sepsis is defined as the acute systemic inflammatory response to infection with a clinical spectrum ranging from hemodynamic changes to multiple organ dysfunction syndrome and even death [ 13 ] .
it is estimated that there are 750,000 cases of sepsis with a mortality rate between 30% and 50% in north america per year .
the incidences of sepsis - associated liver dysfunction and liver failure range from 34% to 46% and from 1.3% to 22% in patients with sepsis , respectively . attenuating liver injury and restoring liver function lower morbidity and mortality rates in patients with sepsis .
the mechanisms involved in sepsis - induced liver injury are diverse , including uncontrolled systemic inflammatory activation , hepatic ischemia , coagulopathy , and deregulated cell apoptosis [ 69 ] . in sepsis ,
inflammatory mediators , such as tumor necrosis factor- ( tnf- )  , interleukin- ( il- ) 1 , il-6 , interferon- ( ifn- )  , il-8 , nitric oxide ( no ) , and reactive oxygen species ( ros ) , are released from liver resident and infiltrated immune cells , which mediate sepsis - induced liver injury .
apoptotic cell death has a prominent role in the evolution of organ damage following sepsis .
animal studies have demonstrated that blocking apoptosis can improve outcome in experimental models of severe sepsis [ 911 ] .
glycogen synthase kinase- ( gsk- ) 3 , a ubiquitously expressed serine / threonine protein kinase , is initially identified as a regulator of glycogen metabolism [ 12 , 13 ] . beyond its role in glycogen metabolism
, recent studies have implicated that gsk-3 plays an important role in regulation of inflammatory response [ 1416 ] .
gsk-3 inhibition reduced the production of tnf- and il-6 and enhanced il-10 production in lipopolysaccharide- ( lps- ) stimulated monocytes [ 14 , 15 ] . furthermore
, gsk-3 inhibition effectively protected mice and rats from endotoxin shock [ 15 , 16 ] or mice from live bacterial infection [ 17 , 18 ] .
additionally , recent studies have demonstrated that inhibition of gsk-3 activity prevents apoptotic cell death .
moreover , gsk-3 blockade significantly reduced hepatic apoptotic cell death in response to d - galactosamine / lps - induced liver injury   or ischemia / reperfusion ( i / r ) injury [ 21 , 22 ] , respectively . although the role of gsk-3 in endotoxin shock and live bacterial infection has been extensively investigated , less information is available on the possible effects of gsk-3 inhibition in polymicrobial sepsis , which is more representative of the clinical condition .
therefore , we aimed to investigate the protective effects of gsk-3 inhibition on polymicrobial sepsis - induced liver injury by a mouse cecal ligation and puncture ( clp ) model and to further explore the possible mechanisms .
male inbred c57bl/6 mice ( 810 weeks old , weighing within 20~22  g ) were purchased from wuhan university center for animal experiment ( wuhan , china ) .
all animals were housed under standard animal care conditions and had free access to water and food .
all procedures were carried out according to the ethical guidelines of the animal care and use committee of huazhong university of science and technology .
mice were completely anesthetized with pentobarbital ( 60  mg / kg , i.p . ) and a midline abdominal incision was performed . after exposing the cecum and manipulating the stool to the tip of the cecum , the cecum was ligated 1  cm from the tip and was perforated by one through - and - through puncture ( two holes ) with a 20-gauge needle to induce polymicrobial peritonitis .
all mice had free access to food and water after recovery from anesthesia . to investigate the role of gsk-3 in clp - induced liver injury , mice were treated with sb216763 ( 25  mg / kg , i.p .
louis , mo ) or vehicle ( 10% dimethyl sulfoxide , dmso , sigma - aldrich ) at 1  h , 6  h , and 12  h following clp .
the dose of sb216763 treatment was selected following previous studies [ 22 , 23 ] .
liver injury , hepatic apoptosis , inflammatory cytokines , transcription factor nuclear factor-b ( nf-b ) activation , and camp response element binding protein ( creb ) activation were analyzed . to assess hepatocellular injury following clp ,
serum alanine transaminase ( alt ) and aspartate aminotransferase ( ast ) were measured using an automated chemical analyzer ( hitachi co. , tokyo , japan ) .
liver tissue was fixed in 4.5% buffered formalin for at least 24  h. paraffin embedding was performed using standard techniques .
after deparaffinization , rehydration , and antigen retrieval , sections were incubated with myeloperoxidase ( mpo ) antibody ( 1  :  50 ; abcam , cambridge , uk ) for 1  h at room temperature , followed by incubation with goat anti - rabbit secondary antibody , and then visualized with a diaminobenzidine kit .
mpo - positive cells were counted in 5 high - power fields ( hpfs)/section under a microscope ( 400 ) , and the number of cells / field was shown .
the murine macrophage cell line raw264.7 was purchased from the type culture collection of the chinese academy of sciences ( shanghai , china ) .
cells were cultured in dmem medium ( invitrogen , carlsbad , ca ) supplemented with 10% fetal calf serum , 2  mm glutamine , penicillin ( 100  iu / ml ) , and streptomycin ( 100  g / ml ) at 37c under a gas phase of air / co2 ( 95  :  5 ) .
cells were plated at a density of 5  10  cells / well in a 24-well plate for cytokine assay or 2  10  cells / well in a 6-well plate for western blotting .
sb216763 ( 10  m ) was used to inhibit gsk-3. relative caspase-3 activity in livers was detected with a caspase-3 colorimetric assay kit ( abcam ) according to the manufacturer 's instructions .
nuclear and cytoplasmic extraction kit was bought from pierce ( thermo pierce , rockford , il ) .
nuclear proteins from liver tissues or raw264.7 cells were isolated according to the instructions of the manufacturer .
levels of nf-b p65 and creb activity in the nuclear extracts were quantified by transam nf-b p65 and transam creb assay kits ( active motif , carlsbad , ca ) according to the manufacturer 's instructions .
the levels of tnf- and il-6 in serum and cultured medium were analyzed using commercially available enzyme - linked immunosorbent assay ( elisa ) kits ( r&d systems , minneapolis , mn ) .
equivalent amounts of protein were separated on 12% gels by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred to polyvinyldifluoride membranes ( ge healthcare , buckinghamshire , uk ) . after blocking , the membranes were incubated with primary antibodies : rabbit anti - phospho - gsk-3 ( ser9 ) ( 1  :  1000 ; cell signaling technology , beverly , ma ) , rabbit anti - gsk-3 ( 1  :  1000 , cell signaling technology ) , rabbit anti - phospho - glycogen synthase ( 1  :  1000 , cell signaling technology ) , rabbit anti - cleaved caspase-3 ( 1  :  1000 , cell signaling technology ) , rabbit anti - cleaved caspase-7 ( 1  :  1000 , cell signaling technology ) , and anti - glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) ( 1  :  20000 ; sigma - aldrich ) , at 4c with gentle shaking overnight .
detection was carried out using horseradish peroxidase - conjugated goat anti - rabbit igg antibody ( 1  :  3000 , abcam ) , followed by ecl western blotting detection reagents ( ge healthcare , buckinghamshire , uk ) .
rochester , ny ) . real time polymerase chain reaction ( pcr ) was performed as described previously .
total rna was isolated by trizol reagent ( invitrogen ) according to the manufacturer 's instruction .
cdna synthesis was performed using the first - strand cdna synthesis kit ( invitrogen ) .
quantitative pcr was performed using a roche light cycle system ( roche , rotkreuz , switzerland ) with sybr green master mix ( qiagen , hilden , germany ) and primers .
amplification conditions were 95c ( 10  min ) followed by 50 cycles of 95c ( 30  s ) and 60c ( 20  s ) .
primers used to amplify a specific mouse gene fragments were as follows : tnf- forward : 5-tgctgggaagcctaaaagg-3 ; reverse : 5-cgaattttgagaagatgatcctg-3 , il-6 forward : 5-tcaattccagaaaccgctatga-3 ; reverse : 5-caccagcatcagtcccaaga-3 , il-1 forward : 5-caggtcgctcagggtcaca-3 ; reverse : 5-cagaggcaaggaggaaacaca-3 , il-10 forward : 5-cacaaagcagccttgcagaa-3 ; reverse : 5-agagcaggcagcatagcagtg-3 , and -actin forward : 5-agagggaaatcgtgcgtgac-3 ; reverse : 5-caatagtgatgacctggccgt-3. relative quantification of target mrna expression was calculated and further normalized to housekeeping gene -actin .
differences between groups were evaluated for significance by one way anova analysis combined with bonferroni post hoc test ( comparison type : versus vehicle - treated group ) .
all tests were performed using sigmastat v3.5 ( systat - software , erkrath , germany ) .
to determine whether gsk-3 inhibition could attenuate liver injury during sepsis , gsk-3 inhibitor sb216763 was administrated to mice at 1  h , 6  h , and 12  h following clp .
as shown in figure 1(a ) , compared with sham controls , the phosphorylated ( serine 9 ) gsk-3 levels in the liver tissue were reduced at 6  h following clp , suggesting gsk-3 was activated by the clp insult .
the inhibition of liver gsk-3 activity in vivo was indicated by the reduced phosphorylation of glycogen synthase , a downstream substrate of gsk-3. as shown in figure 1(b ) , compared with the vehicle - treated group , clp resulted in significant increase in the serum levels of alt and ast .
the histologic examination showed significant liver tissue injury following clp , as indicated by swollen hepatocytes and leukocyte infiltration .
the inhibition of gsk-3 resulted in significant attenuation of these changes ( figure 1(c ) ) . to address whether gsk-3 inhibition was of benefit for septic mice induced by clp , survival
was then assessed at 48  h following clp . as shown in figure 1(d ) , gsk-3 inhibitor sb216763 treatment significantly increased the survival rate of mice .
in contrast , the survival rate of mice treated with sb216763 was 53% . to test whether gsk-3 inhibition could attenuate liver injury during sepsis via inhibition of inflammation ,
the production of tnf- , il-6 , il-1 , and il-10 was analyzed at 6  h following clp .
as shown in figure 2(a ) , clp resulted in significant increase in the mrna levels of tnf- , il-6 , il-1 , and il-10 .
liver tnf- , il-6 , and il-1 mrna levels were significantly attenuated by the administration of sb216763 .
furthermore , the inhibition of gsk-3 significantly reduced the clp - induced production of serum tnf- and il-6 levels ( figure 2(b ) ) .
however , sb216763 administration significantly reduced the clp - induced increase in the infiltration of neutrophils ( figure 3 ) .
these results indicated that inhibition of gsk-3 relieved the inflammation in the liver following clp . to obtain insight into the underlying mechanisms responsible for the anti - inflammatory effect of gsk-3 inhibition ,
however , nf-b activity was significantly inhibited in sb216763-treated animals in response to clp insult compared to vehicle - injected animals .
conversely , activated nuclear creb was enhanced in the liver after the inhibition of gsk-3 ( figure 4 ) . to further gain insight into the underlying signal transduction pathway involved in the regulation of inflammatory response in clp by gsk-3 ,
the effect of gsk-3 inhibition on the production of proinflammatory cytokines in macrophages was assessed in vitro .
as shown in figure 5(a ) , the gsk-3 inhibition significantly reduced the production of tnf- , il-6 , and il-1 but increased il-10 gene induction at 6  h of culture .
similarly , tnf- and il-6 protein levels in the cultured medium were increased dramatically after treatment with lps for 6  h ; gsk-3 inhibition reduced the production of tnf- and il-6 levels . to investigate whether the anti - inflammatory effect of gsk-3 inhibition was mediated via nf-b and creb pathways , we examined the effect of gsk-3 inhibition on the activity of nuclear nf-b and creb .
as shown in figure 5 , gsk-3 inhibition significantly inhibited nf-b activity but increased creb activity . to test whether gsk-3 inhibition could attenuate hepatic apoptosis ,
the caspase-3 activity in the liver was evaluated at 20  h following clp insult .
compared to the activity in the sham operated animals , the caspase-3 activity was significantly increased in the vehicle - treated animals .
the inhibition of gsk-3 significantly reduced the induction of caspase-3 activity by clp ( figure 6(a ) ) .
furthermore , the vehicle - treated animals expressed higher levels of cleaved caspase-3 and cleaved caspase-7 protein , which were significantly reduced by the gsk-3 inhibition ( figure 6(b ) ) .
recent studies suggest that gsk-3 inhibition has protective properties in lps - induced septic shock . however , the role of gsk-3 inhibition in the pathogenesis of live injury induced by polymicrobial sepsis and its regulatory mechanisms remain poorly understood . in the present study , we documented that gsk-3 inhibition protected against clp - induced liver injury via inhibition inflammatory response and hepatic apoptosis .
our data also suggested that nf-b and creb mediated mechanisms are the prominent pathways involved in the protective effects of gsk-3 inhibition in the clp - induced liver injury .
inhibition of gsk-3 suppresses systemic inflammation and confers protection against clp - induced liver injury .
gsk-3 is involved in many intracellular functions including regulating cell activation , differentiation , and survival [ 12 , 13 ] . among the diverse functions that are regulated by gsk-3 ,
recent studies have shown that gsk-3 is a vital factor in the inflammatory process [ 1416 ] .
firstly demonstrated that the gsk-3 inhibition not only suppressed the production of proinflammatory cytokine but also increased the anti - inflammatory cytokine expression , and it provided a survival advantage and attenuated organ injury in lps - induced sepsis models .
documented that the inhibition of gsk-3 suppressed the inflammatory responses and conferred a survival advantage in mice infected with francisella tularensis .
recently , using sb216763 as an inhibitor , chang and colleagues found that gsk-3 inhibition reduced the proinflammatory cytokine expression and improved the survival rate in group a streptococcus - infected mice .
we previously demonstrated that gsk-3 inhibition by lithium reduced liver i / r injury , at least in part via inhibition of inflammation in rats . in agreement with these studies
, we demonstrated that inhibition of gsk-3 reduced inflammation , attenuated liver injury , and improved survival following clp .
these findings suggested that gsk-3 inhibition could protect liver against overwhelming inflammatory response during sepsis .
gsk-3 is known to regulate nf-b signaling , which plays a pivotal role in the pathophysiology of sepsis .
the transcription of proinflammatory mediators , including cytokines , chemokines , and no , occurs mainly via the activation of nf-b . in patients with sepsis , circulating proinflammatory mediators were increased and correlated with the disease severity [ 26 , 27 ] .
blockade of the activity of nf-b using genetic or pharmacologic approaches successfully reduced nf-b - mediated cytokines and inhibited multiple organ inflammation and injury during sepsis and/or endotoxemia , suggesting that inhibition of nf-b could be a promising therapeutic target in sepsis [ 2830 ] .
demonstrated that gsk-3 inhibition reduced the ser536 phosphorylation p65 but had no effect on the nf-b / dna binding activity in the lung and reduced the increase in nf-b p65 activity caused by il-1 in human embryonic kidney cells in vitro .
observed that gsk-3 regulated the production of proinflammatory and anti - inflammatory cytokines by lps - stimulated monocytes via augmenting nuclear creb activity and enhancing its association with creb - binding protein while reducing the interactions between nf-b p65 and cbp . in lithium - treated hepatocytes , schwabe and brenner documented that inhibition of gsk3 impaired nf-b activity , but ib degradation and nf-b dna binding activity were not affected . in this study , we identified gsk-3 regulated clp - induced inflammatory cytokines production by differentially affecting the activation of nf-b and creb .
there is increasing evidence that apoptosis is also involved in sepsis - induced liver dysfunction .
recent studies have demonstrated that gsk-3 inhibition protects renal cell from endotoxemia   and attenuates liver i / r injury   via inhibition of apoptosis .
apoptosis is potentially triggered by infiltrating neutrophil- or macrophage - derived cytokines such as tnf- , ros , and no .
wang et al . observed that gsk-3 inhibitors downregulated lps - induced renal cell apoptosis by reducing the amount of tnf- production .
moreover , results from studies of different types of liver damage also support the hypothesis that the protective effects of gsk-3 inhibition are via antiapoptosis . in a mouse model of liver
i / r injury , xia and colleagues showed that gsk-3 inhibitors increased -catenin accumulation , which further enhanced antiapoptotic signaling through the induction of bcl-2 and survivin expression .
this result was further supported by our previous study , which demonstrated that gsk-3 inhibition by lithium reduced hepatic apoptosis in liver i / r in rats .
moreover , gsk-3 inhibition was shown to be capable of preventing apoptosis in d - galactosamine / lps - induced liver injury by reducing endoplasmic reticulum stress .
in summary , we documented that gsk-3 inhibition could improve the survival of mice with polymicrobial sepsis , ameliorated liver injury , and reduced hepatic apoptosis .
the mechanism appeared to involve its ability to inhibit inflammatory response via modulating nf-b and creb activation .
these findings would suggest that inhibition of gsk-3 may act as an alternative therapeutic strategy beyond antibiotic treatment .
 liver dysfunction has been known to occur frequently in cases of sepsis . 
 excessive inflammation and apoptosis are pathological features of acute liver failure . 
 recent studies suggest that activation of glycogen synthase kinase- ( gsk- ) 3 is involved in inflammation and apoptosis . 
 we aimed to investigate the protective effects of gsk-3 inhibition on polymicrobial sepsis - induced liver injury and to explore the possible mechanisms . 
 polymicrobial sepsis was induced by cecal ligation and puncture ( clp ) , and sb216763 was used to inhibit gsk-3 in c57bl/6 mice . 
 gsk-3 was activated following clp . 
 administration of sb216763 decreased mortality , ameliorated liver injury , and reduced hepatic apoptosis . 
 the inhibition of gsk-3 also reduced leukocyte infiltration and hepatic inflammatory cytokine expression and release . 
 moreover , gsk-3 inhibition suppressed the transcriptional activity of nuclear factor - kappa b ( nf-b ) but enhanced the transcriptional activity of camp response element binding protein ( creb ) in the liver . in in vitro studies , gsk-3 inhibition reduced inflammatory cytokine production via modulation of nf-b and creb signaling pathways in lipopolysaccharide - stimulated macrophages . in conclusion , these findings suggest that gsk-3 blockade protects against clp - induced liver via inhibition of inflammation by modulating nf-b and creb activity and suppression of hepatic apoptosis . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion