myocardial ischemia / reperfusion ( i / r ) injury , a general health problem , is due to blood restoration after a critical period of coronary artery obstruction , which is associated with clinical interventions such as thrombolysis , angioplasty , and coronary bypass surgery . this reperfusion injury involves the activation of an inflammatory cascade and is manifested as functional impairment , arrhythmia , and accelerated progression of cell death in certain critically injured myocytes . the major mediators of reperfusion injury are oxygen radicals , calcium loading , and neutrophils [ 13 ] . currently , great progress has been made in the basic research about the mechanism of myocardial i / r , but symptomatic treatment still remains as the primary therapeutic plan . with the development of molecular biology , pharmacy , and other related disciplines , it will be an important task for scientists in the future to find a safer , more effective therapeutic method . lipoxins ( lxs ) are the most recent addition to the family of bioactive products generated from arachidonic acid . these molecules have both proinflammatory and anti - inflammatory actions   and inhibit neutrophil / granulocyte infiltration and reactive oxygen species ( ros ) production [ 6 , 7 ] . lipoxins are short - lived endogenously produced nonclassic eicosanoids , whose appearance in inflammation signals the resolution of inflammation [ 8 , 9 ] . recent studies showed that lx could protect several organs such as heart , brain , lung , kidney , and stomach from i / r injury [ 6 , 1013 ] . however , there has been little research about their effect on myocardial systemic inflammation , ultrastructure changes , and arrhythmia caused by i / r injury . therefore , we aimed to investigate the effects of lxa4 preconditioning and postconditioning on myocardial i / r injury in rats . in particular , we studied the effects of lxa4 on oxidative stress , inflammatory reaction , na - k - atpase , and connexin 43 ( cx43 ) alternations caused by i / r injury . all animal procedures were approved by wenzhou medical college animal care and use committee , which is certified by the chinese association of accreditation of laboratory animal care ( syxk , zhejiang 2010 - 0150 ) . sprague - dawley ( sd ) male rats ( eight weeks old , 200 to 250  g ) were fed a standard diet and maintained in controlled environment at 25  1c under a 12  h light - dark cycle . briefly , rats were anesthetized by an intraperitoneal injection of 10% chloral hydrate ( 300  mgkg body weight ) and placed in a supine position . next , the animals were intubated for artificial ventilation with 100% oxygen using a small animal breathing machine ( tidal volume 5  ml , frequency 70 per min ) and monitored by ecg . thoracotomy was performed between the sternum and left costa , and then the pericardium was gently opened . myocardial ischemia was induced by ligating the left anterior descending coronary artery ( lad ) using a 30 silk suture with a section of pe tubing placed over the lad and 1  mm from the tip of the normally positioned left atrium , and the coronary artery was occluded by pulling on the suture tightly 10  min later . after 30  min of myocardial ischemia , reperfusion started by releasing the ligature and removing the tube for 120  min . the chest wall was closed , the animal extubated , and body temperature maintained using a 37c warm plate . the indications of successful lad occlusion included elevated st segment ( 0.1  mv ) or sharp rise of t wave , wider and higher qrs wave on ecg , and visual cyanosis of myocardial discoloration . the indications of successful reperfusion were st segment depression ( 1/2 ) of the ecg , gradual falling of t wave , and qrs wave , and myocardial color turning to pink [ 1416 ] . lxa4 ( c20h32o5 , see supplementary figure 1 in the supplementary material available online at http://dx.doi.org/10.1155/2013/231351 ) was purchased from cayman chemical company , ann arbor , usa ( cat . seventy - two sd rats were used in the study and were randomly divided into 6 groups as follows : ( 1 ) applying lxa4 before i / r group 1 ( lx1 ) : femoral vein injection of lxa4 ( 100  g / kg , dissolved in normal saline 2  ml / kg ) before the i / r procedure ; ( 2 ) applying lxa4 after i / r group 2 ( lx2 ) : after 30  min of myocardial ischemia , reperfusion lasted for 30  min before lxa4 ( 100  g / kg , dissolved in normal saline 2  ml / kg ) was injected through femoral vein . after that , reperfusion was resumed for an additional 90  min ; ( 3 ) ischemia / reperfusion group 1 ( i / r1 ) : femoral vein injection of normal saline ( 2  ml / kg ) before i / r ; ( 4 ) ischemia / reperfusion group 2 ( i / r2 ) : after 30  min of myocardial ischemia , reperfusion lasted for 30  min , and then normal saline ( 2  ml / kg ) was injected through femoral vein . after that , reperfusion was resumed for an additional 90  min ; ( 5 ) sham group 1 ( c1 ) : rats underwent a similar operation without myocardial i / r , and other treatment was the same as i / r1 group ; ( 6 ) sham group 2 ( c2 group ) : rats underwent a similar operation without myocardial i / r , and other treatment was the same as i / r2 group . blood samples were collected in each group immediately before thoracotomy and after anaesthetization ( t1 ) or after experiments ( t2 ) . in groups c1 and c2 , t2 was obtained after 150  min of placing surgical suture under lad . for all other groups , t2 blood samples were obtained after 120  min of reperfusion . the heart was removed after obtaining blood samples ( t2 ) , and a portion of myocardial tissue was fixed in 4% formalin . a separate portion of myocardial tissue was frozen in liquid nitrogen and was kept in the freezer at 70c . ecg was recorded throughout the experiment and at a fast speed throughout ischemia and reperfusion . ventricular arrhythmias during ligation of lad to reperfusion ( t3 ) were determined in accordance with the guidelines of the lambeth convention . ventricular tachycardia ( vt ) was defined as a run of four or more consecutive ventricular premature beats . ventricular fibrillation ( vf ) was defined as a ventricular rhythm without recognizable qrs complex , and the signal morphology changed from cycle to cycle . the ecg records for the incidence and total duration of vt and vf during reperfusion were analyzed . in order to evaluate the severity of arrhythmias , ventricular arrhythmia scores ( vas ) were used by giving a certain grade to each rat as follows : 0 = less than 5 times vt ; 1 = more than 5 times vt or other arrhythmias , no vf ; 2 = 11~30  s vt or other arrhythmias , no vf ; 3 = 31~90  s vt or other arrhythmias , no vf ; 4 = 91~180  s vt or other arrhythmias , and/or less than 10  s reversible vf ; 5 = more than 180  s vt or other arrhythmias , and/or more than 10  s reversible vf ; 6 = irreversible vf . for cytokine immunoassay , blood samples were collected by femoral venipuncture at set time points , before thoracotomy ( t1 ) and after reperfusion ( t2 ) , and serum levels of il-1 , il-6 , il-8 , il-10 , tnf- , and ctni were measured using a rat elisa kit ( shanghai boyun biotech , china ) in accordance with the manufacturer instructions . mda content was determined on frozen myocardial tissue by thiobarbituric acid assay kit ( nanjing jiancheng bioengineering institute , china ) . the optical density at 532  nm was measured using a spectrophotometer ( uv-2000 , unico , shanghai ) . myocardial sod activity was determined on frozen myocardial tissue using xanthine oxidase assay kits ( nanjing jiancheng bioengineering institute , china ) and expressed as units per milligram protein . the activity of na - k - atpase was assessed by measuring the release of inorganic phosphate ( pi ) from atp according to the kit protocol ( nanjing jiancheng bioengineering institute , china ) . briefly , frozen myocardial tissue was weighted and normal saline was added at a ratio of 1  :  9 . then , the homogenates were centrifuged to obtain the supernatant , followed by the measurement of protein concentration using bradford method . finally , na - k - atpase activity was determined by measuring the amount of inorganic phosphate with malachite green dye method and expressed as micromoles per milligram protein . the myocardial tissue 2  mm inferior to the ligation of lad was fixed in paraformaldehyde and embedded in paraffin . heart tissues were cut into 5  m sections for histopathological examination by hematoxylin - eosin ( h&e ) staining and for immunohistochemical detection of cx43 . briefly , rehydrated sections were pretreated with pepsin solution for 15  min at 37c . the following protocols were according to the manual of the s - p kit ( zsgb - bio , beijing ) . the myocardium near lad was incubated for 10  min with a peroxidase - blocking solution and goat serum solution for 30  min and then incubated with primary antibody ( rabbit anti - rat cx43 ) ( cell signaling technology , usa ) overnight at 4c . after three rinses with pbs , the sections were incubated with biotinylated secondary antibody ( goat anti - rabbit igg , included in the s - p kit ) for 30  min and further developed with 3,30-diaminobenzidine tetrahydrochloride ( dab ) . for tem examination , samples containing a 2  mm portion from the edge of the incision were immediately fixed in 0.1  m phosphate buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde for 4  h. the samples were then fixed with 1% osmium tetroxide for 2  h , dehydrated through a graded ethanol series , and embedded in epoxy resin . resin - embedded blocks were cut into 60~80  nm ultrathin sections with an ultramicrotome ( pt - xl , rmc , usa ) . the ultrathin sections were placed on carbon coated nickel grids and examined with an h-7500 transmission electron microscope ( h-7500 , tokyo , japan ) operating at 80  kv . total rna was extracted using trizol reagent ( invitrogen , usa ) according to the manufacturer 's instructions . rna ( 1  ng ) was reversed with the cdna synthesis kit ( invitrogen , usa ) . real - time polymerase chain reaction ( pcr ) was performed using the sybr green system ( bio - rad , usa ) . the relative quantification of a target gene was normalized to gapdh and calculated using the absolute quantification standard curve method . melting curve profiles were produced at the end of each pc to confirm the specificity amplifications . equal amounts of protein ( 50  g ) were subjected to sds - page . membrane was blocked with 5% nonfat dry milk in tris - buffered saline , 0.1% tween 20 ( sigma , usa ) , and immunoblotting was performed using rabbit anti - rat na - k - atpase ( 1  :  500 , cell signaling technology , usa ) as described by the manufacturer . anti--actin antibody ( 1  :  200 , santa cruz , usa ) was used as loading control . blots were then developed by incubation with biotinylated anti - rabbit antibody ( 1  :  2000 ; vector laboratories ) , followed by incubation with abc reagent ( ge , usa ) . signal was detected using an ecl luminescence kit ( ge , usa ) and x - ray film . statistical analysis was performed by one - way anova to compare more than two groups or with paired t - test to compare two groups . the effects of different treatments on i / r - induced arrhythmias are shown in table 1 . in c1 and c2 groups , neither vt nor vf was observed and only few ventricular premature beats appeared during the whole procedure . in contrast , almost all rats in i / r1 and i / r2 groups experienced obvious st segment elevation , occurring vt , and high frequency of vf . furthermore , the frequency of arrhythmias was significantly reduced in lx1 and lx2 groups with the exception of 2 rats showing vf in lx2 group . most of the arrhythmias observed occurred in the ischemia ( 30  min ) and in the first 30  min of the reperfusion periods . in addition , the durations of vt and vas were markedly higher in i / r1 and lx1 compared with c1 group , and the same was seen for i / r2 and lx2 groups when compared with c2 group , ( p < 0.05 in both cases ) . finally , vas and the vt durations of lx1 and lx2 groups were significantly ( p < 0.05 ) lower than those in i / r1 and i / r2 groups . interestingly , these parameters in lx1 group were significantly lower than those in lx2 group ( p < 0.05 ) , indicating that lxa4 preconditioning was more effective in preventing i / r - induced arrhythmias than lxa4 postconditioning . serum levels of il-1 , il-6 , il-8 , il-10 , tnf- , and ctni were determined using elisa kits . in contrast , cytokines levels at t2 in i / r1 and lx1 groups were significantly ( p < 0.05 ) elevated compared to c1 controls , and similar results were seen for i / r2 and lx2 groups compared with c2 controls . in the meantime , reduced levels of proinflammatory cytokines such as il-1 , il-6 , il-8 , and tnf- and elevated level of the anti - inflammatory cytokine il-10 were observed in lx1 and lx2 groups compared with i / r1 and i / r2 groups , respectively . these findings indicate that both lxa4 preconditioning and postconditioning could induce the expressions of anti - inflammatory cytokines and inhibit the induction of proinflammatory cytokines . moreover , these changes were more significant in lx2 compared with lx1 group ( p < 0.05 ) , further confirming that lxa4 postconditioning was more effective in preventing i / r injury than lxa4 preconditioning ( figure 1 ) . the production of mda , sod , and na - k - atpase activities in myocardial tissues in response to i / r injury were investigated . compared with c1 group , mda production and sod activity were increased and na - k - atpase activity was reduced in i / r1 and lx1 groups , and the same results were obtained in i / r2 and lx2 groups compared to c2 group ( p < 0.05 ) . furthermore , decreased mda production and increased activities of sod and na - k - atpase in lx1 and lx2 groups were observed compared with i / r1 and i / r2 groups , respectively . similar results were observed when lx2 group was compared with lx1 group , indicating that lxa4 postconditioning was more protective against i / r injury than lxa4 preconditioning ( figure 2 ) . cardiomyocytes were clearly seen in a well - arranged myofilament and intercalated disc manner in sham groups ( c1 and c2 ) , as well as abundant normal mitochondria with no swelling , normal matrix density , and intact cristae . the round endoplasmic reticulum can also be seen between the myofilaments and the cytoplasm ( figures 3(a ) and 3(b ) ) . however , in i / r1 and i / r2 groups , myocardial i / r produced remarkable ultrastructural damages associated with irregularities and edematous separation of myofilaments , hypercontraction , and shortening of sarcomeres . large area of cytoplasmic vacuolization and mitochondrial swelling were evident with decreased matrix density and distortion of cristae ( figures 3(c ) and 3(d ) ) . treatment with lxa4 showed clear protection with relatively parallel arrangement of myofilaments and normal sarcomeres . however , mild cytoplasmic rarefaction with mild edema could still be seen ( figures 3(e ) and 3(f ) ) . to further assess the effect of lxa4 on i / r injury , we analyzed the pathological changes it induced ( figure 3 ) . in the sham groups ( c1 and c2 ) , no epimorphosis , necrosis , or neutrophil infiltration was observed ( figures 3(g ) and 3(h ) ) . in contrast , nontreated i / r groups ( i / r1 and i / r2 ) showed local swelling , myocardial necrosis , disorganized myocardial fibers , and ruptured cells with large number of inflammatory cells in the cytoplasm ( figures 3(i ) and 3(j ) ) . in the lxa4-treated groups ( lx1 and lx2 ) , myocardial cells with normal structure and shape can be seen , though the myocardial fibers were mildly swollen or partially ruptured and slight edema can be seen in the interstitial tissues with small amount of inflammatory cells ( figures 3(k ) and 3(l ) ) . the gap junction protein cx43 is the major component of gap junctions between cardiomyocytes of mammalian ventricular myocardium , including human and rodent hearts . using an immunohistochemical approach , we wanted to know whether lxa4 preconditioning and postconditioning affected cx43 expression . as shown in figure 4 , cx43 expression , demonstrated by a pervasive brown / yellow color in cardiac muscle cells , was observed in myocardial tissue samples from all the groups . the areas of cx43 expression in myocardial tissue samples from sham groups ( c1 and c2 ) were larger than those detected in the i / r groups , which was also confirmed by the quantitative analysis of integral optical density ( iod ) ( figure 4 ) . cx43 was well arranged together at both ends of the intact cardiac myocyte in i / r1 and i / r2 groups , and cx43 staining was significantly reduced when compared with the sham groups . however , in lxa4-treated lx1 and lx2 groups , most cx43 staining was located at the ends of intact cardiac myocytes ( intercalate disc ) and redistributed in both sides of the cell . compared with nontreated i / r1 and i / r2 groups , the iod value was statistically higher in lx1 and lx2 groups , respectively . additionally , cx43 expression of lxa4 preconditioning ( lx1 group ) was significantly ( p < 0.05 ) higher than lxa4 postconditioning ( lx2 group ) ( figure 4 ) . the na - k - atpase and cx43 mrna levels were examined using real - time quantitative pcr to determine if i / r injury influences the na - k - atpase and cx43 expressions and whether this effect is blocked by the different lxa4 treatment ( figure 5 ) . compared with the sham groups ( c1 and c2 ) , mrna levels of na - k - atpase and cx43 in i / r1 , lx1 , i / r2 , and lx2 were remarkably reduced . meanwhile , na - k - atpase and cx43 expressions in lxa4-treated lx1 and lx2 groups were higher than the saline - treated i / r1 and i / r2 groups , respectively ( p < 0.05 ) . furthermore , na - k - atpase mrna level of lxa4 preconditioning ( lx1 ) was lower than that of lxa4 postconditioning ( lx2 ) . however , cx43 expression was higher in lx1 compared to lx2 group ( p < 0.05 ) . the results of na - k - atpase isoforms analyzed by western blotting are shown in figure 6 . compared to the sham c1 group , the expressions of na - k - atpase isoforms 1 , 2 , 3 , and 1 were markedly reduced in i / r1 and lx1 groups and similar results were obtained in i / r2 and lx2 groups , compared to c2 group ( p < 0.05 ) . however , na - k - atpase isoform levels in lxa4-treated lx1 and lx2 groups were higher than the saline - treated i / r1 and i / r2 groups respectively ( p < 0.05 ) . additionally , protein levels of na - k - atpase in lxa4 preconditioning ( lx1 ) were significantly ( p < 0.05 ) lower than those measured in lxa4 postconditioning ( lx2 ) . with the widespread use of cardiac surgery for congenital heart disease and coronary artery bypass grafting under extracorporeal circulation , thrombolytic therapy , percutaneous transluminal coronary intervention , organ transplantation , and cardiopulmonary resuscitation , it has currently become a hot topic for cardiovascular scientists to find safe and effective drugs to treat myocardial i / r injury . in this study , we used rats to induce i / r injury because of the little variation in their cardiovascular system , similarity to human coronary artery territory , less myocardial collateral circulation , and the high probability of occurrence of i / r injury . due to the smaller size of coronary artery in rats , more than 30  min of blocking time can easily lead to permanent occlusion of coronary artery [ 14 , 19 , 20 ] . therefore , we chose 30  min of myocardial ischemia followed by 120  min reperfusion . currently , the effect of i / r injury on myocardial ultrastructure is not entirely clear . reported that lx can inhibit neutrophil aggregation at inflammatory sites and regulate the balance of proinflammatory / anti - inflammatory cytokines . in addition , lxa4 can suppress vascular - endothelial - growth - factor- ( vegf- ) induced inflammatory reaction through the regulation of il-6 , tnf- , il-8 , icam-1 , and il-10 expressions in human umbilical vein endothelial cells . souza et al .   found that reduced production of tnf- following lxa4 treatment of intestinal i / r injury may be due to the increased il-10 expression , and lxa4 could not prevent reperfusion injury in il-10-deficient mice . in our study , we confirmed that i / r resulted in myocardial inflammatory injury through analyzing serum components before and after i / r . although both proinflammatory / anti - inflammatory cytokines were upregulated , the expressions of proinflammatory cytokines were much higher than the anti - inflammatory ones . however , with lxa4 treatment , the expressions of proinflammatory cytokines ( il-1 , il-6 , il-8 , and tnf- ) were reduced while the anti - inflammatory cytokine il-10 was upregulated , leading to an anti - inflammatory state ( figure 1 ) . the results of h&e staining confirmed that lxa4 could alleviate i / r injury as evidenced by reduced swelling , well - organized cardiac muscle , clear striations , and less inflammatory cell infiltration . in accordance with the pathological changes , we found that lxa4 protected the ultrastructures of myocardial tissue as shown by normal matrix density , relatively parallel arrangement of myofilaments , and normal sarcomere mitochondria by tem examination ( figure 3 ) . we found that i / r groups had increased mda levels and sod activity , which indicated enhanced oxidative stress . after lxa4 treatment , sod activity was significantly improved and mda levels were markedly reduced ( figure 2 ) , suggesting that the alleviated i / r injury seen after lxa4 treatment might be due to decreased ros generation . close to 90% of cellular oxygen intake is used in oxidative phosphorylation reactions in the mitochondria , but 1% to 2% of oxygen can escape to form ros . ros can decrease the efficiency of mitochondrial performance and cause swelling of mitochondria by attacking the mitochondrial inner membrane . as we can see from the tem images ( figure 3 ) , the swelling condition of mitochondria has been remarkably alleviated after lxa4 treatment , with clear cristae and well - arranged cardiac fiber ultrastructure . this agrees with the mda levels and the sod activity data and further confirms that reduced i / r injury may be due to the inhibition of oxygen - free radical targeting of mitochondrial membranes . the na - k - atpase is a heterodimer that plays an important role in the maintenance of cell homeostasis by regulating membrane potential and cation transport across the sarcolemmal membrane , which is a reliable indicator of metabolic disorders and tissue injury . i / r injury could affect mitochondrial function and inhibit na - k - atpase activity , leading to intracellular calcium overload , activation of proteases , and release of oxygen - free radicals , which in turn aggravate i / r injury . reported that the maintenance of na - k - atpase cell surface abundance was crucial in myocyte survival after an ischemic attack treated by ouabain preconditioning , and the protection conferred by increased surface expression of na - k - atpase may be independent of ion transport . meanwhile , zheng et al . found that the protection from cardiac injury may be determined by the na - k - atpase activity , involving erk1/2 and pi3  k / akt signal pathways . furthermore , continued intermittent hypobaric hypoxia can reduce the reperfusion injury in guinea pig by increasing myocardial na - k - atpase activity . the na - k - atpase consists of  and  subunits ; the  subunit is responsible for the catalytic activity of the enzyme and includes three isoforms : 1 , 2 , and 3 in the rat heart . the  subunit is responsible for the proper localization and insertion of na - k - atpase in the sarcolemmal membrane . in this study , the expressions of 1 , 2 , and 3 proteins decreased after ischemia / reperfusion injury , which affected the active center and further reduced the activity of the atpase enzyme . the expression of  subunit also declined after i / r injury , and this could cause damage to the cytoskeleton , lead to detachment of na - k - atpase from the cell membrane , and further reduce its activity . the effect of ischemia / reperfusion on the expressions of na - k - atpase 1 , 2 , and 3 subunits were slightly different from earlier report , which may be due to differences in experimental and detection methods . the content and the activity of na - k - atpase can reflect myocardial injury to a certain extent , and i / r injury can also be alleviated by increasing its content and activity . suggested that cardiomyocytes could be protected potentially by reversing the oxidation inhibition of na - k - atpase . and from the our study , we know that lxa4 enhanced na - k - atpase subunits expressions ( figures 5 and 6 ) and reduced ctni concentration , indicating that the cardioprotective effect of lxa4 could be due to increased na - k - atpase content and activity the gap junction ( gj ) plays key roles not only in electrical coupling of cardiomyocytes but also in intercellular transport of biologically active substances . gj is mainly located in the intercalated disc of the cardiac tissue   and participates in decision making for cell survival versus cell death in various types of cells . it has been suggested that reperfusion injury to the heart is partially mediated by gap junctions . cx43 forms gap junctions that facilitate electrical cell - cell coupling and unopposed / nonjunction hemichannels which provide a pathway for the exchange of ions and metabolites between cytoplasm and extracellular milieu . it has also been known that cx43 participates in the process of myocardiac ischemia / reperfusion injury [ 32 , 33 ] . during i / r injury , alterations in cx43 expression , its localization and its phosphorylation status , and changes in gj properties collectively contribute to myocardial infarction and arrhythmogenesis [ 3436 ] . from our research , we found that cx43 was well arranged together in both ends of intact cardiac myocytes ( intercalate disc ) in the sham group , while in the i / r group , cx43 expression was reduced which was associated with damaged cellular ultrastructures ( figures 4 and 5 ) . in addition , increased vas , vtt , and vft duration and reduced cx43 mrna and protein expressions after injury strongly indicate that i / r injury could induce arrhythmia through cx43 pathway . the mechanism of arrhythmogenesis caused by i / r injury has not yet been illustrated clearly . found that this may be due to the structural and functional alternations of cx43 after injury , and danik reported that cx43 knockout mice showed complicated tachyarrhythmia . recently , two lipoxins have been identified , lipoxin a4 ( lxa4 ) and lipoxin b4 ( lxb4 ) . lxa4 treatment in our study showed that this molecule can alleviate cx43 degradation and protect its distribution that limited the damage to the ultrastructure of cardiac tissue caused by i / r injury . at the same time , the mrna transcriptional levels of cx43 were notably increased , which was in accordance with the immunohistochemical results . this indicates that lxa4 plays a protective role in regulating the expressive levels of cx43 protein and gene . in addition , reduced arrhythmia frequency and vas were observed , indicating that the protective effect of lxa4 may be associated with cx43 remodeling , an observation consistent with a previous study showing that cardiac function could be preserved through cx43 remodeling . the pharmacological preconditioning and postconditioning with lxa4 can both attenuate myocardial i / r injury in rats , with lxa4 preconditioning being more effective in preventing the arrhythmogenesis than the lxa4 postconditioning . however , lxa4 postconditioning had a better protective effect on na - k - atpase and the ultrastructure of the myocardium , which may be related to the physicochemical property , duration of the treatment , and the dose of lxa4 . we also observed that most arrhythmias happened in the ischemia ( 30  min ) and the first 30  min of reperfusion , which may be due to the activated compensatory mechanism of the injured body and increased tolerance to the drug . cx43 is also involved in pathological and physiological processes other than arrhythmias during i / r injury [ 41 , 42 ] , and it may cause uncontrolled opening of hemichannels in the plasma membrane   and may be deleterious to the myocardium . blocking hemichannels may confer cardioprotection by preventing ionic imbalance , excessive entry of na and ca , atp leakage , cell swelling , and loss of critical metabolites [ 40 , 45 , 46 ] . therefore , cx43-mediated intercellular communication of the injured tissue can be enlarged into other cells , and this may explain that lxa4 preconditioning induced higher cx43 expression but caused more serious damage to myocardium . at the same time , it indicated that the up - regulation of cx43 is helpless in the myocardial energy metabolism , systemic inflammation , and ultrastructure . the application of pharmacological preconditioning has been limited largely by the unpredictability of i / r injury , and this implicates that lxa4 postconditioning could have a great potential for clinical application in i / r injury in the future .   although there have been reports showing that lxa4 treatment was protective in cerebral i / r injury in rats   and myocardial i / r injury in mice   and rabbits , ours is the first study to report a protective effect of lxa4 preconditioning and postconditioning on myocardial i / r injury in rats . further studies are needed to optimize the dose , the timing , and the route of administration of lxa4 . lxa4 preconditioning and postconditioning in myocardial i / r injury can attenuate the metabolic disturbance in the myocardium through upregulating na - k - atpase expression . meanwhile , both lxa4 preconditioning and postconditioning protected the ultrastructure of the myocardium by inhibiting the inflammatory reaction and oxidative stress . in addition , the upregulation of cx43 expression had a positive effect in preventing arrhythmogenesis caused by i / r injury . all the results showed that lxa4 has great potential for protecting the myocardium from myocardial i / r injury .
this study aims to investigate the pre- and postconditioning effects of lipoxin a4 ( lxa4 ) on myocardial damage caused by ischemia / reperfusion ( i / r ) injury . 
 seventy - two rats were divided into 6 groups : sham groups ( c1 and c2 ) , i / r groups ( i / r1 and i / r2 ) , and i / r plus lxa4 preconditioning and postconditioning groups ( lx1 and lx2 ) . 
 the serum levels of il-1 , il-6 , il-8 , il-10 , tnf- , and cardiac troponin i ( ctni ) were measured . 
 the content and the activity of na+-k+-atpase as well as the superoxide dismutase ( sod ) , and malondialdehyde ( mda ) levels were determined . 
 along with the examination of myocardium ultrastructure and ventricular arrhythmia scores ( vas ) , connexin 43 ( cx43 ) expression were also detected . 
 lower levels of il-1 , il-6 , il-8 , tnf- , ctni , mda content , and vas and higher levels of il-10 , sod activity , na+-k+-atpase content and activity , and cx43 expression appeared in lx groups than i / r groups . 
 besides , h&e staining , tem examination as well as analysis of gene , and protein confirmed that lxa4 preconditioning was more effective than postconditioning in preventing arrhythmogenesis via the upregulation of cx43 . 
 that is , lxa4 postconditioning had better protective effect on na+-k+-atpase and myocardial ultrastructure .