pulmonary edema is a common complication of diabetes mellitus because of the increased capillary permeability .
severely uncontrolled diabetic state may initiate pathologic events leading to the capillary leak of acute respiratory distress syndrome . due to the lower level and impaired binding activity of cell - surface receptors on monocytes , a poorly controlled diabetic state increases susceptibility to infections such as endotoxemia .
the highest incidence of acute respiratory distress syndrome occurred in endotoxemic patients and resulted in lung injury .
however , the severity of lung injury during systemic endotoxemiain type 1 diabetes remains unclear .
endotoxemia is mainly caused by anendotoxin ( lipopolysaccharide ; lps ) from gram - negative bacteria .
endotoxins induce a great amount of alveolar monocytes and macrophages to release tumor necrosis factor - alpha ( tnf- ) , which subsequently damages pulmonary vessels and increases lung vascular / epithelial permeability .
consequently , albumin content in the brochoalveolar lavage fluid increases significantly , contributing to lung edema and poor lung compliance . in unison , the alveolar distortion , atelectasis , and a thick alveolar - capillary membrane produce low efficiency of gas exchange , often resulting in increased mortality .
prior research has demonstrated that heat shock protein72 ( hsp72 ) can be detected in the lungs of exercise trained rats , and that its induction by heat can protect respiratory systems from systemic inflammation .
moreover , hsp72 facilitates the action of an anti - inflammatory cytokine , interleukin ( il)-10 , and alleviates tnf- induced lung damage . in nondiabetic rats ,
in diabetic rats , exercise upregulated hsp72 expression in the heart and alleviated circulatory dysfunction after lps injection .
nevertheless , it remains unknown whether aerobic conditioning via treadmill exercise can attenuate pulmonary injury in diabetic rats receiving endotoxin .
hence , the purpose of this study is to reveal whether endotoxemia - induced lung injury is more marked in type 1 diabetes and to determine whether treadmill exercise conditioning could induce hsp72 overexpression in the lung , alleviate lung injury and proinflammatory cytokines overproduction in endotoxemia , and thus reduce the mortality rate .
male wistar rats ( n = 144 ; 320  20  g ; aged 10 weeks ) were purchased from the animal resource center of the national cheng kung university in taiwan .
rats were housed in groups of four at an ambient temperature of 24  1c .
all experimental procedures were conducted in compliance with the national institutes of health 's guide for the care and use of laboratory animals .
streptozotocin ( stz)-induced type 1 diabetic rats were prepared by intravenous injection with stz ( sigma , st .
louis , mo , usa ) ( 60  mg / kg ) after a 72-hour fast .
this method has been shown to irreversibly destroy pancreatic beta cells , as previously described .
after 1 week of stz administration , rats with blood glucose levels higher than 300  mg / dl along with symptoms of polyuria were considered diabetic .
animals were separated into six groups ( n = 24 in each group ) : ( a ) nondiabetic control rats receiving 0.9% normal saline administration ( ns ) , ( b ) nondiabetic control rats receiving lps administration ( nl ) , ( c ) stz - induced diabetic rats received normal saline administration ( ss ) , ( d ) stz - induced diabetic rats received lps administration ( sl ) , ( e ) stz - induced diabetic rats subjected to exercise training before the injection of normal saline ( ses ) , ( f ) stz - induced diabetic rats subjected to exercise training before the injection of lps ( sel ) .
the exercise training protocol was performed according to a previously described method with some modification . specifically , 1 week after stz injection , the trained rats ran gradually on a treadmill ( treadmill exerciser t510 , diagnostic & research instruments , singa ) for 5 days / week with intensity of 1.0 mile / hr for 3 weeks at room temperature . before the initiation of exercise training
, the rats were acclimated to run 15  min at a time for 3 days . a minor electrical shock ( 1.0  ma ) was used in the beginning to encourage rats to run forward .
