentric muscle contraction ( such as downhill running and resistance exercise ) has been shown to cause muscle inflammation,13 which is also known to be an effective way to increase muscle mass during training.14 in this study , we hypothesized that ( i ) doxorubicin can alter distribution of m1 macrophage and m2 macrophage in skeletal muscle after eccentric exercise ; ( ii ) q10 supplementation can modulate inflammation , prevent muscle loss , and increase survival rate in doxorubicintreated tumourfree rats .
this study was conducted in accordance to guidelines in the care and use of animals conformed to taiwan government law , and was approved by the animal care and use committee at university of taipei ( approval number 102001 ) .
all sprague dawley rats ( aged between 4 and 5  months ) were obtained from lasco corporation ( taipei , taiwan ) .
sixty four male sprague dawley ( sd ) rats ( body weight 534    5  g ) were used for exercise challenge study to trigger muscle inflammation .
fifty six rats ( body weight 485    4  g ) were used for survival experiment , to determine the protective effect of q10 on survival during a longterm doxorubicin treatment .
all rats from above studies were evenly allocated into the following four groups : control ( con ) , doxorubicin ( dox ) , q10 , and dox  +  q10 .
two animals were housed per cage with standard laboratory chow ( pmi nutrition international , brentwood , mo , usa ) and tap water ad libitum .
all animals were kept in an animal room with a 12/12  h light / dark cycle , 23    2  c and 50% relative humidity for approximately one month until experiment . to determine the effect of doxorubicin and q10 on eccentric exerciseinduced muscle inflammation , a dose of doxorubicin ( actavis , italy spa . ,
milano , italy ) at 2.5  mg per kg body weight was intraperitoneal injected according to a method reported elsewhere,15 and a dose of q10 ( pharmaessentia , taipei , taiwan ) at 10  mg per kg body weight was orally delivered by stomach tube in solution.11 
  half of rats from each group were challenged to a downhill treadmill running according to previously published methods ( yu et al . , 2014 ) .
one week prior to experiments , all animals were acclimated to running on a rat treadmill at 10  m / min for 10  min per day .
exercises consisted of 18 sessions ( 5  min / session ) separated by 2  min of rest at 16  m / min for a total of 90  min .
an acute dose of doxorubicin or q10 was delivered 16  h before exercise challenge .
twenty four hours after exercise , rats were anaesthetized with intraperitoneal injection of zoletil ( 50  mg per kg body weight ) .
soleus muscles were surgically collected from both legs and frozen immediately with liquid nitrogen and then stored at 80  c for further histology , immunohistochemistry ( ihc ) , and western blotting analysis .
rats were used to determine whether oral q10 supplementation has beneficial effect for doxorubicin treated rats .
q10 was orally delivered at 0930  am daily , whereas doxorubicin was injected every 3  days .
muscle mass was measured using dualenergy xray absorptiometry ( dxa ) ( lunar idxa , ge medical systems , madison , wi ) , operated with software version 13.60.033 ( encore 2011 ) .
histology and ihc staining were carried out by a professional pathologist at china medical university hospital ( taichung , taiwan ) .
tissue sections were cut in 2 to 5  m slices and transferred onto coated slides ( super frost plus , braunschweig , germany ) .
antigen retrieval occurred in boiled water for 15  min in 0.1  m sodium citrate ( ph  7.2 ) .
these pretreated slides were blocked for 15  min at room temperature with 5% bsa and then incubated at 4  c overnight with primary antibodies : antibodies against rat cd68 ( dilution 1:50 ) ( abcam , cambridge , uk ) and rabbit cd163 ( dilution 1:50 ) ( abd serotec , kidlington , uk ) .
briefly , specific antibody was purchased to perform ihc staining by using horseradish peroxidaseconjugated avidin biotin complex ( abc ) from the vectastain elite abc kit ( vector laboratories , burlingame , ca ) and dab chromogen ( vector laboratories).13 the sections will be counterstained with haematoxylin and mounted .
inflammation grade was evaluated using a standard semiquantitative scoring system , score 0 : none ; score 1 : giant cells , lymphocytes , plasma cells ; score 2 : giant cells , eosinophil , neutrophil ; score 3 : many inflammatory cells , microabscess .
soleus muscle ( ~100  mg ) was homogenized in 1  ml of he s buffer containing 20  mm hepes ( sigmaaldrich , st louis , mo , usa ) , 1  mm edta ( sigmaaldrich , st louis , mo , usa ) , and 250  mm sucrose ( sigmaaldrich , st louis , mo , usa ) .
homogenates containing 50  g of protein were fractionated by sdspage ( 1012% acrylamide ) and transferred to pvdf membranes ( pall life science , ann arbor , mi , usa ) by standard wet transfer methods . after 1  h blocking in pbs containing 5% skim milk , membranes were incubated with primary antibody overnight at 4  c .
primary antibodies against nitrotyrosine were purchased from millipore ( bedford , ma , usa ) ( dilution 1:1000 ) ; antibodies against manganesedependent superoxide dismutase ( mnsod ) ( dilution 1:5000 ) were purchased from enzo life sciences ( ann arbor , mi , usa ) ; and antibodies against gcs ( dilution 1:100 ) were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) .
antibodybound protein was detected using a peroxidaseconjugated antimouse secondary antibody ( sigma , st louis , mo , usa ) or antirabbit igg ( cell signaling technology , beverly , ma , usa ) and enhanced chemiluminescent hrp substrate ( perkinelmer life and analytical sciences , shelton , ct , usa ) . to verify equal protein loading , gapdh ( sigma , st louis , mo , usa ) ( dilution 1:5000 )
western blot bands were quantified using a chemidoc xrs+ system ( biorad , hercules , ca , usa ) .
total rna was isolated using qiagen rneasy fibrous tissue mini kit ( qiagen , valencia , ca , usa ) .
reverse transcription of rna was carried out using iscript cdna synthesis kit ( biorad , hercules , ca , usa ) .
the following primer and probe sequences were used for amplification of target genes : tnf ( nm_012675 ) forward primer : gagtcattgctctgtgag ; reverse primer : ctctgaggagtagacgata ; probe : ctggcgtgttcatccgttctct .
il10 ( l02926 ) forward primer : gatccagagatcttagcta ; reverse primer : ctgaggtatcagaggtaa ; probe : aacctcgtttgtacctctctccaa .
taqman probes contained a fam reporter at the 5end and a tamra quencher at the 3end .
the rtqpcr conditions have been previously described.16 gene relative expression levels were determined by the change in crossing points of reaction amplification ( ct value ) between targets mrna and 18s mrna for each treatment compared with the control group .
analysis of variance ( anova ) was used to compare group difference for all variables .
a twoway anova was used to determine the main effect and interactive effect of treatments . a oneway anova with a repeated measure
was used to compare the mean differences in muscle mass between pre and post during longterm doxorubicin treatment .
fisher 's protected least significant test , which holds the value of type i error to 0.05 for each test , was used to distinguish the differences between pairs of groups .
a level of p  <  0.05 was considered significant on all tests , and all values are expressed as means    standard errors .
this study was conducted in accordance to guidelines in the care and use of animals conformed to taiwan government law , and was approved by the animal care and use committee at university of taipei ( approval number 102001 ) .
all sprague dawley rats ( aged between 4 and 5  months ) were obtained from lasco corporation ( taipei , taiwan ) .
sixty four male sprague dawley ( sd ) rats ( body weight 534    5  g ) were used for exercise challenge study to trigger muscle inflammation .
fifty six rats ( body weight 485    4  g ) were used for survival experiment , to determine the protective effect of q10 on survival during a longterm doxorubicin treatment .
all rats from above studies were evenly allocated into the following four groups : control ( con ) , doxorubicin ( dox ) , q10 , and dox  +  q10 .
two animals were housed per cage with standard laboratory chow ( pmi nutrition international , brentwood , mo , usa ) and tap water ad libitum .
all animals were kept in an animal room with a 12/12  h light / dark cycle , 23    2  c and 50% relative humidity for approximately one month until experiment .
to determine the effect of doxorubicin and q10 on eccentric exerciseinduced muscle inflammation , a dose of doxorubicin ( actavis , italy spa . ,
milano , italy ) at 2.5  mg per kg body weight was intraperitoneal injected according to a method reported elsewhere,15 and a dose of q10 ( pharmaessentia , taipei , taiwan ) at 10  mg per kg body weight was orally delivered by stomach tube in solution.11 

half of rats from each group were challenged to a downhill treadmill running according to previously published methods ( yu et al . , 2014 ) .
one week prior to experiments , all animals were acclimated to running on a rat treadmill at 10  m / min for 10  min per day .
exercises consisted of 18 sessions ( 5  min / session ) separated by 2  min of rest at 16  m / min for a total of 90  min .
an acute dose of doxorubicin or q10 was delivered 16  h before exercise challenge .
twenty four hours after exercise , rats were anaesthetized with intraperitoneal injection of zoletil ( 50  mg per kg body weight ) .
soleus muscles were surgically collected from both legs and frozen immediately with liquid nitrogen and then stored at 80  c for further histology , immunohistochemistry ( ihc ) , and western blotting analysis .
rats were used to determine whether oral q10 supplementation has beneficial effect for doxorubicin treated rats .
q10 was orally delivered at 0930  am daily , whereas doxorubicin was injected every 3  days .
muscle mass was measured using dualenergy xray absorptiometry ( dxa ) ( lunar idxa , ge medical systems , madison , wi ) , operated with software version 13.60.033 ( encore 2011 ) .
histology and ihc staining were carried out by a professional pathologist at china medical university hospital ( taichung , taiwan ) .
tissue sections were cut in 2 to 5  m slices and transferred onto coated slides ( super frost plus , braunschweig , germany ) .
antigen retrieval occurred in boiled water for 15  min in 0.1  m sodium citrate ( ph  7.2 ) .
these pretreated slides were blocked for 15  min at room temperature with 5% bsa and then incubated at 4  c overnight with primary antibodies : antibodies against rat cd68 ( dilution 1:50 ) ( abcam , cambridge , uk ) and rabbit cd163 ( dilution 1:50 ) ( abd serotec , kidlington , uk ) .
briefly , specific antibody was purchased to perform ihc staining by using horseradish peroxidaseconjugated avidin biotin complex ( abc ) from the vectastain elite abc kit ( vector laboratories , burlingame , ca ) and dab chromogen ( vector laboratories).13 the sections will be counterstained with haematoxylin and mounted .
inflammation grade was evaluated using a standard semiquantitative scoring system , score 0 : none ; score 1 : giant cells , lymphocytes , plasma cells ; score 2 : giant cells , eosinophil , neutrophil ; score 3 : many inflammatory cells , microabscess .
soleus muscle ( ~100  mg ) was homogenized in 1  ml of he s buffer containing 20  mm hepes ( sigmaaldrich , st louis , mo , usa ) , 1  mm edta ( sigmaaldrich , st louis , mo , usa ) , and 250  mm sucrose ( sigmaaldrich , st louis , mo , usa ) .
homogenates containing 50  g of protein were fractionated by sdspage ( 1012% acrylamide ) and transferred to pvdf membranes ( pall life science , ann arbor , mi , usa ) by standard wet transfer methods . after 1  h blocking in pbs containing 5% skim milk
primary antibodies against nitrotyrosine were purchased from millipore ( bedford , ma , usa ) ( dilution 1:1000 ) ; antibodies against manganesedependent superoxide dismutase ( mnsod ) ( dilution 1:5000 ) were purchased from enzo life sciences ( ann arbor , mi , usa ) ; and antibodies against gcs ( dilution 1:100 ) were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) . antibodybound protein was detected using a peroxidaseconjugated antimouse secondary antibody ( sigma , st louis , mo , usa ) or antirabbit igg ( cell signaling technology , beverly , ma , usa ) and enhanced chemiluminescent hrp substrate ( perkinelmer life and analytical sciences , shelton , ct , usa ) . to verify equal protein loading , gapdh ( sigma , st louis , mo , usa ) ( dilution 1:5000 )
western blot bands were quantified using a chemidoc xrs+ system ( biorad , hercules , ca , usa ) .
total rna was isolated using qiagen rneasy fibrous tissue mini kit ( qiagen , valencia , ca , usa ) .
reverse transcription of rna was carried out using iscript cdna synthesis kit ( biorad , hercules , ca , usa ) .
the following primer and probe sequences were used for amplification of target genes : tnf ( nm_012675 ) forward primer : gagtcattgctctgtgag ; reverse primer : ctctgaggagtagacgata ; probe : ctggcgtgttcatccgttctct .
il10 ( l02926 ) forward primer : gatccagagatcttagcta ; reverse primer : ctgaggtatcagaggtaa ; probe : aacctcgtttgtacctctctccaa .
taqman probes contained a fam reporter at the 5end and a tamra quencher at the 3end .
the rtqpcr conditions have been previously described.16 gene relative expression levels were determined by the change in crossing points of reaction amplification ( ct value ) between targets mrna and 18s mrna for each treatment compared with the control group .
analysis of variance ( anova ) was used to compare group difference for all variables .
a twoway anova was used to determine the main effect and interactive effect of treatments .
a oneway anova with a repeated measure was used to compare the mean differences in muscle mass between pre and post during longterm doxorubicin treatment .
fisher 's protected least significant test , which holds the value of type i error to 0.05 for each test , was used to distinguish the differences between pairs of groups .
a level of p  <  0.05 was considered significant on all tests , and all values are expressed as means    standard errors .
all rats treated with doxorubicin ( at dose of 2.5  mg / kg per body weight every 3  days ) died within 40  days ( figure 
 1a ) .
no protective effect of q10 supplementation ( 10  mg per kg body weight ) was found during the entire treatment period .
muscle mass in doxorubicintreated rats decreased significantly below those in salineinjected control rats at day 14 of the treatment ( figure 
 1b ) .
q10 supplementation failed to prevent accelerated death ( a ) and muscle loss ( b ) of rats during doxorubicin treatment ( one injection every 3  days ) .
doxorubicin treatment resulted in a significant muscle loss in only 14  days , and q10 supplementation failed to reverse this loss . *
significant difference against pre , p  <  0.05 ;  significant difference against con group , p  <  0.05 .
abbreviation : con , control ( n  =  14 ) ; q10 , coenzyme q10 ( n  =  14 ) ; dox , doxorubicin ; q10  +  dox ( n  =  14 ) , coenzyme q10  +  doxorubicin ( n  =  14 )
. an acute bout of downhill running resulted in increases in inflammation score ( figure 
 2a ) and necrotic fibres ( figure 
 2b ) of soleus muscle , observed 24  h after the challenge .
centronucleation ( a marker of muscle fibre regeneration ) of soleus muscle was not changed in normal rats observed 24  h following eccentric exercise .
however , a significant decreased centronucleation of exercised soleus muscle was observed in doxorubicintreated rats ( figure 
 2c ) .
an acute injection of doxorubicin abolished inflammation of soleus muscle 24  h after eccentric exercise .
inflammation score was determined by pathologist based on haematoxylin and eosin ( h&e ) staining ( a ) .
score 0 : none ; score 1 : giant cells , lymphocytes , plasma cells ; score 2 : giant cells , eosinophil , neutrophil ; score 3 : many inflammatory cells , microabscess .
an acute injection of doxorubicin inhibited necrosis ( b ) and lowered centronucleation ( c ) of soleus muscle 24  h after eccentric exercise .
representative histochemistry analysis of muscle sections from soleus muscle with h&e staining ( left side of bar chart ) .
original magnification was 400 , scale bar 50  m . * significant difference against con group , p  <  0.05
. abbreviation : con , control ; q10 , coenzyme q10 ; dox , doxorubicin ; q10  +  dox , coenzyme q10  +  doxorubicin .
ihc results on cd68 cell staining show a similar trend to inflammation score among four groups ( figure 
 3a ) .
exercise increased cd163 cells , but this increase was not depleted in doxorubicin rats ( figure 
 3b ) .
tnfalpha mrnas in nonexercised soleus muscle of q10 , doxorubicin , and doxorubicin  +  q10 groups were significantly increased above saline control group ( figure 
 4a ) , in the absence of increased leukocyte infiltration .
no additive effect of doxorubicin and q10 on mrnas of both inflammatory mediators ( tnfalpha and il10 ) was found .
exercise increased tnfalpha mrna in muscle of both doxorubicintreated and salinecontrol rats above nonexercised control level . decreased il10 mrna in exercised muscle below nonexercised control level occurred in both doxorubicintreated and salinetreated rats ( figure 
 4b ) .
nitrotyrosine ( figure 
 5a ) and gcs ( figure 
 5b ) levels of soleus muscle were changed in similar to inflammation score among groups . in particular , exerciseinduced increases in nitrotyrosine and gcs
antioxidant enzyme mnsod was not different among four groups in both exercised and nonexercised muscles ( figure 
 5c ) .
immunohistochemical staining shows a selective depletion of m1 macrophage ( cd68 ) in acute doxorubicin treatment ( a ) but not m2 macrophage ( cd163 ) ( b ) . *
* significant difference against con group , p  <  0.05 ;  significant difference against q10 group , p  <  0.05 .
abbreviation : con , control ; q10 , coenzyme q10 ; dox , doxorubicin ; q10  +  dox , coenzyme q10  +  doxorubicin .
protein nitration , manganesedependent superoxide dismutase ( mnsod ) , and gammaglutamylcysteine synthetase ( gcs ) .
an acute injection of doxorubicin abolished increased in nitrotyrosine ( a ) and gcs ( b ) levels of soleus muscle induced by eccentric exercise , but mnsod remained unaffected by both treatments ( c ) . *
abbreviation : con , control ; q10 , coenzyme q10 ; dox , doxorubicin ; q10  +  dox , coenzyme q10  +  doxorubicin .
inflammation is a woundhealing mechanism in living multicellular organisms.4 during muscle inflammation , macrophages localize in damaged tissues.9 , 17 two subpopulations of macrophage take part during inflammation : ( i ) the earlyinvading , phagocytic macrophages ( m1 ) reaches their highest concentration around 24  h following challenge and rapidly declines in 48  h9 ; ( ii ) the nonphagocytic macrophage ( m2 ) that are distributed near regenerative fibres for several days .
the major finding of this study is the inhibition of exerciseinduced muscle inflammation by an acute dose of doxorubicin administration .
ihc staining analysis confirms that m1 macrophage ( cd68 ) was selectively depleted in exercised soleus muscle , 24  h after eccentric exercise challenge .
inability to clear unhealthy muscle fibres in exercised muscle because of m1 macrophage depletion may explain the observed lower ratio of regenerative muscle fibre with doxorubicin treatment .
the suppressive effect of doxorubicin on inflammation is further supported by nitrotyrosine data.18 inhibiting nitric oxide production is expected to decrease stem cell recruitment to damaged tissue during regenerative phase of inflammation,19 which may also contribute to the reduced ratio of regenerative fibres after eccentric exercise .
doxorubicin is an inhibitor for cell proliferation , which has been widely used to inhibit proliferating tumour cells in cancer patients .
therefore , systemically inhibition of cell proliferation decreases muscle mass as doxorubicin treatment continues . in adults , cell turnover in muscle tissue
is continuously occurring.20 from our centronucleation results , approximately 1.33% of muscle fibre is renewing in eccentrically contracted soleus muscle .
longterm systemic inhibition on cell proliferation will lead to tissue atrophy to most cell types in the body . cells with high turnover rate , such as gastrointestinal epithelial cells , will particularly suffer and thus deteriorate nutrient absorption.21 this may add to the devastating consequence of muscle loss during chronic doxorubicin treatment .
another novel finding of the study is that exerciseinduced increase in tnfalpha mrna and decrease in il10 mrnas occur in the absence of increased leukocyte infiltration during acute doxorubicin treatment , suggesting that inhibiting leukocyte infiltration to exercised muscle sustains expression of inflammatory mediators in muscle tissue .
it is possible that inhibiting cell regeneration by doxorubicin will cause population ageing of cells in muscle tissue .
a substantial amount of muscle fibre is renewed during daily life of humans , evidenced by c dna measurement of skeletal muscle from two individuals aged 37 and 38  years old exhibiting an actual average muscle age of 15.1  years.20 if cell regeneration mechanism is systemically blocked by doxorubicin , number of aged cell population will accumulate , which leads to increase tnfalpha mrna.22 doxorubicin has been shown to increase tnfalpha mrna levels of mice heart.23 inflammation is a process to trigger regeneration when cells produce dangerassociated molecular pattern ( likely the aged and unhealthy cells).24 because no significant increase in leukocyte infiltration in muscle tissue was found , the changes in tnfalpha and il10 may simply mirror an imbalance between healthy vs. unhealthy ( young vs. old ) cell population resided in skeletal muscle caused by doxorubicin .
an unexpected new finding of the study is the increased baseline nitrotyrosine and tnfalpha mrna of skeletal muscle following q10 supplementation , suggesting that q10 is proinflammatory to skeletal muscle .
however , existing literatures on q10 supplementation show an attenuated protein nitration level25 under increased oxidative stress induced by ischemic reperfusion and a suppressed tnfalpha level in hyperglycemic heart.26 because none of those previous studies were conducted in normal ( unchallenged ) physiological condition , we speculate that q10 may exert a hormesis effect by modulating inflammation to facilitate cell turnover ( renewal ) .
we have previously found that pretreatment of proinflammatory components of ginseng eliminates exerciseinduced muscle inflammation and protein nitration.13 furthermore , in this study , we hypothesized that q10 supplementation can protect animals from lethal action of doxorubicin treatment , based on previous reports demonstrating its benefits on eliminating toxic action of doxorubicin on renal and testicular damage.11 however , the results of current study fail to support this hypothesis .
the major limitation of this study is the lack of mechanistic insight into the role of mitochondria in the doxorubicininduced muscle degeneration .
mitochondria dysfunction has been suggested as an underlying cause of doxorubicininduced muscle degeneration and weakness.27 doxorubicin can intercalate into mitochondrial dna28 that may inhibit mitochondria biogenesis and results in accumulation of aged mitochondria.29 because mitochondrial turnover occurs at much faster pace compared with normal cells,30 it is possible that increased reactive oxygen species because of accumulation of dysfunctional mitochondria is a cause of catabolic outcome of doxorubicininduced muscle atrophy.31 , 32 
  previous study in tumourfree model1 has documented a significant muscle loss during doxorubicin treatment , which is apparently unrelated to tumour growth .
given the fact that muscle loss in both humans and animals are closely associated with increased mortality in both cancer patient2 and elderly,33 , 34 development of other adjuvant intervention to preserve muscle mass , other than q10 supplementation , should be regarded as a primary goal of cancer patients under doxorubicin therapy .
this is the first study demonstrating that doxorubicin blocks muscle inflammation induced by eccentric muscle contraction .
our results provide a new clue to implicate that imbalanced m1 and m2 macrophages in challenged muscle are involved with the development of muscle loss during antiproliferationbased chemotherapy . furthermore , although q10 supplementation is proinflammatory to skeletal muscle , it can not overcome muscle loss and accelerated death under longterm doxorubicin treatment .
chihyang huang has received research grants from taiwan ministry of health and welfare clinical trial and research center of excellence .
shengchih huang , jinfu wu , suchada saovieng , mingfen hsu , weihorng chien , shinda lee and xiaofei li declare that they have no conflict of interest .
 abstractbackgrounddoxorubicin , a widely used antitumour drug , is known to cause muscle loss in cancer patients.methodsfollowing an acute dose of doxorubicin injection ( 2.5  mg / kg per body weight ) , we examined macrophage distribution in rat soleus muscle challenged by eccentric exercise ( downhill running ) . 
 longterm doxorubicin treatment ( one injection every 3  days ) on muscle mass and survival were also determined.resultsunder nonexercised condition , increased tumour necrosis factor ( tnf)alpha mrna and decreased il10 mrna were observed in soleus muscle of doxorubicintreated rats , compared with salinetreated control rats . 
 however , increases in inflammation score ( leukocyte infiltration ) , nitrotyrosine level , and m1 macrophage ( cd68 + ) invasion in exercised soleus muscle were absent in doxorubicintreated rats , whereas increased m2 macrophage ( cd163 + ) localization in exercised muscle was less affected by doxorubicin . 
 despites coenzyme q ( q10 ) supplementation significantly elevated tnfalpha mrna , nitrotyrosine , and antioxidant gammaglutamylcysteine synthetase ( gcs ) levels in nonexercised soleus muscle , these proinflammatory responses were also abolished in doxorubicintreated rats . 
 results from longterm doxorubicin treatment show a significant muscle loss followed by an accelerated death , which can not be reversed by q10 supplementation.conclusions(i ) doxorubicin impairs inflammation mechanism by depleting m1 macrophage in exercised skeletal muscle ; ( ii ) muscle loss and accelerated death during prolonged doxorubicin treatment can not be reversed by q10 supplementation . 
Introduction
Methods
Animals
Doxorubicin and Q10
Exercise challenge
Survival experiment
Body composition
Histology and immunohistochemistry (IHC)
Western blotting analysis
Quantitative polymerase chain reaction (qPCR)
Statistical analysis
Results
Discussion
Conclusions
Conflict of interest