late - stage diabetes is always associated with multiple chronic complications that lead to 
 extremely high rates of mortality and disability .
the disease has recently been regarded as a chronic 
 low - grade inflammatory disease whose onset is characterized with persistent low - grade 
 inflammation1 .
the latest studies 
 confirm the significant elevation of tumor necrosis factor - alpha ( tnf- ) , interleukin 
 ( il)-6 , c - reactive protein ( crp ) , etc . in diabetes2 , 
 3 .
in addition , diabetes patients 
 exhibit abnormal expression of adipokines such as leptin , resistin , and adiponectin , 
 indicating chronic low - grade inflammation3 .
furthermore , inflammatory factors involved in the genesis of insulin 
 resistance ( ir ) also contribute to the pathogenesis and progression of diabetes .
those 
 findings strongly indicate a close association between low - grade inflammations and type 2 
 diabetes mellitus ( t2 dm ) .
regular exercise improves 
 glycolipid metabolism and ir , thereby reducing diabetes - associated complications . 

exercise can reduce systemic low - grade inflammation and thus 
 effectively improve chronic inflammatory diseases such as obesity , metabolic syndrome , 
 diabetes mellitus , ageing , etc.5 , 6 .
it can decrease the levels of inflammatory 
 factors such as tnf- , il-6 , crp7 , 8 , and leptin9 while increasing the levels of anti - inflammatory factors such as 
 il-4 , il-10 , and adiponectin in diabetes patients10 , 
 11 .
exercise also reduces the 
 inflammation level and improves ir by enhancing anti - stress and anti - oxidative effects . 

nevertheless , the anti - inflammatory mechanism induced by exercise is complex and 
 incompletely understood .
toll - like receptor 4 ( tlr4 ) is a major member of tlr family , which acts as a core mediator 
 of innate immunity .
tlr4 is an important receptor for the chronic inflammatory response 
 because it is closely related to the inflammatory response .
it substantially increases the 
 expressions of inflammatory factors such as tnf- , il-6 , and il-1 by regulating the 
 activities of transcription factors such as nf-b12 ; this creates a persistent low - level chronic inflammatory response 
 and ir in vivo . under oxidative stress ,
nf-b also activates its inhibitor 
 and increases the serine phosphorylation of insulin receptors and insulin receptor 
 substrate , leading to ir13 .
thus , tlr4 
 and nf-b play pivotal roles in generating an inflammatory response in 
 vivo .
therefore , they might play critical roles in the pathogenesis and 
 progression of diabetes . accordingly
, the present study investigated the long - term effects 
 of a combination of aerobic and resistance exercises for 12 weeks on tlr4 , nf-b , and 
 inflammatory factors in diabetes patients .
thus , we aimed to provide a theoretical basis to 
 encourage exercise therapy for patients with diabetes .
from march 2012 to march 2014 , 42 patients with non - insulin - dependent t2 dm were selected 
 from among the outpatients visiting the department of cardiovascular rehabilitation , xiangya 
 hospital , central south university , changsha , china .
the patients were randomized to the 
 conventional therapy group ( ct group , n = 20 ) or the intensive therapy 
 group ( it group , n = 22 ) .
the ct group received regular drug treatment and 
 diet guidance , while the it group additionally performed aerobic and resistance exercises 
 for 12 weeks .
the inclusion 
 criteria were as follows : ( 1 ) meeting the diagnostic criteria proposed by the world health 
 organization in 1999 , patients who presented diabetes symptoms , serum glucose level  
 11.1  mmol / l when detected randomly , fasting blood glucose  7.0  mmol / l , and oral glucose 
 tolerance test ( ogtt ) showing a 2-hour postprandial blood glucose ( pbg )  11.1 
mmol / l ; ( 2 ) 
 no severe complications such as acute infectious diseases , numbness of limbs , decreased 
 visual acuity , and diabetic nephropathy , retinopathy , or foot ; ( 3 ) no severe cardiovascular 
 or cerebrovascular diseases such as severe arrhythmia , or acute myocardial infarction or 
 stroke , or severe joint disease leading to poor exercise tolerance ; and ( 4 ) no frequent 
 exercise ( exercise frequency less than twice per week for less than 15 minutes ) .
the study procedures were 
 approved by the institutional review board of central south university , changsha , china .
cardiopulmonary exercise testing ( cpet ) was performed on all participants before the 
 intervention in a laboratory with 5055% humidity and at a temperature of 2425c . the 
 maximum oxygen consumption ( vo2max )
was defined as the highest value of oxygen 
 consumption measured during the exercise period . using an incremental and symptom - limited 
 protocol
, participants made their efforts to their maximal capacity on a cycle ergometer 
 ( schiller cs-200 , switzerland ) . during the exercise ,
the initial workload was 1020 w , and then increased by 1020 w / min . 

finally , the test was terminated when any of the following symptoms occured , such as 
 fatigue , dyspnea , angina , adverse changes in blood pressure , musculoskeletal complaint , and 
 electrocardiographic evidence of ischemia or arrhythmia .
participants 
 were also asked to self report the intensity of exercise by the borg scale of rate of 
 perceived exertion ( rpe)14 .
all the subjects in the it group received a combined home - based exercise program and 
 supervised exercise training at the department of cardiovascular rehabilitation , xiangya 
 hospital , central south university .
these subjects took part in at least 310 supervised 
 training sessions at hospital at the beginning of the program .
each subject received 
 in - person instructions on how to exercise at a moderate - intensity level , how to monitor 
 heart rate , how to use the resistant band , and how to acquire an individualized home - based 
 exercise program .
the intervention was gradually shifted to home - based exercise for the rest 
 time of total 12 weeks , as the subjects were familiar with the whole exercise procedure .
the 
 exercise program mainly consisted of the aerobic exercise and the resistance training . 

aerobic exercise was conducted for a total of 4060 minutes per session , 3 sessions per 
 week . the exercise intensity for each patient set at 4060% of his / her vo2 max . 

as the 
 weeks progressed , the exercise intensity was increased to 60%vo2max , while the 
 duration was increased to 30 minutes over the period of 1 month .
the aerobic exercise 
 session generally consisted of a 10-minute warm - up period and a 15-minute cool - down period . 

rpe ( 1213 on the borg scale ) was also used to assist in 
 estimating the moderate exercsie intensity16 .
the patients also performed a moderate - intensity ( i.e. , 5060% of 
 the one repetition maximum , 1rm ) resistance training program 23 times per week .
the subjects performed all resistance exercises with 810 
 repetitions in two sets by using an elastic band ( thera - band ; hygenic corporation , akron , 
 oh , usa ) . resistance training intensity commenced at 50% of 1rm and increased to 60% in the 
 fourth week of the program .
in addition , the rpe ( 1114 on the borg scale ) was also used to 
 monitor the training intensity .
the patients were instructed to avoid the valsalva maneuver 
 during the resistance training program .
in addition to the training , routine diet guidance and 
 psychological counseling were provided .
all the subjects had to visit the hospital to make 
 sure the right home - based exercise program every month .
furthermore , the telephone 
 consultation was applied to enhance the compliance of these subjects and check out the 
 individual feedback during the exercise procedure .
the telephone consultation lasted for 
 510 minutes and was offered once a week for 3 months18 .
the ogtt was performed before and after the intervention , and the results were recorded in 
 detail .
plasma glucose concentrations 
 were analyzed by glucose oxidase method , and insulin levels were measured using ria kit 
 ( beijing north institute , beijing , china ) .
serum total cholesterol ( tc ) , low - density lipoprotein 
 ( ldl ) , high density lipoprotein ( hdl ) , and triglyceride ( tg ) concentrations were determined 
 by using a cobas fara robot ( roche , switzerland ) .
insulin resistance index ( iri ) was 
 calculated using the method of homeostatic model assessment ( homa)19 .
venous blood ( 15  ml ) was drawn from each participant and 
 subjected to ficoll - hypaque density gradient centrifugation in order to separate mononuclear 
 cells ; 5  ml venous blood was used for quantitative polymerase chain reaction ( qpcr ) to 
 detect the expression levels of tlr4 and nf-b mrna , while the remaining 10  ml was used to 
 detect the expression levels of tlr4 and nf-b proteins .
sera were analyzed by elisa to 
 determine the expression levels of il-18 , il-33 , ptx3 , and ykl40 .
the mononuclear cells were treated with lysis buffer , and 30  g total protein was loaded 
 and separated on a 10% sds - page gel before transfer to a nitrocellulose membrane . 

-actin 
 was used as a loading control . after incubating with the recommended horseradish peroxidase 
 secondary antibodies ,
chemiluminescence fluorescence image analysis system was used to scan fluorescence images 
 and various optical density value ( od value ) was calculated by bandscan image analysis 
 software .
the relative expression of target genes was determined by the the ratio of od 
 value of the target genes to that of internal reference gene .
total rna was extracted from the mononuclear cells using trizol reagent ( invitrogen , 
 waltham , ma , usa ) according to the manufacturer s instructions .
the expressions of tlr4 and 
 nf-bp65 mrna were detected by sybr green qpcr assay ( bio - rad , hercules , ca , usa ) .
the 
 specic primers were as follows : tlr4 forward : 5- tgggtaaggaatgagctagtaaa -3 , reverse:5- 
 gctgggacaccacaacaa -3 ; -actin forward : 5- catcctgcgtctggacctgg-3 , reverse : 
 5-taatgtcacgcacgatttcc-3 ; nf-bp65 forward:5- -cccatcgggttcccataaag-3 , reverse : 
 5-gcctgaagcaaatgttggcgta-3. all reactions were performed in triplicate .
the biochemical analyses of serum il-18 , il-33 , ptx3 , and ykl40 were performed by elisa 
 ( abcam , ma , usa ) ; human il-18 , il-33 , ptx3 , and ykl40 were detected quantitatively .
all 
 factors were quantified using polyclonal antibodies that recognized native human il-18 , 
 il-33 , ptx3 , or ykl40 and a series of plates containing wells coated with predetermined 
 amounts of recombinant human il-18 , il-33 , ptx3 , or ykl40 .
continuous variables are 
 expressed as mean  standard deviation and were compared using the student - newman - keuls 
 test .
the insulin resistance index ( iri ) showed a skewed distribution and a natural 
 logarithmic transformation was performed befor statistical analysis .
the diabetes groups did not differ significantly from the normal control group with respect 
 to age ( 52.59  11.43 vs. 51.20  11.34  years ) or gender ( f30/m12 vs. f13/m7 ) .
compared to 
 the ct group , fasting blood glucose ( fbg ) , postprandial blood glucose ( pbg ) , fasting plasma 
 insulin ( fin ) , postprandial plasma insulin ( pin ) , hba1c , ln homa - ir , tg , tc , hdl , and ldl 
 levels were not significantly different between the ct and it groups ( all p > 0.05 , table 1table 1.changes of serum glucose , insulin , homa - ir , and blood lipidsvariablesct group ( n = 20 ) it group ( n = 22 ) control group ( n = 20)pre - treatmentpost - treatmentpre - treatmentpost - treatmentfbg ( mmol / l)7.14  1.976.37  1.40**6.84  1.425.78  0.48**5.65  0.41pbg ( mmol / l)11.08  2.639.00  1.91**11.30  2.177.30  0.986.26  1.04fin ( u / ml)12.81  6.1611.65  5.2613.40  5.119.04  3.19**9.19  3.73pin ( u / ml)94.54  46.0569.67  31.30**83.66  30.7536.16  18.2634.50  11.74hba1c ( % ) 6.88  0.766.44  0.65**6.85  1.145.89  0.615.73  0.31homa - ir4.23  2.993.32  1.734.11  1.982.33  0.922.30  0.94ln homa - ir1.26  0.601.08  0.501.33  0.400.77  0.400.74  0.43tg ( mmol / l)1.99  1.071.75  0.762.20  1.271.17  0.35 * * 1.13  0.31tc ( mmol / l)4.62  1.314.45  1.125.53  1.654.77  1.03 * 3.91  0.56hdl ( mmol / l)1.27  0.311.31  0.301.38  0.291.43  0.261.38  0.19ldl ( mmol / l)2.58  1.002.46  1.023.22  1.472.54  1.22
p < 0.05 , * * p < 0.01 vs. 
 pre - treatment ; p < 0.05 , p 
 < 0.01 vs. conventional therapy group .
fbg : fasting blood 
 glucose ; fin : fasting insulin ; hba1c : glycated hemoglobin ; homa - ir : the index for 
 homeostasis model assessment of insulin resistance ; ldl : low - density lipoprotein ; pbg : 
 postprandial blood glucose ( 2-h ) ; pin : postprandial insulin ( 2-h ) ; tc : total 
 cholesterol ; tg : triglycerides ) .
data are mean  sd . * p < 0.05 , * * p < 0.01 vs. 
 pre - treatment ; p < 0.05 , p 
 < 0.01 vs. conventional therapy group .
fbg : fasting blood 
 glucose ; fin : fasting insulin ; hba1c : glycated hemoglobin ; homa - ir : the index for 
 homeostasis model assessment of insulin resistance ; ldl : low - density lipoprotein ; pbg : 
 postprandial blood glucose ( 2-h ) ; pin : postprandial insulin ( 2-h ) ; tc : total 
 cholesterol ; tg : triglycerides in the ct group , fbg , pbg , pin , and hba1c decreased significantly after treatment 
 ( p < 0.05 , < 0.01 )
. meanwhile , fbg , pbg , fin , pin , hba1c , and ln 
 homa - ir decreased significantly in the it group , ( all p < 0.01 ) .
pbg , 
 pin , hba1c , and ln homa - ir were significantly lower in the it group than the ct group 
 ( p < 0.01 , < 0.05 , table 1 ) . before treatment , the mrna and protein expression levels of tlr4 and nf-bp65 , and serum 
 il-18 level were significantly higher in the ct and it groups than the control group , while 
 il-33 level were significantly lower in the ct and it groups ( p < 0.05 , 
 < 0.01 ) . after the exercise intervention , the mrna and protein 
 expressions of tlr4 and nf-bp65 were significantly lower in the it group than the ct group 
 ( p < 0.05 , < 0.01 ) .
the melting and amplification 
 curves of tlr4 and nf-b mrna detected by qpcr are shown in fig .
serum il-18 level was significantly lower in the it group than the ct group 
 ( p < 0.05 ) .
meanwhile , the serum il-33 level was significantly higher 
 in the it group than the ct group but lower than that in the control group ( p 
 < 0.05 ) .
there was no significant difference in the expression levels of ptx3 
 or ykl40 among the three groups ( table 
 2table 2.effects of exercise intervention on the inflammatory factors in diabetes 
 patientsitemsct group ( n = 20)it group (
n = 22)control group ( n = 20)pre - treatmentpost - treatmentpre - treatmentpost - treatmenttlr4 mrna1.68  0.29**0.77  0.181.85  0.31**0.54  0.63**1.05  0.18nf-bp65mrna1.28  0.26**1.01  0.111.41  1.77**0.86  0.660.86  0.12tlr4 protein0.95  0.34**0.67  0.33 * 0.86  0.25**0.41  0.160 .
. 28  0.090.27  0.11il-180.80  0.12**0.72  0.12 * 0.81  0.09**0.51  0.100.59  0.31ptx30.63  0.130.63  0.140.64  0.150.58  0.080.54  0.78il-330.40  0.09**0.65  0.12 * 0.42  0.14**0.85  0.14 * 1.38  0.19data are mean  sd , * p < 0.05 , * * p < 0.01 vs. 
 control group ; p < 0.05 , p 
 < 0.01 vs. conventional therapy group .
ct group : the 
 conventional therapy group ; it group : the intensive therapy group ; il-18 : 
 interleukin-18 ; il-33 : interleukin-33 ; nf-bp65 : nuclear factor-bp65 ; ptx3 : 
 pentraxin - related protein 3 ; tlr4 : toll - like receptor 4 ; ykl40 : human cartilage 
 glycoprotein 39 and fig . 2fig .
2.changes in the protein expressions of tlr4 and nf-bp65 before and after the 
 intervention . a : control group
; b : conventional therapy group before intervention ; c : 
 conventional therapy group after intervention ; d : intensive - therapy group before 
 intervention ; e : intensive therapy group after intervention .
nf-bp65 : nuclear 
 factor-bp65 ; lr4 : toll - like receptor 4 ) . melting and amplification curves of tlr4 mrna and nf-b mrna detected by qpcr .
: toll - like receptor 4 data are mean  sd , * p < 0.05 , * * p < 0.01 vs. 
 control group ; p < 0.05 , p 
 < 0.01 vs. conventional therapy group .
ct group : the 
 conventional therapy group ; it group : the intensive therapy group ; il-18 : 
 interleukin-18 ; il-33 : interleukin-33 ; nf-bp65 : nuclear factor-bp65 ; ptx3 : 
 pentraxin - related protein 3 ; tlr4 : toll - like receptor 4 ; ykl40 : human cartilage 
 glycoprotein 39 changes in the protein expressions of tlr4 and nf-bp65 before and after the 
 intervention . a : control group
; b : conventional therapy group before intervention ; c : 
 conventional therapy group after intervention ; d : intensive - therapy group before 
 intervention ; e : intensive therapy group after intervention .
many studies show 
 aerobic exercise including home - based exercise decreases blood glucose , lipid , and hba1c 
 levels as well as improves insulin sensitivity , hypercoagulability state , quality of life , 
 and physical condition in t2 dm patients20,21,22,23,24,25 . in the present study ,
t2 dm patients 
 performed a combination of aerobic exercises and resistance training for 12 weeks .
the 
 results show significant improvements in the fasting and postprandial blood glucose , hba1c , 
 tg , tc , ldl cholesterol , and ir in the intensive - therapy group after 12 weeks of therapy 
 compared to conventional therapy .
balducci et al.26 report that compared to conventional therapies , a 1  year exercise 
 intervention significantly reduced the blood glucose , tg , tc , and ldl cholesterol levels of 
 patients with t2 dm while significantly increasing hdl cholesterol level .
tokmakidis et 
 al.27 report that a 4-month exercise 
 intervention significantly reduced the fasting and ( 2-hour ) postprandial blood glucose , 
 insulin , and hba1c levels of diabetes patients .
thus , the present results are concordant 
 with those of balducci et al . and tokmakidis et al .
among diabetes patients , regular 
 exercise has a better blood glucose - controlling effect , because it improves insulin 
 sensitivity .
moreover , regular exercise enhances blood pressure and blood lipid control , 
 thus reducing the risks of large vascular and microvascular complications . despite the attention of many researchers for a long time ,
the latest studies indicate a close 
 association between the pathogenesis of diabetes mellitus and a chronic low - grade 
 inflammatory response . in the present study ,
the mrna and protein expression levels of tlr4 
 and nf-b in mononuclear cells as well as serum levels of the inflammatory factor il-18 were 
 higher in t2 dm patients than healthy controls .
however , the expression level of the 
 protective cytokine il-33 was lower in t2 dm patients than control group . meanwhile
, the 
 serum inflammatory factors ptx3 and ykl40 did not differ significantly between t2 dm patients 
 and controls .
these results indicate tlr4 , nf-b , il-18 , and il-33 may play important roles 
 in the pathogenesis of diabetes mellitus .
previous studies report that patients with 
 diabetes mellitus have higher tlr4 mrna and protein expression in mononuclear cells and that 
 these changes are closely associated with blood glucose and hba1c levels28 .
nf-b 
 subsequently activates a series of inflammatory factors including tnf- , il-1 , il-6 , and 
 il-8 and induces the expressions of several adherence factors and chemokines by mononuclear 
 macrophages and endothelial cells30 .
this 
 cascade ultimately results in ir and secretary dysfunction of insulin in the body .
il-18 is 
 mainly secreted by mononuclear macrophages and may be important for generating an 
 inflammatory response31 .
some studies 
 report diabetes patients have elevated plasma il-18 expression , which is closely associated 
 with fasting blood glucose and hba1c levels32 .
in addition , elevated il-18 expression is reported to be an 
 independent risk factor for the development of diabetes mellitus33 .
il-33 , a member of the il-1 cytokine family , is released 
 after cell damage or as a negative regulator of nf-b gene transcription ; it not only 
 enhances cytokine production by t helper cells , but also reduces the activities of natural 
 killer cells and the generation of oxidation products ; therefore , it is recognized as a 
 protective factor against ir , diabetes mellitus , and obesity .
elevated il-33 expression 
 improves fasting blood glucose and glucose tolerance34 , 35 . on the contrary
, the 
 removal of il-33 may increase the cytotoxicity of natural killer cells and trigger 
 inflammatory factors such as tnf- , possibly resulting in ir36 .
although some studies show ptx3 and ykl40 are associated with the onset and 
 progression of diabetes37 , 38 , we found no significant increases of their expression 
 levels .
this discrepancy is probably due to the variation in the duration and type of 
 disease of our patients .
the present results indicate diabetes patients exhibit increased 
 expressions of some inflammatory factors and decreased expressions of protective cytokines , 
 indicating a close relationship between diabetes mellitus and a chronic low - grade 
 inflammatory response .
long - term regular exercise can reduce chronic low - level inflammation in humans , and an 
 exercise - containing lifestyle may also delay the genesis and development of t2dm3 , 39 . 

there was research also demontrated that the home - based exercise improved the metabolic 
 asset and reduced the systemic inflammation of sedentary people40 .
another study proved that home - based exercise could 
 improve clinical features and systemic inflammation of patients with chronic obstructive 
 pulmonary disease41 .
in the present 
 study , the intensive intervention program for diabetes patients included a 12-week 
 moderate - intensity workout program consisting of aerobic exercise and resistance training 
 ( i.e. , the it group ) .
after exercise training , the it group had significantly lower tlr4 and 
 nf-b mrna and protein expressions as well as serum il-18 levels than the ct group . 

meanwhile , the serum level of il-33 ( a protective cytokine ) was significantly higher in the 
 it group than the ct group but lower than that in the control group .
exercise can 
 significantly reduce tlr4 expression on the surface of mononuclear cells in non - diabetes 
 patients42 , 43 .
lambert et al . also report a similar reduction in elderly obese 
 patients following long - term exercise therapy44 .
other studies also demonstrate that exercise reduces il-18 levels 
 in elderly people and patients with metabolic syndrome45 , 46 .
furthermore , regular 
 exercise increases the expressions of heat shock proteins and il-33 by vascular endothelial 
 cells .
the present 
 study suggests the anti - inflammatory mechanism of long - term medium - intensity exercise may be 
 associated with decreased mrna and protein expressions of tlr4 and nf-b , and the 
 inflammatory factor il-18 as well as elevated levels of the anti - inflammatory factor il-33 , 
 all of which improve ir and the outcome of diabetes .
as the sample size was relatively 
 small , our results may be affected by some biases .
moreover , the mechanism by which exercise 
 intervention alters the gene expressions of inflammatory factors remains unclear .
therefore , 
 further animal and cellular experiments must be conducted to clarify the underlying 
 mechanism of exercise intervention . in conclusion
, the present study demonstrates the existence of low - grade chronic 
 inflammation in diabetes patients and that long - term medium - intensity exercise can not only 
 decrease the mrna and protein expressions of tlr4 and nf-b , but can also reduce the level 
 of the inflammatory factor il-18 and increase the level of the anti - inflammatory factor 
 il-33 .
thus , long - term moderate - intensity exercise can resolve inflammatory response , 
 improve ir , and achieve better blood glucose control in t2 dm patients .
 [ purpose ] to investigate the effects of combined aerobic and resistance training on 
 glycolipid metabolism and inflammation levels in type 2 diabetes mellitus patients . 
 [ subjects and methods ] 
 forty - two diabetes patients were randomized to the conventional 
 therapy group ( n = 20 ) or intensive therapy group ( n = 
 22 ) . 
 the control group contained 20 healthy people . 
 the conventional therapy group 
 received routine drug therapy and diet control , while the intensive therapy group 
 additionally underwent combined aerobic and resistance training for 12 weeks . the oral 
 glucose tolerance test and cardiopulmonary exercise testing were performed . 
 toll - like 
 receptor 4 and nf-bp65 protein and mrna expressions were determined by qpcr and western 
 blotting . 
 elisa was used to determine the expression levels of interleukin-18 , 
 interleukin-33 , pentraxin - related protein 3 , and human cartilage glycoprotein 39 . 
 
 [ results ] after exercise training , the intensive therapy group had significantly lower 
 postprandial blood glucose , postprandial insulin , and glycated hemoglobin level and 
 insulin resistance index than the conventional therapy group . 
 the intensive therapy group 
 had significantly lower toll - like receptor 4 and nf-bp65 protein and mrna expressions , 
 and serum interleukin-18 levels but significantly higher serum interleukin-33 levels . 
 
 [ conclusion ] combined aerobic and resistance training can improve glycolipid metabolism 
 and reduce low - grade inflammation in patients with diabetes mellitus patients . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION