sepsis refers to the systemic inflammatory response by the immune system towards invading foreign pathogens . this multipronged defense response is the third leading cause for death in the united states . during this response , multiple organ failure has the potential to be elicited , where manifestation of brain dysfunction during sepsis can lead to sepsis - associated encephalopathy ( sae ) [ 2 , 3 ] . sae is defined as diffuse cerebral dysfunction during sepsis , without explicit evidence of microbial infection within the central nervous system , intracranial defects , or encephalopathy induced by hepatic or renal factors . the incidence of sae is reported to be as high as 70% and the clinical symptoms may vary from cognitive dysfunction and delirium to deep coma . the presence of sae is seen to occur in parallel with extensive treatment time in the intensive care unit ( icu ) and a higher mortality rate . despite the overwhelming amounts of deaths associated with sae , an effective therapy against sae is still lacking . majority of the treatments directed towards sae patients are focused towards controlling the systemic spread of infection , while providing supportive therapy . the etiologies contributing towards sae , as concluded by gofton and young , include inflammatory cytokines , microscopic brain injury , blood - brain barrier ( bbb ) compromise , altered cerebral metabolism , and neurotransmission and cerebral microcirculation . microglia are heavily activated during sae , which are dependent on neural , humoral pathways or bbb damage . at this time , patients also experience an increase in the secretion of cytokines , nitric oxide , and reactive oxygen species ( ros ) in the brain parenchyma [ 3 , 5 ] . il-1 is a vital cytokine that mediates brain dysfunction by activating type 1 il-1 receptor , ultimately preventing long - term potentiation deficiency in the hippocampus of septic mice [ 68 ] . resveratrol is a natural phenol that is extracted from the skin of grapes , which are commonly used in red wine production , where they are advertised to have antiaging benefits for consumers [ 9 , 10 ] . the biological role of resveratrol is to initiate the activation of sirtuin 1 ( sirt1 ) , which is a class of deacetylase that epigenetically modifies and inactivates the acetylation of inflammatory proteins . though the role of resveratrol in sepsis prognosis remains to be controversial , some studies suggest that resveratrol might be able to improve renal and myocardial functions , in murine model of sepsis [ 11 , 12 ] . moreover , resveratrol was also reported to be a potential therapy for neurodegenerative diseases through the inhibition of microglia activation [ 13 , 14 ] . therefore , our study attempted to investigate the role of resveratrol in development of sae and nlrp3/il-1 axis in microglia . male c57bl/6 mice , aged 68 weeks , were provided by the experimental animal center at the second military medical university . all experimental animal studies were approved by the local animal care and use committee of the second military medical university . sepsis was induced by the cecal ligation and puncture ( clp ) , as per procedures previously described by our laboratory [ 15 , 16 ] . in general , all mice were subjected to sevoflurane anesthesia , in supine position , after a week worth of acclimatization to the lab environment . after skin sterilization , the skin and abdominal membrane were opened using scissors to expose the cecum , which was further ligated at half the distance to the end with a 1 - 0 prolene thread . the ligated cecum was then punctured once with a 20  g needle and intestinal content was pushed out of the cecum . then , the abdominal membrane and skin were closed in two layers . in the sham - operated mice , the cecum was exposed in a similar fashion to the clp procedure , without ligation and puncture . after surgery , all mice were provided with free access to water and food after recovery from anesthesia . in the animal experiments , mice were randomly allocated to one of four groups : sham group , clp group , res 30  mg / kg group , and res 10  mg / kg group ( n = 6 for each group ) . in both res groups , the mice were treated with four abdominal infusions of 30  mg / kg and 10  mg / kg resveratrol ( winherb medical tech . , shanghai , china ) in dimethyl sulfoxide ( dmso ) ( sigma - aldrich , st . louis , usa ) saline solution at 1 hour prior to surgery and again at 6  h , 12  h , and 18  h after surgery . the sham and clp groups were treated with four times of dmso infusions at the same concentration . the mouse bv2 cell lines were purchased from the tongpai biological technology ( shanghai , china ) as an alternative to investigate microglia in vitro . bv2 cells , at passages 1015 , were cultured in dulbecco 's modified eagle medium / f-12 ( dmem / f-12 , gibco , langley , usa ) with 10% fetal bovine serum ( sigma - aldrich , st . louis , usa ) , 1% penicillin , and streptomycin ( gibco , langley , usa ) . in the first set of in vitro experiments , the cell populations were allocated into one of four groups : control group , lipopolysaccharide ( lps ) group , res1 group , and res2 group . cells in the control group were treated with dmso + adenosine triphosphate ( atp ) ( 100  m , sigma - aldrich , st . louis , usa ) , dmso + lps ( 100  ng / ml , sigma - aldrich , st . louis , usa ) + atp ( 100  m ) in the lps groups , resveratrol ( 30  m ) + atp ( 100  m ) + lps ( 100  ng / ml ) in the res 30  m group , and resveratrol ( 15  m ) + atp ( 100  m ) + lps ( 100  ng / ml ) in the res 15  m group . in the second set of in vitro experiments , the cell populations were allocated into one of four groups : control group , lps group , res group , and nam group . the cells in the control group were treated with dmso + atp ( 100  m ) , dmso + lps ( 100  ng / ml ) + atp ( 100  m ) in the lps groups , resveratrol ( 30  m ) + lps ( 100  ng / ml ) + atp ( 100  m ) in the res group , and nicotinamide ( 10  mm , sigma - aldrich , st . louis , usa ) + resveratrol ( 30  m ) + lps ( 100  ng / ml ) + atp ( 100  m ) in the nam group . morris water maze task was performed in a circular pool with a diameter of 100  cm and a height of 50  cm , in an isolated environment ( jiliang software , shanghai , china ) . different shapes were marked on the inner walls of the pool to recognize the relative position of the mouse . water ( 21.5  0.5c ) containing food - grade titanium dioxide ( jianghu taibai , shanghai , china ) was filled into the pool to a height of the three quarters of the wall . the pool was also divided into four quadrants and monitored with a video camera on the top . a platform with a diameter of 7 cm was place into one of the four quadrants , 1  cm below the water surface . clp or sham - operated mice were trained for morris water maze task from the 4th day to 7th day after surgery . the mice were place on the platform for a total of 10 seconds and were then removed from the pool . in the subsequent training session , the mice were individually placed into each of the 4 quadrants and were allowed to search the platform for a period of 60 seconds . if the mice were unable to reach the platform within 60 seconds , they would be placed on the platform for an additional 10 seconds . the escape latency , distance of swimming , and time spent in the target quadrant were recorded for each training process . half an hour after the last training session on the 7th day , the mice were subjected to the probe trial with the platform removed from the pool . all mice were monitored for 60 seconds in order to observe the distance of swimming , time spent in the target quadrant , and frequency of crossing the platform . tunel assay was performed to determine the apoptotic rates of the cell , in the hippocampus region of the mouse using the in - site cell death detection kit ( roche , basel , switzerland ) . in general , the hippocampus region was isolated from the brain tissue at 24 hours after surgery and was fixed using 4% paraformaldehyde for 24 hours , allowing sections to be prepared . the sections were deparaffinized with xylene , hydrated with ethanol , and pretreated with proteinase k. the endogenous peroxidase was blocked with 3% h2o2 , after which sections were incubated in terminal deoxynucleotidyl transferase ( tdt ) reaction mixture . in the negative control group , the sections were then incubated with streptavidin - hrp , colorized diaminobenzidine ( dab ) , and counterstained with bis - benzamide . the apoptotic rate was represented by the average number of tunel - positive cells in each field . after deparaffinization and rehydration , the sections were incubated in citrate buffer to retrieve antigen by heat - mediated method . the sections were then blocked with 3% bovine serum albumin ( sigma - aldrich , st . louis , usa ) for 30 minutes and incubated in the primary antibody diluted at 1  :  100 ( rabbit anti - mouse anti - iba-1 antibody , abcam , cambridge , uk ) at 4c overnight and lastly in cy3 labeled secondary antibody diluted at 1  :  400 at room temperature for 60 minutes . then the slides were incubated in the primary antibody against iba-1 antibody ( 1  :  100 , abcam , cambridge , uk ) at 4c overnight and in alex488 labeled secondary antibody diluted at 1  :  400 . the nucleus was stained with hoechst ( beyotime , nantong , china ) . finally , the slides were dehydrated and mounted for detection under the inverted fluorescence microscope ( leica , wetzlar , germany ) . hippocampus tissue harvested at 24 hours after surgery was homogenized in 500  l trizol ( invitrogen , carlsbad , usa ) and the total rna were purified by chloroform / isopropanol method . reverse transcriptase was performed using the primescript rt reagent kit ( takara , dalian , china ) and cdna was stored at 80c . the quantitative real - time pcr was performed using the sybr advantage pcr premix ( takara , dalian , china ) in a 20  l system . the primers for nlrp3 , il-1 , and gapdh were as follows :  nlrp3 : forward 5-atgctgcttcgacatctcct-3 , reverse 5-gtttctggaggttgcagagc-3 ;  il-1 : forward 5-gcccatcctctgtgactcat-3 , reverse 5-aggccacaggtattttgtcg-3 ;  gapdh : forward 5-acccagaagactgtggatgg-3 , reverse 5-cacattgggggtaggaacac-3. nlrp3 : forward 5-atgctgcttcgacatctcct-3 , reverse 5-gtttctggaggttgcagagc-3 ; il-1 : forward 5-gcccatcctctgtgactcat-3 , reverse 5-aggccacaggtattttgtcg-3 ; gapdh : forward 5-acccagaagactgtggatgg-3 , reverse 5-cacattgggggtaggaacac-3. the hippocampus tissue was homogenized while the cells were lysed in lysis buffer with protease inhibitor ( sigma - aldrich , st . 30  g of protein was loaded for each sample , which was then subjected to 10% sds - page electrophoresis . the gel was then transferred to a polyvinylidene fluoride membrane ( bd bioscience , franklin lakes , usa ) . after blocking with 5% skimmed milk , the membrane was incubated on a rocker with primary antibody at 4c overnight . membranes were then treated with ecl reagent ( thermofisher , waltham , usa ) to detect protein expression , using the uvp bioimaging system ( upland , usa ) . the densitometry was analyzed using the image - pro plus software ( media cybernetics , bethesda , usa ) . the antibodies for western blot included the rabbit anti - mouse anti - il-1 antibody ( 1  :  1000 , abcam , cambridge , uk ) , rabbit anti - mouse anti - iba-1 antibody ( 1  :  1000 , abcam , cambridge , uk ) , rat anti - mouse nlrp3 antibody ( 1  :  1000 , r&d , minneapolis , usa ) , rabbit anti - mouse -actin ( 1  :  1000 , abcam , cambridge , uk ) , hrp - conjugated goat anti - rabbit igg antibody ( 1  :  4000 , cell signaling technology , danvers , usa ) , and hrp - conjugated goat anti - rat igg antibody ( 1  :  4000 , r&d , minneapolis , usa ) . all statistical analyses were performed using the graphpad prism 5.0 software ( la jolla , usa ) . the latency , distance , and time during the water maze training were compared using the two - way anova test with bonferroni 's correction . the other comparisons among four groups were performed by the one - way anova test , followed by lsd post hoc analyses . morris water maze training was performed for a total of 4 days , after clp or sham surgery . in comparison to the sham mice , clp mice demonstrated significantly prolonged escape of latency from the fifth day after surgery ( figure 1(a ) ) . clp mice treated with a high dose ( 30  mg / kg ) of resveratrol had shorter latency periods at days 3 and 4 of training ( p < 0.05 ) , while those treated with a lower dose of resveratrol ( 10  mg / kg ) exhibited shorter latency periods on the last day of training ( p < 0.05 ) . it should be noted that the distance and time spent in the target quadrant were similar over the four days of training across all four groups ( figures 1(b ) and 1(c ) ) . after the water maze training , the platform was removed to test the distance , time spent in the target quadrant , and frequency of crossing . the distance travelled was similar among the four groups ( p > 0.05 ) ( figure 1(d ) ) . clp mice spent the shortest amount of time in the target quadrant ( 6.47  4.63  s ) , which was significantly less than that of the sham - operated ( 41.89  7.53  s ) ( p < 0.05 ) and resveratrol treated clp mice ( res1 : 23.17  4.96  s ; res2 : 16.35  6.19  s ) ( p < 0.05 for both ) . also , the two doses of resveratrol did not affect the time spent in the target quadrant ( p > 0.05 ) . the time spent in the target quadrant in both resveratrol groups was shorter than in sham group , while it was longer than in clp group ( p < 0.05 ) ( figure 1(e ) ) . the frequency of crossing the platform was reduced in clp mice ( 0.17  0.41 ) in comparison to the sham - operated mice ( 2.67  0.82 ) ( p < 0.05 ) , while resveratrol significantly increased the frequency , in a dose response manner ( res1 : 2.17  0.75 ; res2 : 1.00  0.63 ) ( res1 versus clp , p < 0.05 ; res2 versus clp , p < 0.05 ; res1 versus res2 , p < 0.05 ) ( figure 1(f ) ) . pathological changes in septic mice were determined by measuring the rates of apoptosis and microglial activation in the hippocampus . tunel assay showed that the apoptotic rates of cells were significantly increased during sepsis , 17.0  1.0 versus 1.67  0.58 per field ( p < 0.05 ) . resveratrol was seen to effectively inhibit significant increases in apoptosis rates , 4.0  1.0% in res 30  mg / kg group and 9.7  0.6% in res 10  mg / kg group ( p < 0.05 for comparisons between res1 or res2 group versus clp or sham group ) ( figure 2 ) . relative expression of iba-1 in the hippocampus region was detected by both if and western blot assay . iba-1 fluorescence intensity as shown by if assay was significantly increased in clp mice , while 30  mg / kg resveratrol reduced the intensity significantly ( p < 0.05 ) . il-1 was also inhibited significantly by 30  mg / kg resveratrol ( p < 0.05 ) ( figure 3(a ) ) . resveratrol reduced the expression of iba-1 at both doses , but there were no significant changes between the two res groups ( p < 0.05 for all comparisons between sham group and other groups , p < 0.05 for comparisons between clp group and both res groups , p > 0.05 between res1 and res2 groups ) ( figure 3(b ) ) . the nlrp inflammasome is a type of nod - like receptor ( nlr ) , which is known to activate caspase-1 , which promotes the cleavage of il-1 . we detected the expression of both nlrp3 and il-1 at both mrna and protein levels in the hippocampus . at the mrna level , resveratrol reduced the nlrp3 and il-1 expression in a dose dependent manner , while the nlrp3 level in res 30  mg / kg group was reduced to a similar level to that in sham group ( figure 4(a ) ) . nlrp3 protein levels were reflective and followed the expression levels found at the mrna level . resveratrol was able to significantly reduce the expression of nlrp3 , in a dose dependent manner . interestingly , the il-1 levels were also significantly reduced in res1 group at the level of cleaved protein but not at the pro - form level ( figure 4(b ) ) . 
 in vitro study of microglia bv2 cells were stimulated with atp to prime the nlrp3 ( lps ) and two doses of resveratrol . in the presence of lps and atp , the nlrp3 expression significantly upregulated and the cleavage of caspase-1 and il-1 were enhanced ; however , in the presence of resveratrol , we were able to significantly inhibit such processes ( figure 5 ) . in order to investigate if the protective role of resveratrol was dependent on sirt1 , bv2 cells were blocked with the sirt1 inhibitor , nam . the usage of the nam inhibitor completely abolished the inhibition of nlrp3 expression and il-1 cleavage , which was induced by resveratrol ( figure 6 ) . in this study we have demonstrated that resveratrol improves the spatial learning and memory capacity of septic mice . these conclusions were made based on the attenuation of neuronal apoptosis and microglial activation , after resveratrol administration . moreover , resveratrol was demonstrated to inhibit the nlrp3/il-1 axis at both in vivo and in vitro levels . recent literature reports indicate that resveratrol has been considered for optimal usage for neuroprotective effects . it was shown that resveratrol preconditioning reduced the infarction volume , while improving the neurological score in the mouse after middle cerebral artery occlusion , thereby providing an extended ischemia tolerance in the brain [ 19 , 20 ] . resveratrol was also reported to protect against poststroke depression through the regulation of the hypothalamus - pituitary - adrenal axis in rats . additional studies regarding the neuroprotective effect were focused on exploring the antiaging and antineurodegerative effect [ 14 , 22 ] . resveratrol was demonstrated to reduce p53 expression , nuclear factor-b activation , and tau phosphorylation and improve cognitive function in the senescence - accelerated prone mouse , a model with accelerated aging and cognitive decline . in another common neurodegenerative disease , parkinson 's disease , resveratrol was able to increase the motor coordination skills in rat models . but most importantly , in humans , a controlled randomized , pair - matched , placebo - controlled trial showed that continuous intake of resveratrol for a period of 26 weeks showed improvements in memory performance , which included memory retention , delayed recall , recognition , learning ability , and fifth learning trial . the above - mentioned studies provide strong evidence for resveratrol 's ability to provide protection against neuronal impairment caused by stroke , neurodegenerative factor , and aging . therefore , not surprisingly , our data demonstrated that resveratrol was protective against sae according to the spatial learning training and test , as well as the neuronal apoptosis in the hippocampus . microglia activation in sae has been well documented in an autopsy study , which showed a significantly increase in the expression of cd68 in both the cortex and white matter of patients dying from sepsis . michels et al .   demonstrated that minocycline , an inhibitor for microglia activation , reduced oxidative stress and inflammatory burden in the hippocampus , while improving the long - term cognitive behavior of clp mice . another study showed that il-1 expression was increased by the microglia of aged mice that were challenged with peripheral lps administration . the ihc and western blot assays in our present study showed that resveratrol administration reduced the expression of both iba-1 and il-1 , which may potentially be a pivotal mechanism involved in the protective effect . il-1 secretion is controlled by the activation of nlrp3 inflammasomes , which recognize the pathogen - associated molecular pattern ( pamp ) or danger - associated molecular pattern ( damp ) , such as ros . nlrp3 activation promotes the cleavage of procaspase-1 , in the presence of the adaptor protein called asc . mature caspase-1 then promotes the proteolytic cleavage of pro - il-1 at asp116 into il-1. nlrp3/caspase-1 axis played a central role in the pathogenesis of alzheimer 's disease . the accumulation of amyloid- can activate nlrp3 in microglia , while the increased maturation of il-1 further hampered the clearance of amyloid- by microglia . our data suggest that nlrp3/il-1 might also participate the development of sae and that resveratrol ameliorates sae through the reverse activation of nlrp3 . the role of resveratrol on nlrp3 activation has also been demonstrated in macrophages and microglial cells in different cell or disease models [ 30 , 31 ] . we believe that there are several mechanisms involved in the reduction of nlrp3 expression and il-1 maturation by resveratrol . sirt1 was reported to deacetylate nuclear factor-b , thereby inhibiting the translocation of the p65 subunit to the nucleus [ 32 , 33 ] . therefore , our findings can be considered parallel to those in literature as resveratrol was observed to decrease the expression of nlrp3 and il-1 mrna . sirt1 is a protein that assists in the clearance of ros , which may potentially downregulate the activation of nlrp3 . therefore , we can potentially correlate the decreased activation of nlrp3 with a reduction in ros , which was dependent on resveratrol administration . furthermore , misawa et al .   found that resveratrol might block the assembly of nlrp3 on endoplasmic reticulum and asc on mitochondria , thereby inhibiting the activation of nlrp3 inflammasome . firstly , the exact role of nlrp3/il-1 axis in the protective effects of resveratrol against sae was not fully established . in order to determine the causal relation of nlrp3 inflammasome and protective role of resveratrol , nlrp3 knockout mice might be required to detect the effect of resveratrol on sae in nlrp3 deficient mice . secondly , which step of nlrp3 priming and activation did the resveratrol inhibit was not clarified . resveratrol might inhibit nlrp3 activation by the above - mentioned mechanisms , but it may also inhibit the nlrp3 and il-1 priming via sirt1 , since sirt1 might prevent the transcriptional level of il-1 . finally , nam is an inhibitor for nad dependent enzymes and sirt1 is one of the enzymes that can be inhibited by nam . thus the specificity issue may limit the conclusion that protective role of resveratrol is dependent on sirt1 . resveratrol can also inhibit the nlrp3/il-1 axis in hippocampus of sae mice and microglial cells stimulated by lps and atp , which is critical for the development of sae . the exact mechanism underlying in the protective role of resveratrol against sae remains to be further investigated .
sepsis - associated encephalopathy ( sae ) is characterized as brain dysfunction associated with sepsis . in this study we sought to investigate the effects of resveratrol in mice with sae , as well as its effects in nlrp3 inflammasome and il-1 , which were critical in the pathogenesis of sae . 
 sae was induced in mice via cecal ligation and puncture ( clp ) , and resveratrol was administered at two doses after surgery . 
 spatial learning memory functions were evaluated by morris water maze testing . 
 apoptosis in the hippocampus was quantified using tunel assay . 
 inflammation in the hippocampus was quantified by measuring the levels of microglial activation , nlrp3 , and il-1. clp mice treated with resveratrol demonstrated a better spatial memory during water maze training . 
 the tunel assay demonstrated significantly attenuated rates of apoptosis , in resveratrol treated mice , while decreasing the number of iba-1 positive microglia in the hippocampus region . 
 nlrp3 expression and il-1 cleavage were well inhibited by resveratrol dose - dependently . 
 the in vitro results showed that in the bv2 cell lines resveratrol prevents atp induced nlrp3 activation and il-1 cleavage , which were reversed by the sirtuin 1 inhibitor , nicotinamide . in conclusion 
 , resveratrol improves the spatial memory in mice with sae and inhibits the nlrp3/il-1 axis in the microglia .