the duration of the exercise was progressively increased , with the rats running for 30  min / day during the first 2 weeks , and 60  min / day during the last week of training .
the intensity of exercise was maintained throughout the training period at approximately 7580% of maximal oxygen consumption [ 15 , 18 ] .
same exercise training protocol is used in all training groups , close to 90% of the trained rats were able to finish the training program .
rats which could not achieve the intensity and duration of this training protocol were withdrawn from this experiment .
however , the withdrawal rate was indistinguishable between nondiabetic and diabetic group . at the 24th hr after the last training session , the rats received an intravenous injection of lipopolysaccharide ( lps , 15  mg / kg ) or saline .
louis , mo ) was used as a fresh solution in phosphate buffered saline ( ph 7.40 ) at a concentration of 10  mg / ml . after administration of lps , the survival time of each rat was continuously monitored .
different parts of the animals were used for three experiments ( n = 8 for each part in each group ) : ( i ) determination ofsurvival time and survival rate in sedentary and exercised rats after receiving an injection of lps ; ( ii ) determination of hsp72 expression in sedentary and exercised rats ; and ( iii ) determination of arterial blood gas , lung injury , and tnf- , il-6 , and il-10 levels in bronchoalveolar lavage at 240  min after normal saline or lps injection .
the albumin content in bronchoalveolar lavage fluid was determined to assess the damage of endothelial cells within the lung capillaries .
the rat 's trachea was intubated and a bronchoalveolar lavage sample was collected by perfusing saline ( 15  ml ) from the endotracheal tube at 240  min after the administration of lps or saline .
the lavage samples were centrifuged for 20  min ( 15000  rpm , 4c ) to remove cells .
the supernatant of lavage was analyzed by a bio - rad protein assay system ( bio - rad hercules , ca ) with bovine serum albumin as the standard .
the albumin content in lavage was calculated by dividing albumin by the dried weight of the lung to evaluate pulmonary capillary / endothelial cell permeability .
the concentrations of tnf- , il-6 , and il-10 in lavage fluid were assayed using double - antibody sandwich elisa ( r&d systems , minneapolis , mn , usa ) according to the manufacturer 's instructions .
the optical density of each well was determined by a microplate photometer ( multiskan ex , thermo fisher scientific inc , waltham , ma , usa ) . at 240  min after administration of lps
, both whole lungs were removed , wiped clean , and weighed as the wet weight .
dry weight was measured after the lungs were dried at 63c for 48  hrs . in order to assess degree of lung edema
, the wet / dry weight ratios of lung were calculated by dividing the wet weight by dry weight . at 240  min after injection of lps
the systemic circulation was perfused with 250  ml of 0.9% saline ( 163.2  cm - h2o ) while the pulmonary circulation was perfused with 50  ml of 0.9% saline ( 40.8  cm - h2o ) .
subsequently , the same amounts of 4% formalin were instilled into systemic and pulmonary circulation .
right upper lobes were embedded in paraffin blocks and serial sections ( 4  m in thickness ) were stained with hematoxylin - eosin for histopathologic evaluation .
the characteristics of lung damage include vascular congestion , hemorrhage , polymorphonuclear leukocytes ( pmn ) infiltration , and edematous changes of alveolar wall .
each characteristic was scored ( 0 : normal ; 1 : mild ; 2 : moderate ; 3 : severe ) by a pathologist and overall lung injury was further calculated according to the sum of the scores . in order to determine the arterial ph , arterial partial pressure of o2 ( pao2 ) , co2 ( paco2 ) , and o2 saturation ( so2 ) of rats ,
0.4  ml of arterial blood was sampled from the femoral artery by a heparin - rinsed syringe at 240  min after administration of lps .
