intestinal ischemia / reperfusion ( ii / r ) injury was the common pathological process of multiple clinical severe diseases .
ii / r not only caused the intestinal local damage , but also led to the injury of distant organs , especially acute lung injury ( ali ) .
ali induced by ii / r has high morbidity and mortality in clinic [ 4 , 5 ] .
previously , the study indicated that the excessive elevation of proinflammatory cytokines is a major contributor in the occurrence and development of ali caused by ii / r . however , the regulated mechanism of proinflammatory cytokines involving in interleukin-1 ( il-1 ) in ali induced by ii / r is waiting to be elucidated .
il-1 , a member of inflammatory factors , could interact with central endogenous opioid neurotransmitter systems and then induce or perpetuate pathological states such as persistent pain syndromes , depression , substance use disorders , and their comorbidity .
it has been reported that one of the most biologically active cytokines in the early phases of ali is il-1 , which is elevated in plasma , and that il-1 is a potent inducer of lung and causes release of a variety of proinflammatory chemokine , such as monocyte chemotactic protein-1 , macrophage inflammatory protein-1 , il-6 , and il-8 with subsequent recruitment of inflammatory cells into the air spaces as well as being able to alter endothelial - epithelial barrier permeability and fluid transport leading to edema [ 711 ] .
although the expression of il-1 has been detected in lung tissues of rats from liver ischemia reperfusion injury , and il-1 was found in lung tissues of rats with ali induced by ii / r , also the expression of il-1 in the ischemic jejunum and the lung tissues treated with ii / r was increased [ 1 , 13 ] . whether il-1 may be considered as a candidate molecule for regulating ali induced by ii / r and underlying regulating mechanism involved in p38 need to be elucidated . in this study
then , by administration of sb239063 , an inhibitor of p38  and p38  mitogen activated protein kinase ( mapk ) , we investigated whether il-1 expression could be regulated by p38 mapk signal , so as to determine role of il-1 in ali induced by ii / r and relative molecular signal regulation in lung tissues subjected to ii / r .
all adult male sprague - dawley ( sd ) rats , weighing 200250  g , were purchased from animal center of sichuan university . the guidelines of caring laboratory animals have been described as follows .
briefly , all animal care , breeding , and testing procedures were conformed to the principle of guidance suggestion for the care and use of laboratory animals promulgated by national institutes of health .
rats were housed in cages with a 12-h light / dark cycle and have access to food and water . what is more , following intestinal ischemia / reperfusion ( ii / r ) experiment , animals were placed in warm condition ( 36.537c ) using a heat lamp in order to keep their body temperature .
adult male sd rats were randomly divided into sham group and intestinal ischemia / reperfusion ( ii / r ) groups ( n = 2 ) at 8  h and 16  h postoperation , described in table 1 .
rats were anesthetized using 3.6% chloral hydrate by intraperitoneal injection and then fixed by prone position .
after the skin of rat was disinfected using povidone , then abdomen cavity was opened , then the superior mesenteric artery ( sma ) and coeliac artery ( ca ) were clamped for 40  min at the same time , followed by intestinal reperfusion as described by previous reports [ 14 , 15 ] .
the sham group was performed with similar laparotomy but did not clamp sma and ca .
adult male sd rats were randomly divided into two groups : ordinary ii / r ( injury ) group ; inhibitor + ii / r ( inhibitor ) group .
inhibitor + ii / r ( inhibitor ) group accepted p38  and p38  inhibitor sb239063 ( 10  mg / kg ) injection at 1 hour after operation .
the injury groups were performed with similar processes but were not injected by p38  and p38  inhibitor .
animals performed for surgery 1 were deeply anesthetized at 8 hours of postoperation ( hpo ) and 16  hpo , respectively . then , lung tissues were obtained and stored at 80c for further use .
moreover , animals subjected to surgery 2 were sacrificed at 16  hpo ; then , their lung tissues were harvested and stored at 80c for further use . wet / dry ratio of lung was used for testing the level of pulmonary edema .
the right lung was placed at 90c drying oven for 24  h after it was weighed .
wet / dry ratio of lung was calculated by dividing the wet weight by the dry weight .
the lung tissues were sectioned in a freezing microtome ( leica cm1990 , germany ) , and then the sections were fixed for 30  s and washed for 2  s. the sections were stained using hematoxylin at 60c for 60  s and then washed in running water for 10  s. then , the sections were incubated in 1% hydrochloric acid alcohol for 3  s. this was followed by a blue staining using promoting blue liquid for 10  s and then washed using running water for 30  s. afterwards , 0.5% eosin for 60  s was added to the sections and the sections were washed using distilled water for 2  s. 80% ethyl alcohol for 2  s , 95% ethyl alcohol for 2  s , alcohol for 2  s , phenolic xylene for 3  s , xylene ( i ) for 3  s , and xylene ( ii ) for 3  s were successively added to the sections . in the end , the sections were mounted using neutral balsam .
morphologic examinations were performed using light microscopy , and ali scoring was evaluated blindly according to mikawa 's method .
ali scores were evaluated by alveolar congestion , hemorrhage , neutrophilic infiltration , and thickness of the alveolar wall and hyaline membrane formation .
the severity of lung injury was scored from 0 to 4 ( 0 , minimal damage ; 1 , mild damage ; 2 , moderate damage ; 3 , severe damage ; 4 , maximal damage ) . in each animal ,
4 separate lung sections were graded to generate the mean score [ 16 , 17 ] .
the lung tissues were harvested and dehydrated by 30% sucrose overnight and then sectioned at 10  m thickness in a freezing microtome ( leica cm1990 , germany ) .
the sections were washed three times in 0.01  mol / l pbs for five minutes , and 5% goat serum with triton x-100 was added on the sections for 30  min to block nonspecific binding site .
after the sections were incubated at 4c in a moist chamber over 18  h with anti - il-1 antibody ( rabbit , pa90563rao1 , 1  :  100 ) and anti - p38 antibody ( rabbit , sc7149 , 1  :  200 ) , respectively , they were washed three times in 0.01  mol / l pbs for 5 minutes
. then sections were incubated with fluorescence - labeled secondary antibody alexa594 ( anti - rabbit , invitrogen , 1  :  200 ) at 37c for 1  h in dark place .
after the sections were washed three times in 0.01  mol / l pbs for 5 minutes in dark condition , the nucleus was counterstained with dapi . in the end
, sections were observed by the immunofluorescent microscope , and the immunostaining images were obtained using leica af6000 , to observe the location of il- and p38 in lung was observed .
total rna was isolated from lung tissues after 8  h and 16  h ii / r , and lung tissues from sham groups using the trizol reagent ( invitrogen ) produce cdna , respectively .
then , quantitative rt - pcr ( qrt - pcr ) analysis was performed to quantify the level of il-1 mrna .
qrt - pcr of cdna was performed using the forward and reverse primer sequences of il-1 , forward : gagctgaaagctctccacct ; reverse : ttccatcttcttctttgggt .
then the reaction was performed at 95c for 2  min and circulated 40 times at 95c for 20  s , 50c for 30  s , and 60c for 40  s. the fluorescence was collected and recorded after finishing 40 cycles .
data were analyzed using a relative critical threshold ( ct ) method where the amount of target normalized to the amount of inner control .
1  ml precooling protein extraction reagent which contained 98% ripa lysis buffer ( beyotime , jiangsu , china ) and 2% cocktail pill ( roche ) was added to lung tissues and lung tissues were homogenized on ice using in situ homogenate machine so that tissue mass was invisible to the eyes .
after the mixture was ice - bathed for 30  min and blended every 10 minutes , the mixture was quashed for 5  s every 5  s , total 10 times in ultrasonic cell breaking machine .
the lysate was centrifuged at 12000  g for 15  min at 4c ; then , the supernatant was collected , and the concentration of protein was curtained by bca protein assay kit ( beyotime institute ) .
after that , the precipitated proteins ( 80  g ) were separated on a sds - page gel at 350  ma for 2  h and transferred to pvdf membranes at 350  ma for 2  h. sds - page consisted of 10  ml 15% separation gel and 6  ml 5% spacer gel .
10  ml 15% separation gel consisted of 2.3  ml ddh2o , 5  ml 30% polyacrylamide , 2.5  ml 1.5  mol / l tris ( ph8.8 ) , 0.1  ml 10% sodium dodecyl sulfate ( sds ) , 0.1  ml 10% ammonium persulfate ( ap ) , and 0.004  ml temed .
6  ml 5% spacer gel consisted of 4.1 ddh2o , 1.0  ml 30% polyacrylamide , 0.75  ml 1.5  mol / l tris ( ph6.8 ) , 0.06  ml 10% sds , 0.06  ml 10% ap , and 0.006  ml temed .
after the transfer was finished , the pvdf membranes were washed in 1  tbs , then placed in 5% nonfat dried milk seen as sealed liquid , and slowly swayed for 2  h on horizontal pendulum table . then the pvdf membranes were incubated with primary anti - il-1 antibody ( rabbit , pa90563rao1 , 1  :  400 ) , anti - p38 antibody ( rabbit , sc7149 , 1  :  200 ) , and anti - p - p38 antibody ( rabbit , sc-101759 , 1  :  400 ) overnight at 4c , respectively . after the pvdf membranes were rapidly washed three times in 1
 tbst for 5 minutes on horizontal pendulum table , the pvdf membranes were incubated with the secondary antibody abexcel ( anti - rabbit , abcam , 1  :  1000 ) for 2  h with bobble at room temperature on horizontal pendulum table . afterwards , the pvdf membranes were washed three times in 1  tbst for 5 minutes on horizontal pendulum table and scanned in alpha innotech ( biorad ) with ecl .
the experimental data was in the form of the mean  standard deviation ( sd ) and analyzed using spss 13.0 .
moreover , data were analyzed by one - way analysis of variance ( anova ) .
wet / dry ratio of lung was measured to detect the degree of pulmonary edema .
all results showed that rats with ii / r or not have a different wet / dry ratio of lung , which revealed a statistical significant uptrend with more treatment time . and rats with ii / r have the highest wet / dry ratio of lung at 16  hpo , compared with 8  hpo rats and sham groups , which have significant difference ( figure 1(a ) ) .
these data confirmed that the level of pulmonary edema at 16  hpo enhanced greatly than 8  hpo and sham groups .
the results demonstrated that there was normal morphology in sham group , in which the structure of alveoli was clear and intact with the epithelium and microblood vessel distribution .
almost no evidence of lung injury was seen in the sham group . however , following the ii / r , severe lung injury was found , in which the presence of extensive interstitial edema , severe alveolar hemorrhage , and extensive inflammatory cell infiltration could be seen ( figure 1(b ) ) .
the assessment of ali scores showed that ii / r increased the lung histological score .
ali scores in rats with ali induced by ii / r at 8  hpo and 16  hpo were higher than that in sham group ( figure 1(c ) ) .
immunofluorescent staining was performed to detect the distribution of il-1 and p38 mapk in lung tissues , which showed that , in the lung tissue , the localizations of il-1 and p38 mapk were mainly found in both epithelia and macrophage ( figures 2(a ) and 2(b ) ) .
western blotting was used for verifying the expression level of il-1 and the level of phosphorylated p38 mapk in lung tissues .
the results showed that rats with ii / r are different from sham one on the level of il-1 expression .
rats with ii / r have the highest il-1 translation level in lung tissues at 8  hpo and 16  hpo , compared with sham group ( figure 3(a ) ) .
moreover , the phosphorylation of p38 mapk was the highest at 16  hpo compared with 8  hpo and sham group ( figure 3(b ) ) .
p38  and p38  inhibitor sb239063 , designed and purchased from company , was applied for interfering in the protein expression of p38  and p38 . the results showed that , in rats injected by p38  and p38  inhibitor ( inhibitor group ) , low expression level of p38  and p38  could be detected , when compared with those of no injection one ( injury group ) ( figure 4(a ) ) . as a result , the mrna expression for il-1 was correspondingly decreased in rats subjected to p38  and p38  inhibitor administrated rats ( inhibitor group ) , when compared with only ii / r groups ( injury group ) ( figure 4(b ) ) .
moreover , the protein expression of il-1 revealed similar result with mrna expression of il-1 ( figure 4(c ) ) .
the effects of p38  and p38  inhibitor on the histopathological changes of lungs in rats with ii / r were shown in figures 4(d ) and 4(e ) .
morphological study showed the lung tissues of rats were badly damaged in injury group , with severe interstitial edema , severe alveolar hemorrhage , and extensive inflammatory cell infiltration , whereas only mild lung edema , hemorrhage , and inflammatory cell infiltration were observed in inhibitor group ( figure 4(d ) ) .
the results of ali scores showed that after administration of p38  and p38  inhibitor , a drastic reduction in pathological score was shown in inhibitor group compared with that in injury group ( figure 4(e ) ) .
in this study , ali can be caused by ii / r , which was similar as in the previous study [ 18 , 19 ] .
the results of immunofluorescent staining demonstrated that il-1 and p38 mapk were detected in both epithelia and macrophage in lung tissues after ii / r .
moreover , the results of western blotting showed that the raising levels of the expression of il-1 and p38 mapk in lung tissues after ii / r were exhibited .
previously , upregulation of il-1 was detected in lung tissues , but the activation of p38 mapk in lung tissues after ii / r was reported by a few . in this study , we confirmed activation of p38 mapk exhibited similar change tendency with il-1. importantly , the level of il-1 in lung tissues after ii / r was decreased with the administration of p38  and p38  inhibitor , which indicated that the decrease of the level of il-1 expression may be correlated with inhibition of p38 mapk signal pathway .
previous study showed obstruction of thoracic lymphatic flow before ii / r decreased the ability of cultured lung tissue explants to release il-1 , il-10 , and vegf , and diminished neutrophil recruitment , as well as reduced concentration of tnf- and il-1 level .
there were different changes in the level of protein and gene expression for il-1 after treatment and phospholipase a(2 ) inhibitor chloroquine or cyclooxidase inhibitor indomethacin after injury , respectively .
il-1 increase may be the result of the lung injury induced by ii / r , whereas , in this study , significantly , we found p38 inhibitor is useful to downregulate il-1 , which is an important finding to elucidate the mechanism of p38 mapk signal pathway involving in il-1 regulation .
although it has well been known p38 mapk plays a crucial role in organ damage after ii / r [ 2325 ] , no report showed that inhibition of p38 can influence il-1 in rats with ali after ii / r .
our results provide the novel information , in which p38 inhibition to suppress il-1 may be a useful strategy for the treatment of ali .
previously , most of views thought that il-1 could induce p38 mapk activation in gastric adenocarcinoma , and pretreatment of cells with tangeretin could inhibit il-1-induced p38 mapk in human lung carcinoma cells .
moreover , other opinions indicated that p38 mapk can activate nuclear factor- ( nf- ) b to induce il-1 production in rat pulmonary interstitial macrophages . comparing these findings , we found inhibitor of p38 mapk decreased effectively the level of il-1 expression and significantly improved the lung injury , which may be important to give a new cure by administration of p38 mapk to interfere ali after intestinal ischemia reperfusion .
it has been known that il-1 is a bad molecule that is not convenient recovery [ 2931 ] ; therefore , decreased il-1 expression should be better for the recovery of lung injury . to our knowledge , this is the first study to show a protective effect of inhibition of p38  and  mapk on ali induced by ii / r .
our results give important implication to explore the mechanism of lung injury in rats subjected to ii / r , which is involved in p38 mapk regulating il-1. this is different from previous report that il-1 directly regulates p38 . in conclusion
, ii / r can increase the levels of expression of il-1 and p38 mapk in ali , and the level of il-1 expression and the severity of ali can be decreased by p38  and  inhibitor sb239063 .
this fundamental information may provide a potential strategy to target directly p38 or il-1 for the treatment of reducing ali induced by ii / r in the future clinical trial .
 acute lung injury ( ali ) induced by intestinal ischemia / reperfusion ( ii / r ) has high incidence and mortality , in which il-1 was essential for the full development of ali . 
 however , the detailed regulating mechanism for this phenomenon remains to be unclear . 
 the purpose of this study was to investigate whether inhibition of p38 mapk could downregulate the expression of il-1 to protect lung from acute injury in ii / r rats . here , we found that the level of pulmonary edema at 16 hours after operation ( hpo ) was obviously enhanced compared to that in 8hpo and sham groups . 
 immunofluorescent staining demonstrated that il-1 and p38 mapk were detected in lung tissues . 
 and rats with ii / r have the highest translation level for il-1 and phosphorylation of p38 mapk in lung tissues at 16hpo compared with 8hpo and sham groups . 
 moreover , administration of sb239063 , an inhibitor of p38  and  , could effectively downregulate the expressions of il-1 and protects lung tissues from injury in ii / r rats . 
 our findings indicate that the inhibition of p38  and  may downregulate the expression of il-1 to protect lung from acute injury in ii / r , which could be used as a potential target for reducing ali induced by ii / r in the future clinical trial . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion