dai is one of the most common and important pathologic features of traumatic brain injury ( tbi ) , and it is associated with high mortality and morbidity rates . the major cause of damage in dai is the disruption of axons , which can lead to the development of a secondary injury cascade , cellular stress , inflammation , changes in bbb permeability , and apoptosis . secondary injury can be activated at later time points and may be more amenable to targeting . thus , an accurate investigation of the pathological changes in the brain after dai is of utmost importance for the clinical treatment of this widespread axonal damage . the inflammatory response is regarded as a key factor in the secondary injury cascade after tbi . activation of the inflammatory cascade is mediated by the release of pro- and anti - inflammatory cytokines . recent studies indicated that inflammation plays a crucial role in the pathology of dai , thus representing a potential target to preserve the tissue surviving the initial impact of dai . recent study found that there are two patterns of inflammation , pathogen - associated molecular pattern ( pamp ) and damage - associated molecular pattern ( damp ) , which can both lead to the inflammatory response . pamps are molecules associated with groups of pathogens , which are recognized by cells of the innate immune system , while damp are host molecules that can initiate and perpetuate a noninfectious inflammatory response , which include the chromatin - associated protein hmgb1 . hmgb1 functions as a proinflammatory factor when released into the extracellular milieu and acts on several cells to trigger inflammatory responses by promoting the expression of inflammatory cytokines . these proinflammatory effects have been found in focal cerebral ischemia , sah , and tbi [ 69 ] . moreover , emerging evidence indicated hmgb1 inhibitors as potential therapeutic agents for sepsis and ischemic injury . in this study , we investigated the inflammatory response after dai , and we further explored the protecting effects of gl ( a hmgb1 inhibitor ) on the brain injury after dai . eighty - seven healthy adults sd rats weighing 250300  g were purchased from the experimental animal center of xi'an jiaotong university . animals were housed and fed in a temperature and humidity controlled environment with a standardized light / dark cycle ( 12  h day / night ) for 1 week . all animal procedures were in accordance with the guidance suggestions for the care and use of laboratory animals , formulated by the ministry of science and technology of china . thirty - six rats were randomly divided into sham group , dai 3  h group , dai 6  h group , dai 12  h group , dai 24  h group , and dai 48  h group ( n = 6 per group : 3 were used for western blot analysis and 3 for tunel staining ) . forty - eight rats were randomly divided into control group ( sham ) , dai group , dai + gl group , and dai + vehicle group ( normal saline ) ( n = 12 per group : 3 were used for bwc evaluation , 3 for tunel staining and immunohistochemistry , 3 for evans blue detection , and 3 for western blot analysis ) . primary antibodies anti - hmgb1 , anti - mmp-9 , anti - il-1 , and anti - tnf- were purchased from abcam ( cambridge , uk ) ; primary antibodies against caspases 3 and 9 , cleaved caspases 3 and 9 , primary antibodies against bcl-2 , and phosphorylated bcl-2 were purchased from cell signaling technology ( ma , usa ) ; primary antibodies against -app were purchased from merck millipore ( billerica , ma , usa ) ; primary antibody against -actin , secondary antibody , fitc - labelled anti - rabbit igg , and dapi were purchased from santa cruz biotechnology ( ca , usa ) ; tunel staining kit was purchased from roche ( basel , switzerland ) ; glycyrrhizic acid was purchased from sigma aldrich ( st . a rat dai model was established using a lateral head rotation device as previously described . rats were anesthetized by intraperitoneal injection of 10% chloral hydrate ( 30  mg / kg ) , and then , the rat head was horizontally secured to the rotation device by two lateral ear bars , a head clip , and an anterior teeth hole , with its body 30 oblique to the top of the laboratory table . when the trigger was pressed , the rat head was rapidly rotated by 90 , involving sudden acceleration and deceleration . control rats ( sham group ) underwent anesthesia and fixation to the device and were not subjected to injury . rats that died after the injury were excluded and replaced by new rats . gl ( 10  mg / kg ) was diluted in normal saline ( ns ) and intravenously administered to the dai + gl group 30  min before the induction of dai , while , in dai + ns group , the same volume of ns was intravenously administered . sections were stained with mallory ptah staining kits ( rongbai biological technology co. , shanghai , china ) according to the manufacturer 's instruction , then followed by dehydration , hyalinization , and fixation , and observed by microscopy . total protein was isolated from rat injured brain tissues using ice - cold ripa buffer . total protein concentrations were measured using the bca protein assay kit ( thermo fisher scientific , ma , usa ) . protein samples ( 30  g total proteins per lane ) were separated using sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes . proteins were detected by incubation with primary antibodies such as rabbit monoclonal hmgb1 , mmp-9 and tnf- ( 1  :  2000 ) , and rabbit polyclonal il-1 ( 1  :  1000 ) followed by the secondary antibody ( goat anti - rabbit igg , horseradish peroxidase conjugated , 1  :  1000 ) . -actin , used as the loading control , was detected with a 1  :  3000 antibody . briefly , brains were formalin - fixed , paraffin - embedded , and cut into 10  m serial sections . endogenous peroxidase activity was blocked using 3% h2o2 for 5  min , followed by citric acid buffer ( ph 6.0 ) microwave antigen retrieval . sections were incubated with primary mice -app monoclonal antibody ( 1  :  1000 ) overnight at 4c , followed by a 15  min wash in pbs . next , sections were incubated with horseradish peroxidase- ( hrp- ) conjugated igg ( 1  :  500 ) for 1  h at room temperature . dab ( 30  mg dab and 200  l h2o2/100  ml pbs ) for 15  min at room temperature in the dark was used as chromogen and counterstaining was made using hematoxylin . triton x-100 in pbs for 5  min , and 5  min wash in pbs for 3 times . sections were placed in 10  mmol / l citrate buffer ( ph 6.0 ) and heated in the microwave oven at 95c for 30  min . sections were incubated with primary rabbit hmgb1 monoclonal antibody ( 1  :  200 ) overnight at 4c , followed by 5  min wash in pbs for 3 times . sections were incubated with fitc - labelled anti - rabbit igg for 2  h at room temperature . next , sections were incubated with 4,6-diamidino-2-phenylindole ( dapi ; 1  g / ml ) for 15  min at room temperature . in order to assess apoptosis , dna fragmentation in the nuclei was determined using terminal deoxynucleotidyl transferase - mediated dutp nick end - labeling ( tunel ) reaction . first , tissue sections were deparaffinized with xylene , rehydrated through descending concentrations of ethanol , rinsed for 10  min in 0.1  m pbs , and treated with 20  g / ml of proteinase k for 20  min at room temperature . samples were treated with 3% h2o2 in methanol for 10  min to inactivate endogenous peroxidase . after washing with pbs , sections were incubated in the labeling reaction mixture containing terminal deoxynucleotidyl transferase and the deoxynucleotide at 4c overnight . after incubation , the sections were rinsed in pbs and incubated with horseradish peroxidase ( pod , 1  :  500 ) for 30  min at room temperature . next , the sections were extensively washed with pbs for 3  min and treated with dab for 15  min at room temperature in the dark . after washing under running water , the sections were counterstained with hematoxylin for 1  min . finally , the sections were dehydrated in increasing graded ethanol , cleared in xylene , and mounted with a cover slip . apoptotic cells in the brain cortex were counted under a light microscope with a 40x objective by two independent investigators . the number of apoptotic cells was quantified by the count of positive cells in 5 randomly selected sights . the permeability of the bbb was determined by measuring the penetration of evans blue ( sigma aldrich , st . evans blue ( 2% in saline ; 4  ml / kg body weight ) was intravenously administered via the tail vein 1 hour before measurement , and then the rats were transcardially perfused with saline , 200  ml per rat . each brain was quickly removed , weighed , and homogenized with 400  l pbs and then precipitated by 50% trichloroacetic acid overnight . the samples were centrifuged for 30  minutes at 3000  g to pellet the brain tissue . absorption of the supernatant was measured at a wavelength of 610  nm using a plate reader ( biotek , winooski , vt ) . the extravasation of evans blue was quantified as microgram / gram brain tissue using an evans blue standardized curve . brain water content ( bwc ) was evaluated using the standard wet - dry method after dai . brains were then dried in an oven at 105c for 72  h and weighed again to determine the dry weight and the bwc was calculated by the formula bwc = [ ( wet weight  dry weight)/wet weight ] . a modified neurologic severity score ( mnss ) , which includes motor , sensory , reflex , and balance tests , was used to evaluate the neurological deficits of each animal before sacrifice by two observers blinded to the treatment . statistical differences among the groups were analyzed by one - way anova and followed by student - newman - keuls test for multiple comparisons . in order to evaluate our dai model , ptah staining was used to observe the cerebral histopathology . the control group showed healthy axons , neuronal cells normally arranged without any neuronal loss , and morphological changes . on the other hand , ptah staining in the dai group showed irregular cells arrangement , and axons clearly twisted and ruptured with axonal bulbs formation ( figure 1 ) . these results confirmed that the dai model used in this study was reliable since it could effectively simulate the clinical pathological characteristics of the dai . hmgb1 protein level showed a significant decrease in the first 48  h after dai compared with sham group , except for at 12  h , indicating the extracellular release of hmgb1 after dai ( figure 2(a ) ) . no apoptotic cells were found in the sham and dai group at 3  h , while a significant increase in neuronal apoptosis occurred at 6  h after dai in the dai group compared with the sham group , reaching the peak level at 24 and 48  h ( figure 2(b ) ) . caspases 3 and 9 expression were significantly increased at 6  h and 12  h after dai compared with the sham group , while they both decreased at 24 and 48  h after dai . however , the cleaved caspase-3 began to increase at 6  h , and cleaved caspase-9 at 3  h after dai . bcl-2 and phosphorylated bcl-2 significantly increased at 6  h after dai compared with the sham group ( figure 2(c ) ) . these results indicate that dai could induce the activation of the apoptotic pathway , leading to neuronal apoptosis . previous studies suggested that gl could significantly alleviate apoptotic injury by inhibiting the activity of hmgb1 on rat cerebral ischemia - reperfusion . therefore , we further detected neuronal apoptosis in rat brains that underwent gl pretreatment , at 24  h after dai . tunel staining showed that the pretreatment with gl significantly decreased the neuronal apoptosis induced by dai in the dai + gl group compared with the dai + ns group ( figure 3(a ) ) . moreover , the expression of bcl-2 and caspases 3 and 9 were increased in dai group , while the expression of the phosphorylated bcl-2 and cleaved caspases 3 and 9 were significantly decreased in the dai + gl group compared with the dai + ns group ( figure 3(b ) ) . the immunofluorescence staining showed that the cytosolic translocation of hmgb1 was also clearly suppressed by gl pretreatment ( figure 3(c ) ) . the mnss test was performed to assess the short - term functional outcome of dai . the results showed that the pretreatment with gl significantly improved the neurological outcomes ( figure 4(a ) ) . to investigate the ability of gl to inhibit the release of hmgb1 induced by dai and evaluate its therapeutic potential in the treatment of dai , evans blue was used to detect the destruction of bbb , bwc was used to indicate the brain edema , and -app was used to evaluate the axonal injury severity after dai . our results showed that dai could significantly lead to brain edema that was significantly inhibited by gl pretreatment ( figure 4(b ) ) in the dai + gl group compared with the dai + ns group . indeed , no evans blue diffusion was detected in the control group , while the level of evans blue significantly increased after dai , and the pretreatment with gl clearly decreased the evans blue diffusion , indicating the improved integrity of bbb ( figures 4(c ) and 4(d ) ) . in healthy brain , -app is not detected in the brain tissue due to its low level . however , -app accumulation occurs in the proximal and distal axonal segment to a detectable degree as a consequence of axonal injury . thus , -app was considered as the indicator for axonal injury . in the control group , however , -app was clearly accumulated in the cytosol and axons after dai , while the accumulation of -app decreased with gl pretreatment in the dai + gl group compared with the dai + ns group ( figure 4(e ) ) . our western blot results showed that hmgb1 inhibition induced by gl significantly suppressed the expression of proinflammatory factor tnf- , il-6 , and mmp-9 in the dai + gl group compared with the dai + ns group ( figure 5 ) . it is a nuclear protein that , following extracellular release , has endogenous cytokine - like activity , promoting inflammation and participating in the pathogenesis of several cerebral diseases , including cerebral ischemia and subarachnoid hemorrhage [ 15 , 16 ] . however , most of these studies are related to focal cerebral injury due to head trauma . in contrast to focal injury , studies on the inflammatory reaction due to dai , a diffuse form of tbi , are still limited . previous studies have indicated that hmgb1 can be released from neuronal cells after cerebral ischemia [ 18 , 19 ] ; thus the inhibition of the proinflammatory function of hmgb1 confers a robust neuroprotection in the postischemic brain . in our research , we used a rat dai model inducing a hmgb1 dynamic expression in the first 48  h after dai . previous work has shown that hmgb1 significantly decreased during the first 3 days after cerebral ischemia and gradually increased to above the basal level at day 4 . our results also showed a reduced hmgb1 level below the basal level at the first 48  h after dai , with a slight increase at 12  h. these results indicated that hmgb1 could be released into cytosol and intracellular space , which was consistent with previous studies [ 18 , 19 ] . hmgb1 release can activate microglia , which are the markers of brain inflammation [ 7 , 21 ] . hmgb1 also activates macrophages / monocytes to release the proinflammatory cytokines tnf- , il-1 , and il-6 [ 22 , 23 ] , and tnf- and il-1 could induce the synthesis and release of hmgb1 , as demonstrated by in vitro studies [ 24 , 25 ] . thus , it could be possible that neurons release hmgb1 in response to stimuli triggering to a crosstalk of neuroinflammatory signaling between neurons and glia . our present results supported the notion that positive feedback loops of neuroinflammation in brain may require hmgb1 activation of microglial tlr4 , resulting in a production of microglia - released proinflammatory mediators such as tnf- and il-1 , which further increase the release of hmgb1 from neurons and other cells [ 21 , 26 , 27 ] . thus , we concluded that hmgb1 was massively released into the extracellular milieu after dai , which may result in the decreased hmgb1 level , but at the late stage after dai , astrocyte and microglia may be activated and produce the proinflammatory cytokines . it is reported that gl is a hmgb1 inhibitor , since it can directly bind to hmgb1 and interact with two shallow concave surfaces formed by the two arms of both hmg boxes , thus inhibiting the phosphorylation and secretion of hmgb1 . this gl - hmgb1 bond significantly protects the brain by inhibiting the inflammatory response after cerebral ischemia . after cerebral ischemia , hmgb1 could significantly trigger mmp-9 upregulation in neurons and astrocytes predominantly via tlr4 pathway . furthermore , hmgb1 may enhance the disruption of the bbb through the activation of mmp via t - pa . in our present study , we also found a remarkable disruption of the bbb after dai , while the inhibition of hmgb1 induced by gl could clearly decrease the evans blue leakage , indicating the protective effect of gl on the integrity of bbb after dai . moreover , mmp-9 can damage myelin basic protein , which is the major protein component of the myelin sheath . thus , we further detected the accumulation of -app , which could only be detected after structural axonal injury . our results showed that gl pretreatment significantly decreased the accumulation of -app , which indicated the improved axonal injury . hmgb1 can promote the expression of proinflammatory cytokines , and inhibition of hmgb1 with hmgb1 antibodies can remarkably protect the brain by suppressing the inflammatory response after tbi . in our present study , the inhibition of the hmgb1 induced by gl significantly suppressed the expression of tnf- and il-1 , thus significantly alleviating the brain edema and neuronal apoptosis . although the anti - inflammatory effects of gl have been confirmed with increasing evidence , molecular mechanisms underlying the inhibition of hmgb1 by gl are unclear . as we discussed above , direct binding between gl and hmgb1 appears to be critical , which may disturb direct interactions between hmgb1 and various other partner molecules . however , recent work by xie et al . has demonstrated that suppressing the activation of pla2 and production of aa metabolites may be involved in the anti - inflammatory properties of gl . these findings suggest that the mechanisms underlying the neuroprotective effects of gl include the hmgb1-dependent and hmgb1-independent mechanisms , which need further study . compared to focal injury models , a dai experimental animal model is relatively difficult to set up . however , most focal injury models not only cause axonal injury but also result in focal contusion ; thus the results could not fully clarify the pathological change due to dai . in this study , we first evaluated our dai model , which was leading to a clear axonal injury without focal contusion , thus resulting similar to the clinical pathology of dai . by using this dai model these results indicated that hmgb1 is likely relevant to the expression of inflammatory factors and inhibition of hmgb1 release can alleviate the brain injury after dai . this may help to better understand dai - induced inflammatory responses and they could potentially provide methods to develop efficient therapies against dai .
high - mobility group box 1 ( hmgb1 ) , a nuclear protein that has endogenous cytokine - like activity , is involved in several neurological diseases by mediating inflammatory response . in this study , 
 a lateral head rotation device was used to establish a rat diffuse axonal injury ( dai ) model . 
 the dynamic expression of hmgb1 , apoptosis - associated proteins , and proinflammatory cytokines were detected by western blot , and neuronal apoptosis was observed by tunel staining . 
 the extracellular release of hmgb1 and the accumulation of -app were observed by immunofluorescence and immunohistochemistry , respectively . 
 the brain injury was indicated by modified neurological severity score ( mnss ) , brain water content ( bwc ) , and the extravasation of evans blue . 
 we showed that hmgb1 level obviously decreased within 48  h after dai , accompanied by neuronal apoptosis , the activation of caspases 3 and 9 , and the phosphorylation of bcl-2 . 
 inhibiting hmgb1 with glycyrrhizic acid ( gl ) can suppress the activation of apoptosis - associated proteins and inhibit the expression of proinflammatory cytokines , which ameliorated motor and cognitive deficits , reduced neuronal apoptosis , and protected the integrity of blood brain barrier ( bbb ) and axonal injury after experimental dai in rats . 
 thus , hmgb1 may be involved in the inflammatory response after dai , and inhibition of hmgb1 release with gl can notably alleviate the brain injury after dai .