the sample was analyzed by a blood gas analyzer ( synthesis1725 , diamond diagnostics inc , hollinton , ma , usa ) . for purposes of quantifying hsp72 expression in the lungs , rats with or without exercise preconditioning
lung tissue was homogenized and denatured in a sds sample buffer ( 0.5  m tris - hcl ( ph 6.8 ) , 10% sds , 0.1% bromphenol blue , 2-mercaptoethanol , and glycerol ) .
protein contents were assayed using the bio - rad kit ( bio - rad , hercules , ca , usa ) and an elisa reader ( multskan ex , thermo , ma , usa ) at 630  nm .
after equal amounts of protein extract were loaded and separated by 10% sds - polyacrylamide gel electrophoresis ( sds - page ) , the proteins were transferred to polyvinylidene fluoride ( pvdf ) membranes ( millipore , bedford , ma , usa ) .
the membrane was incubated with a mouse monoclonal anti - hsp72 antibody ( spa 810 ; stressgen biotechonologies , victoria , bc , canada ) .
immunodetection for hsp72 was performed using the enhanced chemiluminescence protocol using a renaissance reagent ( nen life science products , boston , ma , usa ) .
quantification of blot band was performed using an optical scanner and imagemaster totallab 1d elite software ( version 2.01 ; amersham pharmacia , piscataway , nj , usa ) .
the wilcoxon rank - sum test was used for the analysis of survival time , while the kaplan - meier test was used for survival rates .
after induction of diabetes , the body weight of diabetic rats was lower than nondiabetic ones ( figure 1 ) .
exercise training decreased the body weight of nondiabetic rats and increased the body weight of diabetic rats .
in other words , exercise moves the body weight of rats toward a healthier status .
after lps administration , we found that the colonic temperature is slightly elevated within 10  mins and it returned to baseline level at 20 minutes ( figure 2 ) .
the survival time of the sl group was markedly shorter than that of the nl group ( 316.4  39.6  min versus 420.78  38.99  min , resp . ;
. additionally , survival time of the sel group was significantly longer than that of the sl group ( 489.2  23.5  min versus 316.4  39.6  min , resp . ;
p < 0.05 ) , suggesting that exercise preconditioning significantly improved survival rate of diabetic rats during endotoxemia ( figure 3 ) . presented in another way ,
by 420  min after administration of lps , 100% of the sel group was still alive in contrast to only 11.1% survival among the sl group . 

figure 4 shows that all groups treated with lps displayed significantly raised tnf- ( 120.34  14.11  pg / ml in nl group and 197.43  30.11  pg / ml in sl group ) and il-6 ( 142.11  124.83 
pg / ml in sl group ) levels in lavage fluid at 240  min after treatment . however , lavage tnf- level was statistically higher in the sl group as compared to the nl group ( p < 0.05 ) .
additionally , the increases of tnf- and il-6 were attenuated significantly by exercise preconditioning ( 25.5  3.72  pg / ml in tnf- and 97.52  58.40 
pg / ml in il-6 ) , while levels of il-10 were actually higher in exercise - treated groups ( 68.50  23.14  pg / ml ) .
the lung wet / dry weight ratios and albumin content of the lungs increased significantly after administration of lps ( figure 5 ) .
these parameters were not significantly different between the nl and sl groups , but the values were significantly decreased in rats with exercise preconditioning .
even though the albumin content in the sel group was significantly lower than that in the sl group ( 0.76  0.07  mg  ml  g versus 1.64  0.22  mg  ml  g , resp . ; p < 0.05 ) , the value of the sel group was still significantly higher than that reported among the ses groups ( 0.76  0.07  mg  ml  g versus 0.20  0.05  mg  ml  g , resp .
; p < 0.05 ) . histopathologic examination of the lungs and quantitative analyses of histology were shown in figures 6 and 7 .
the lung injury score was significantly increased in nl , sl , and sel groups compared with that of ns groups . although exercise preconditioning diminished these changes , the values of lung injury score are indistinguishable between sl and sel groups .
after administration of lps , the values of paco2 decreased significantly , whereas the value of pao2 and sao2 increased significantly , indicating that hyperventilation had occurred . nevertheless , values of paco2 , pao2 , and sao2 were indistinguishable between the sl and sel groups ( table 1 ) .
while levels of hsp72 expression in the lung were increased in rats with exercise as compared to those without exercise , the increase of expression was less pronounced in the diabetic groups ( figure 8) . a negative correlation between lung hsp72 level and lavage tnf- level
in this study , we demonstrated that after lps challenge , the tnf- level in lung lavage fluid is higher in diabetic rats than that in nondiabetic ones .
we also found overexpression of hsp72 in the lung and higher il-10 level in lavage among exercised diabetic rats .
thus , our data suggest that preconditioning with treadmill exercise reduces lung edema , albumin content , tnf- and il-6 levels , and consequently , the mortality rate , after administration of lps . lung injury is associated with elevated cytokines .
tnf- is believed to play an important role in the pathogenesis of endotoxin - induced multiple organ failure .
previous findings showed that lps treatment resulted in higher lung tnf- levels , as well as increased lung edema and pulmonary albumin .
however , hsp72 could facilitate the action of an anti - inflammatory cytokine , il-10 , thereby decreasing tnf--induced lung damage .
several studies also demonstrated that hsp72 could actually inhibit the production of tnf-   and tnf--induced inflammatory responses .
after lps injection , we found that the colonic temperature is slightly elevated within 10  mins and it returned to baseline level at 20 minutes .
hsp72 also plays an important role in thermotolerance and cytoprotection against damage from stresses such as ischemia and cytokines [ 10 , 17 , 25 ] . in the present study ,
nevertheless , exercise - induced hsp72 may provide cellular protection during the lps - induced raised temperature period and lessen tissue inflammation and damage .
hsp72 is induced in multiple organs by heat stress or exercise [ 10 , 26 ] and plays a critical role in the protection of cellular damage from such stressors .
more specifically , after exercise activates its generation , hsp72 interacts with denatured proteins causing refolding activity that intensifies the structure of normal proteins to prevent their degradation in response to a subsequent stress .
additionally , hsp72 induced by heat was found to protect respiratory systems from systemic inflammation .
previous study has demonstrated that the levels of other heat shock proteins , such as heat shock protein90 and heme oxygenase-1 , are also changed in stz - induced diabetic rats .
heat shock protein90 levels were increased in heart and decreased in liver of stz - induced diabetic rats . however , exercise training significantly increased the expression of hsp72 , but not heat shock protein90 and heme oxygenase-1 , in heart , liver , and muscles of stz - induced diabetic rats . therefore , we focus on whether exercise training increases the expression of hsp72 in lung of stz - induced diabetic rats in the present study
a prior study has reported that long - term exercise has an anti - inflammatory effect that is partially mediated by il-6 .
after long - term exercise , il-10 is stimulated by il-6 in systemic circulation , which then causes a reduction in the tnf- level .
furthermore , after 12 weeks of aerobic exercise , il-6 level was found to be decreased while il-10 level was increased in type 2 diabetic patients   and the patients with coronary heart disease . in nondiabetic rats , recent studies documented that treadmill exercise attenuates septic responses and
swimming trained rats also showed a lower pulmonary edema index after lps challenge . in the present study of diabetic rats ,
treadmill exercise increased the lung expression of hsp72 , but this induction was less pronounced than in the nondiabetic controls .
these data are consistent with prior reports indicating that induction of diabetes decreased hsp72 expression in heart , liver , and muscles .
also , we found that exercised diabetic rats had lower inflammatory response during endotoxemia as well as a negative correlation between lavage tnf- level and the hsp72 level in the lungs .
a prior study has shown that activation of stress protein response caused intracellular expression of hsp72 in lung endothelial , epithelial , and macrophage cells and that this activation has immunomodulatory effects .
hence , the attenuation of lavage cytokine levels during endotoxemia may be , in part , related to the higher expression of hsp72 in the lungs .
hence , we have demonstrated that lps - induced pulmonary dysfunction is more marked in type i diabetic rats , but endurance training may upregulate lung hsp72 expression and counterbalance some of the negative effects of diabetes .
however , since lps injections affect multiple organs , the effect on survival time may not only be explained by the reduced severity of acute lung injury .
in fact , our previous study has indicated that exercise could increase hsp72 expression in heart and nts as well and attenuate cardiovascular dysfunction in diabetic rats during endotoxemia .
the present results showed that paco2 decreased and pao2 increased significantly in diabetic rats during endotoxemia .
lps can induce many mediators , such as nitric oxide , reactive oxygen species , and tnf- , all of which are associated with respiratory muscular dysfunction .
furthermore , pulmonary morphology alterations can be involved in the changes of blood gas levels .
our results are similar to those of fakioglu et al . , who documented that hyperventilation occurred during endotoxemia , but inconsistent with findings from another study .
the controversial findings may be explained by different dosage of lps injection in each study .
in addition , some studies have demonstrated that alloxan - induced diabetes may be associated with a reduced risk of acute lung injury in response to intratracheal instillation of endotoxin .
the conflicting outcome in our study may result from the lung injury being induced by intravenous infusion of endotoxin and the use of a different animal model of diabetes .
our results suggest that regular exercise could prevent the occurrence of a rapidly lethal infection or endotoxemia even among those with poorly - controlled diabetes .
although exercise prolonged the survival time only by hours in this acute septic shock model , it could provide more opportunities for providing enough emergency treatments and critical care within these hours for the diabetic patients and reduce the higher mortality .
the tissue responses to exercise training may be different in chronic / prolonged diabetic state .
a direct relationship between lung hsp72 content and related damage can not be inferred since this study assessed the consequences separately .
such a question may be addressed by injecting hsp72 antisense or shrna into the exercising rats to further clarify the effect of exercise - induced hsp72 expression on endotoxemia .
although the increase of tnf- and il-6 levels in exercised rats was alleviated after lps challenge , a much broader panel of pro- and anti - inflammatory cytokines , chemokines , inflammatory mediators , and oxidative stress bio - markers also need to be measured in the lavage fluid as well as in other tissues and in systemic circulation .
since these data are derived from a rapidly lethal model of endotoxemia , the present results may not be extrapolated to less severe models . in conclusion ,
lps - induced lung inflammation and damage are more marked in type 1 diabetic rats .
exercise could alleviate lung damage and confer significant protection against the high mortality risk in diabetic rats during endotoxemia .
 endotoxemia induces a series of inflammatory responses that may result in lung injury . however , heat shock protein72 ( hsp72 ) has the potential to protect the lungs from damage . 
 the objective of this study was to determine whether prior exercise conditioning could increase the expression of hsp72 in the lungs and attenuate lung damage in diabetic rats receiving lipopolysaccharide ( lps ) . 
 streptozotocin was used to induce diabetes in adult male wistar rats . 
 rats were randomly assigned to sedentary or exercise groups . 
 rats in the exercise condition ran on a treadmill 5 days / week , 3060 min / day , with an intensity of 1.0 mile / hour over a 3-week period . 
 rats received an intravenous infusion of lps after 24  hrs from the last training session . 
 elevated lavage tumor necrosis factor - alpha ( tnf- ) level in response to lps was more marked in diabetic rats . 
 hsp72 expression in lungs was significantly increased after exercise conditioning , but less pronounced in diabetic rats . 
 after administration of lps , exercised rats displayed higher survival rate as well as decreased lavage tnf- level and lung edema in comparison to sedentary rats . 
 our findings suggest that exercise conditioning could attenuate the occurrence of inflammatory responses and lung damage , thereby reducing mortality rate in diabetic rats during endotoxemia . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion