ouabain , a steroid capable of inhibiting nak - atpase , is an adrenal and hypothalamus - derived hormone , present in mammalian plasma and tissue , which appears to be produced in response to volume expansion , angiotensin ii , and/or adrenocorticotropic hormone stimulation [ 14 ] . it has also been suggested that endogenous ouabain levels are modulated by stress conditions [ 5 , 6 ] . moreover , elevated levels of ouabain were found in hypertensive patients and normotensive individuals after physical exercise [ 5 , 7 ] . ouabain is able to modulate several immunological functions , inhibiting in vitro proliferation of lymphocytes induced by different mitogens [ 811 ] , increasing the expression of the molecule cd69 on the thymocyte surface , and decreasing the levels of phosphorylated mapk p38 and nuclear factor of activated t cells ( nfatc1 ) . the combination of ouabain plus corticoids produced a synergic effect inducing thymic atrophy in vivo . additionally , ouabain was shown to modulate function and cytokine production of monocytes in vitro   and dendritic cells . ouabain negatively modulated acute peritoneal inflammatory response induced by leishmania amazonensis infection in swiss mice . it was also reported that ouabain presented anti - inflammatory and antinociceptive effects , but the mechanisms involved were still unclear . it has also been described , using different models , that ouabain is capable of increasing the activation of nf-b , vcam-1 , and inos expression , no , and inflammatory cytokines levels [ 1922 ] . during inflammation , a complex process of intracellular signal transduction and transcription events , driven by multiple proinflammatory mediators and cytokines , is activated . acute inflammation is characterized by exudation of protein - rich fluid , edema , vasodilatation , and cell migration , into the site of injury . intraperitoneal injection of zymosan , a polysaccharide component of cell wall from saccharomyces cerevisiae , represents a self - resolving model of acute inflammation and has been widely used for the quantification of particular cell types and inflammation - related soluble factors [ 2426 ] . when administered in mouse peritoneal cavity , zymosan induces an increased vascular extravasation , which is one of the primary signs of inflammation . this is a key step in inflammatory exudate formation and is followed by a time - dependent recruitment of migratory cells , specially neutrophils and monocytes / macrophages [ 2632 ] . therefore , to better understand the anti - inflammatory effect of ouabain , we aimed to assess its action in vascular leakage , leukocyte migration , as well as apoptosis of leukocytes , cytokine production , and endocytosis . female swiss albino mice ( 2 months old ) were obtained from thomas george animal house of centro de biotecnologia ( cbiotec , ufpb , joo pessoa , brazil ) . animals were kept under standard laboratory conditions on a constant 12 h light / dark cycle with temperature ( 21  1c ) . all procedures adopted in this study were approved by institutional ethics committee of centro de biotecnologia ( protocol : 0504/11 ) . in all in vivo experiments , 0.56  mg / kg ouabain ( sigma aldrich ) [ 14 , 17 , 18 ] or phosphate buffered saline ( pbs ) was given intraperitoneally ( i.p . ) for three consecutive days . mice were randomly divided into four groups ( n = 6 ) and each group of mice was administrated intraperitoneally ( i.p . ) once a day for 3 consecutive days with either vehicle ( pbs ) or ouabain ( 0.56  mg / kg ) . one hour after the last injection on day 3 , mice were injected i.v . into the caudal vein with evans blue dye suspended in saline at 10  mg / ml ( 0.3  ml / mouse ) . the animals were euthanized after 30  min and the peritoneal cavity was lavaged with 1.5  ml of saline . lavage fluid was then centrifuged for 10  min at 350  g. the absorbance of cell - free lavage fluid was measured at 650  nm . mice were randomly divided into four groups ( n = 8) , and each group received i.p . the same volume ( 200  l ) of vehicle ( pbs ) or ouabain ( 0.56  mg / kg ) zymosan a was freshly prepared ( 2  mg / ml ) in sterile 0.9%  w / v saline and 0.5  ml was injected i.p . one hour after the last injection on day 3 . at either four or twenty - four hours after stimulus , animals were euthanized by cervical dislocation . the peritoneal cavity was then lavaged with 1  ml of cold pbs . exudates were pooled and the number of leukocytes present was determined by optical microscopy in hemocytometer , using turk 's solution ( 0.01% crystal violet in 3% acetic acid ) . finally , the exudates were centrifuged and an aliquot of the supernatant was collected and stored at 20c for cytokine analysis . analysis of the cell populations in the peritoneal cavity was performed by flow cytometry , based on size and granularity parameters , as well as on gr-1 ( ly-6 g and ly-6c ) expression and mac-3 expression , to identify neutrophils and macrophages , respectively . leukocytes collected from peritoneal lavage were stained with the antibodies kits ( ebioscience ) according to the manufacturer 's protocol . briefly , 1  10  cells / ml were incubated with a saturating amount of anti - gr-1 pe - conjugated and anti - mac-3 fitc - conjugated ( ebioscience ) for 30  min at 4c . analyses of gr-1 and mac-3 expression were performed using facscalibur flow cytometer . in these experiments , tnf- , il-1 , il-6 , and il-10 levels in the peritoneal fluid were evaluated by mouse specific sandwich elisa , according to the manufacturer 's instructions ( ebioscience ) . optical density was read using a microplate spectrophotometer ( microplate reader versamax , tunable , bn 2529 , molecular devices ) . apoptotic leukocytes were identified quantitatively by annexin v - pe apoptosis detection kit ( bd bioscience ) that enables cell staining with annexin v and propidium iodide ( pi ) . annexin v binds to phosphatidylserine exposed on the outer leaflet of the plasma membrane of apoptotic dying cells while pi is a vital dye that enters any necrotic cell . leukocytes collected from peritoneum were stained with this kit , according to the manufacturer 's protocol . briefly , cells were washed twice with cold pbs and resuspended in binding buffer ( 10  mm hepes , 140  mm nacl , and 25  mm cacl2 ) . then , 2.5  l of annexin v - pe and 10  l of pi ( 50  g / ml ) were added to cell suspensions ( 5  10  cells/100  l binding buffer ) and incubated for 15  min at room temperature in the dark . thioglycolate injection , animals were euthanized by cervical displacement and the peritoneal cavity was washed with 8  ml of complete rpmi-1640 medium ( gibco ) ( streptomycin : 10  mg / ml , penicillin : 6  mg / ml , and kanamycin : 2  mg / ml ) , supplemented with 10% fetal bovine serum ( fbs ) ( gibco ) . cell suspension obtained from peritoneal lavage was centrifuged at 350  g for 15 minutes ( 4c ) . supernatant was discarded and the pellet was resuspended in 1  ml of complete rpmi medium . viable cells were counted with a neubauer chamber ( hemocytometer l. optik atc-111020 ) using trypan blue solution ( merck ) . for that , viable peritoneal cells were seeded in 96-well plates at a concentration of 4  10  cells / well in a final volume of 200  l and incubated for 2  h with fbs - free culture medium in an atmosphere of 5% co2 at 37c . remaining cells were further incubated for 24  h with complete rpmi medium , in the presence and absence of different concentrations of ouabain ( 1 , 10 , and 100  nm ) . for that , 200  l of rpmi medium containing 20  l of mtt solution ( 5  mg / ml of mtt in pbs ) was added to each well . after 4  h of incubation , mtt - containing medium was removed and the precipitate was solubilized in dmso solution ( 200  l ) . the optical density was read at 570  nm using a microplate spectrophotometer ( spectramax ; molecular devices , sunnyvale , ca ) . resident peritoneal cells were placed in sterile 24-well plates , at the concentration of 2  10  cells / ml ( final volume = 0.5  ml ) , and incubated for 2  h with rpmi-1640 medium containing 10% fcs in an atmosphere of 5% co2 and 37c . following the incubation period , remaining cells were further incubated in a final volume ( 500  l ) of rpmi-1640 medium containing 10% fcs with 0.5  mg / ml of 40  kda fitc - dextran ( sigma aldrich ) , in the presence or absence of different concentrations of ouabain ( 1 , 10 , and 100  nm ) , for 1  h at 37c or 4c ( control endocytic activity ) , or in rpmi-1640 medium containing 10% fcs , in the presence or absence of different concentrations of ouabain , for 24  h at 37c followed by the addition of rpmi-1640 medium containing 10% fcs with fitc - dextran ( 0.5  mg / ml ) for 1  h at 37c or 4c . cells were then washed with ice - cold pbs and fluorescence measurement was performed using a bd facscalibur flow cytometer . fluorescence signs corresponding to dextran - fitc uptake were analyzed by summit v4.3 software ( dako , usa ) using a macrophage / neutrophil gate , which was determined according to cell size and complexity parameters . in a manner somewhat similar to the previous article from teixeira and rumjanek , results are presented both as percentage of cells that endocytosed dextran and as mfi ( median fluorescence intensity ) a measurement that , in the present work , is representative of the amount of fluorescence per cell . the control group consisted of resident peritoneal cells obtained from animals injected , once a day , in the three previous days with pbs and the experimental pool obtained from animals injected with ouabain ( 0.56  mg / kg ) once a day for 3 consecutive days . chemotaxis was measured by migration through a polycarbonate filter of 5  m pore size in 24-well transwell chambers ( corning costar , cambridge , ma ) . dmem containing 0.5% fcs ( 500  l ) plus lipopolysaccharide ( 100  ng / ml ) , or medium alone as control for spontaneous migration , was added to the lower chambers . 10 cells ( 100  l ) were added to the upper chambers and were incubated at 37c in a 5% co2 humidified atmosphere . after 4  h , migration was defined by counting the cells that migrated to the lower chambers by flow cytometry ( bd facscalibur flow cytometer ) . nuclear extracts of peritoneal cells were prepared based on an adapted version of rong and baudry . briefly , cells were lysed in cold phosphate buffered saline ( pbs ) [ supplemented with 2  g / ml leupeptin , 2  g / ml antipain , and 0.5  mm pmsf ( all obtained from sigma aldrich ) ] and centrifuged at 4c for 2  min at 12,000  g. pellets were resuspended in lysis buffer [ 10  mm hepes ph 7.9 , 1.5  mm mgcl2 , 10  mm kcl , 0.1  mm edta , 0.5  mm pmsf , 2  g / ml leupeptin , 2  g / ml antipain , 3  mm sodium orthovanadate , 30  mm sodium fluoride , and 20  mm sodium pyrophosphate ( all obtained from sigma aldrich ) ] and incubated on ice for 15  min . after that , np-40 ( sigma aldrich ) ( final concentration of 0.5% ) was added , and samples were vigorously mixed and centrifuged for 30  s at 12,000  g. pellets were then resuspended in extraction buffer [ 20  mm hepes , ph 7.9 , 25% glycerol , 1.5  mm mgcl2 , 300  mm nacl , 0.25  mm edta , 0.5  mm pmsf , 2  g / ml leupeptin , and 2  g / ml antipain ( all obtained from sigma aldrich ) ] , incubated for 20  min on ice , and centrifuged for 20  min at 13,000  g at 4c . protein concentration was determined using the bio - rad ( richmond , ca , usa ) colorimetric assay . electrophoretic mobility shift assay ( emsa ) for nf-b was performed using gel shift assay kit from promega ( madison , wi , usa ) . nfb double - stranded consensus oligonucleotide ( 5-agttgaggggactttcccaggc-3 ) was end - labeled with -32p - atp using t4 polynucleotide kinase . unincorporated nucleotides were removed by passing the reaction mixture through a sephadex g-25 spin column ( amersham - pharmacia , uppsala , sweden ) . purified 32p - labeled probe ( 25,000  cpm ) was incubated in 20  l with 5  g of nuclear extracts in a binding reaction mixture containing 50  mm nacl , 0.2  mm edta , 0.5  mm dtt , 4% glycerol , 10  mm tris hcl ( ph 7.5 ) , and 0.05  g of poly ( di  dc ) for 30  min at room temperature . dna - protein complexes were separated by electrophoresis using a 6% nondenaturing acrylamide : bisacrylamide ( 37.5  :  1 ) gel in 0.5 x tris - borate / edta ( tbe ) for 2  h at 150  v. gels were vacuum dried and analyzed by autoradiography . autoradiographs were visualized with a photo documentation system dp-001-fdc ( vilber lourmat , marne la valle , france ) and quantified in nih imagej software ( bethesda , md , usa ) . all data were expressed as mean  sem and analyzed by graphpad prism 5.0 software using one - way analysis of variance ( anova ) followed by tukey 's test . figure 1 shows that zymosan administration led to an increase ( 90% ) on early vascular permeability , measured at 30  min , when compared to pbs control group . pretreatment with ouabain ( 0.56  mg / kg i.p . ) for three consecutive days reduced ( 33% ) plasma extravasation induced by zymosan . as expected , pretreatment for three consecutive days with pbs without ouabain had no effect on vascular permeability . as shown in figure 2 , administration of ouabain had no effect on the number of resident peritoneal cells . however , zymosan treatment led to an increase in total cell number in the peritoneal cavity at 4 and 24  h ( 66% and 67% , resp . ) . ouabain was able to inhibit zymosan - induced leukocyte number in the peritoneal cavity at 4  h ( 46% ) , but not at 24  h , when compared to the zymosan group . once we determined that ouabain reduces peritoneal exudate leukocyte number , flow cytometry was used to identify which cell subpopulations were affected at 4 and 24  h after zymosan treatment . as expected , no significant difference in cell subpopulations was observed between pbs and ouabain groups ( figures 3(a ) , 3(b ) , 3(c ) , and 3(d ) ) . injection , there was a significant increase ( 90% ) in neutrophils ( gr1 ) at the site . also , ouabain pretreatment was able to reduce neutrophil cell population ( 53% ) when compared to zymosan group ( figure 3(b ) ) . monocyte ( gr1mac3 ) and macrophage ( mac3 ) cell numbers were only enhanced significantly 24  h after zymosan injection ; at this time - point , ouabain pretreatment was not able to inhibit monocyte or macrophages cell number ( figures 3(c ) and 3(d ) ) . reduced neutrophil migration promoted by ouabain could be a consequence of the modulation of cytokines levels ( tnf- , il-1 , il-6 , and il-10 ) at 4 and 24  h after zymosan stimulation . figure 4 indicates that animals stimulated with zymosan showed increased levels of tnf- , il-1 , il-6 , and il-10 ( 96% , 97% , 99% , and 72% , resp . ) in the peritoneal cavity at 4  h , but these levels returned to baseline values at 24  h. ouabain pretreatment for three consecutive days reduced significantly il-1 ( 58% ) and tnf- ( 57% ) levels ( figures 4(a ) and 4(b ) ) at 4  h , but not at 24  h , when compared to the zymosan group . on the other hand , ouabain did not affect il-6 and il-10 levels when compared to zymosan group at both time points studied ( figures 4(c ) and 4(d ) ) . to investigate a possible modulation of apoptosis by ouabain at 4 and 24  h , the percentage of apoptotic and necrotic leukocytes in the peritoneum was accessed by flow cytometry . mice peritoneal cavity injected with pbs contained a low percentage of apoptotic and necrotic cells , and these numbers were increased by zymosan injection at 4 and 24  h compared with pbs group ( figure 5 ) . our results demonstrated that preinjection with ouabain does not interfere in the death of these cells at the times evaluated ( figure 5 ) . our results show that ouabain ( 1 , 10 , and 100  nm ) did not interfere with macrophages viability ( figure 6 ) . additionally , we tested ouabain effect on fitc - dextran endocytosis by resident peritoneal cells . no significant difference was observed on the percentage of dextran - fitc cells or on the mean fluorescence intensity of endocytic cells , indicating that ouabain did not affect the number of endocytic cells or the amount of dextran particles endocytosed by them ( figure 7 ) . also , no significant difference was observed on fitc - dextran endocytosis by peritoneal cells from thioglycolate - elicited mice ( data not shown ) . the diminished number of cells in the peritoneal cavity of zymosan injected mice pretreated with ouabain could be a result of an effect on the migratory capacity of cells from ouabain - treated animals . to investigate this possibility , our preliminary experiment shows a reduction on the number of migrating peritoneal cells of ouabain pretreated mice group when compared to control group ( pbs - injected mice ) . as an in vitro attractant lps ( 100  ng / ml ) was used in the lower chamber of transwell plates . the number of migrating peritoneal cells from untreated mice towards culture medium was 19  10 and towards culture medium plus lps was 35  10 , whereas the number of migrating peritoneal cells from ouabain pretreated mice towards culture medium was 6  10 and towards culture medium plus lps was 11  10 . finally , we investigated whether ouabain could modulate nf-b activity in peritoneal cells of mice exposed or not to zymosan ( figure 8) . to verify dna - binding activity of nf-b our results show that the binding activity of nf-b was increased ( 85% ) in peritoneal cells from zymosan - treated mice at 4  h when compared to control group animals . moreover , ouabain by itself also increased ( 73% ) nf-b binging activity in peritoneal cells when compared to pbs group . in previous studies , our group demonstrated , using intraplantar injection of different phlogistic agents , an in vivo anti - inflammatory and analgesic potential of ouabain , which might be related to prostaglandin e2 as well as to opioid mechanisms . it was also demonstrated that ouabain negatively modulated acute peritoneal inflammatory response induced by leishmania amazonensis infection by decreasing tnf- and ifn- levels . in order to better understand the mechanisms involved in the anti - inflammatory effect of ouabain , a zymosan - induced peritonitis model was used in the present work . in accordance with what was previously reported , ouabain produced an anti - inflammatory effect which involves a reduction of il-1 and tnf- levels , reduced neutrophil migration , and decreased vascular permeability . intraperitoneal injection of zymosan increases vascular permeability , one of the primary signs of inflammation , within 30 minutes after the inflammatory stimulus due to the activation of resident macrophages and mast cells , which release prostaglandins , particularly histamine and leukotrienes [ 26 , 2830 ] . the reduction of the increase in vascular permeability promoted by ouabain appears to be related to mast cell degranulation and action pge2 , since ouabain treatment was capable of decreasing paw edema induced by compound 48/80 and pge2 . furthermore , in accordance with our findings , in vitro studies demonstrated that ouabain inhibited histamine release from mast cells . zymosan injection in swiss mice induces massive polymorphonuclear leukocytes ( pmn ) influx with maximal cell accumulation at the 4th hour of peritonitis [ 27 , 28 , 32 ] . in the present study , ouabain pretreatment for three consecutive days produced anti - inflammatory effects , decreasing the number of intraperitoneal pmns at the 4th hour . these data are consistent with earlier studies by our group , where ouabain reduced the number of pmns in models of peritonitis induced by concanavalin a and leishmania amazonensis [ 17 , 18 ] . our results show a reduction of the number of peritoneal cells of ouabain pretreated mice group , suggesting that inhibition of cell migration may be a key event for the ouabain - induced decrease in the number of peritoneal leukocytes observed by us . furthermore , our preliminary in vitro study using a transwell model indicated that peritoneal cavity cells from ouabain treated mice migrate less than those of control animals . in addition , other studies have demonstrated that ouabain inhibits in vitro neutrophil migration , by interfering with il-8 receptor recycling . besides that , oleandrin , another cardiac glycoside , potentially inhibited il-8-mediated biological responses in diverse cell types . furthermore , in studies with lung tumor cells , cardiotonic glycosides , ouabain , and odoroside a inhibited icam-1 adhesion molecule expression . inflammatory signaling in response to zymosan , which can be also recognized by dectin and complement receptors [ 39 , 40 ] , requires tlr2/tlr6 heterodimerization and subsequent recruitment to the zymosan phagosome . nf-b is a pivotal regulator in the expression of many proinflammatory cytokines such as tumor necrosis factor- ( tnf- )  , interleukin- ( il- ) 1 , il-6 , il-10 chemokines , and no [ 42 , 43 ] leading the leukocytes infiltrating zymosan - inflamed peritoneum such as neutrophils and macrophages . we investigated whether the inhibitory effect of ouabain preinjection was related to modulation of cytokine levels and nf-b activation . in this model , our results demonstrated that treatment with ouabain inhibited tnf- and il-1 production induced by zymosan , but not il-6 and il-10 levels . however , other groups described that ouabain suppressed the production of the proinflammatory cytokines il-6 and tnf- stimulated by lps both in vitro and in vivo . the difference between our results and those cited above may be due to a different nature of the inflammatory agent ( fungal ) , the source of investigated samples ( peritoneal exudates versus serum ) , and ouabain concentrations . in addition , it has been reported that cardiac glycoside drugs inhibit tnf-/nf-b signaling pathway , which is a central common regulator for inflammatory process . in the present study , when ouabain was associated with zymosan it significantly decreased the nf-b activation observed with zymosan alone . however , similar to a study using dendritic cells   ouabain by itself was capable of increasing nf-b activation . therefore , the decrease in tnf- and il-1 levels observed by us correlates with an inhibitory effect of ouabain on nf-b binding activity . furthermore , other studies with lps - stimulated pbmc cells indicated that digitalis including ouabain , proscillaridin a , digoxin , digitoxin , and lanatoside c reduced tnf- , il-1 , and il-6 production by inhibiting nf-b signaling pathway . moreover , ouabain was able to inhibit il-1 release in primary astrocytes stimulated with lps . another cell signaling pathway involved in the production of proinflammatory cytokines , the p38 mapk pathway , is also regulated by ouabain [ 13 , 46 ] . zymosan peritonitis represents a model of acute resolving inflammation [ 25 , 26 , 28 , 31 , 42 ] . a fundamental paradigm of inflammatory research has postulated that neutrophil apoptosis and its subsequent clearance by macrophages are the major mechanisms promoting resolution of inflammation [ 47 , 48 ] . these data may be associated with ouabain 's ability to reduce tnf- production , which is important for apoptosis induction of this cell . furthermore , it is known that digitalis inhibits tradd receptor protein coupling , which leads to tnfr1 inhibition . the process of clearance involves endocytosis . in the present work it was possible to observe that ouabain did not alter in vitro endocytosis of dextran particles by peritoneal cells . thus , we suggest that the anti - inflammatory effects observed by us are not related to events involved in resolution ; nevertheless more detailed studies should be performed to confirm this hypothesis . ouabain modulated acute inflammatory responses induced by zymosan , reducing il-1 and tnf- levels and the number of peritoneal cells , possibly due to a decrease in cell migration . moreover , ouabain inhibited nf-b activation , which is related to the production of tnf- and il-1 , cytokines important for leucocyte extravasation . taken together , our results provide new evidences for the mechanisms related to the anti - inflammatory effects of ouabain in vivo .