| BACKGROUD:Traumatic brain injury (TBI) is one of the most common emergency in neurosurgery and a major cause of morbidity and mortality in children and young adults. Especially, medium and severe TBI can significantly deteriorate the patients’survival time and neurological function, it also can cause severe dysfunction of other systems and lead to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). So, it is a heavy burden to the family and the whole society. However, basic and clinical research of traumatic brain injury has made a great progress in past decades, and new theories are being discovered and development constantly, but it is still not have an effective treatment can reduce mortality and morbidity. The pathophysiological process after TBI is still not entirely clear, we only know that the brain injury followed the TBI can be divided into two phases:the initial stage is the injury itself, it caused by direct head impact, acceleration or deceleration injury, these injury factors will lead to compression of brain tissue, cerebral cortical contusion, edema, hemorrhagic and ischemia. The second phase from injury beginning to several days or weeks after injury, and the reaction in physiology, cellular and molecular level will occur in this delayed phase, if these reaction are not well controlled will lead to secondary injury. How do these cascade of events leading to the secondary brain injury are not yet entirely clear, we just know that primary injury will trigger a series of pathological cascades including the activation of the inflammatory response, oxygen free radicals and excitotoxic neurotransmitter release, intracellular enzyme activation, lead to cells structural damage and activation of apoptotic pathways.The inflammatory response has an important role in secondary brain injury followed the TBI. Nuclear factor kappa B (NF-κB) as a nuclear transcription factor, it was proved has an important role in regulation of the downstream inflammatory factors, chemokines, acute phase proteins, apoptosis/anti-apoptotic gene activation and transcription. Multiple signaling pathways involved in the regulation of NF-κB DNA binding activity, and one of them is toll-like receptors (TLRs)/myeloid differentiation primary response protein88(Myd88)/NF-κB pathway. As a key adapter protein of the TLRs, Myd88-mediated signaling pathways mainly stimulate the activation of NF-κB. NF-κB binding activity and downstream pro-inflammatory cytokines expression was significantly decreased after inhibited Myd88. TBI induced a rapid and persistent up-regulation interleukin-1β (IL-1β), IL-6, tumor necrosis factor-a (TNF-a), intercellular adhesion molecule1(ICAM-1), et al, and these pro-inflammatory cytokines can promote the inflammatory cells such as neutrophil accumulation and infiltration from the circulation to the brain tissue. Neutrophils accumulate into the central nervous system followed the TBI have been well addressed by numerous experiment studies. Infiltrate neutrophils can be activated and release free radicals, inflammatory cytokines or destructive enzymes, which enzymes include neutrophil elastase (NE), proteinase3(PR3), cathepsin G (CG), abnormal and excessive NE release into the tissue and can lead to enhanced acute inflammation and chronic inflammatory diseases.NE belongs to the chymotrypsin superfamily, and have multiple effects in central nervous system (CNS) injury which include destruct the blood-brain barrier (BBB) leading to vasogenic brain edema, promote the inflammatory cells infiltrate into the brain perivascular and parenchyma, induce apoptosis, et al. But the specific mechanisms related to the NE to fulfill these roles is not very clear. Furthermore, ONO-5046, a specific neutrophil elastase inhibitor, previous studies have well confirmed it also has a protective effect against central nervous system damage induced by ischemia and trauma. Thus, understand the role of NE in brain injury followed the TBI can further clarify the mechanism of secondary brain injury, and can also provide the theoretical basis for the development of new drugs.AIM:By analyze the mRNA and protein expression of Myd88, NF-κB binding activity, transcription of downstream pro-inflammatory cytokines such as TNF-α、 IL-1β、ICAM-land infiltration of neutrophils in mice brain cerebral contusion cortex after administrated ONO-5046, clarified the potential mechanism of NE secondary brain injury followed the TBI.METHODS:1. Groups and model.54Adult male C57BL/6mice (25～30g) were randomly divided into control groups (n=18), TBI groups (n=18) and TBI+ONO-5046groups (n=18). Closed head injury model was induced by weight drop (333g metal rod dropping from2.5cm height). Control sham-operated mice underwent procedures without subjected to the impact of the weight drop. ONO-5046(Ono Pharmaceutical Co., Ltd., Osaka, Japan) was dissolved in phosphate-buffered saline (PBS) and administered third times (intraperitoneally just after TBI, post-injury24h and48h) to groups of mice at doses of0(vehicle) and50mg/kg. NSS score was performed at1h after TBI. At72h after injury, randomly selected12mice in each group, each mouse was perfused transcardially with40ml phosphate-buffered saline through the left ventricle. Upon decapitation, the5mm surrounding brain tissue of the injured cortex was dissected on ice and stored at-80℃.6mice were used to western blot analyze and real-time PCR, and another6mice were used to myeloperoxidase (MPO) activity assays. The remaining mice in each group were perfused transcardially with30ml4%buffered formalin. Upon decapitation, embedded in paraffin after dehydration and stored at4℃until used.2. Cytosolic/nuclear protein extraction.The brain tissue was homogenized in1ml ice-cold buffer A composed of10mM HEPES (pH7.9),2mM MgCl2,10mM KCl,0.1mM EDTA,1mM dithiothreitol (DTT) and0.5mM phenylmethylsulfonyl fluoride (PMSF)(all from Sigma, MO, USA). The homogenate was incubated on ice for20min, and then30μl of10%Nonidet P-40solution was added (Sigma, MO, USA); the mixture was vortexed for30sec and spun by centrifugation for10min at5,000×g,4℃. The cytosolic protein extracts were collected and stored at-80℃for Western blot analysis. The total protein concentration was determined by Bradford method. The crude nuclear pellets were suspended in200μl ice-cold buffer B containing20mM HEPES (pH7.9),25%(v/v) glycerol,1.5mM MgCl2,20mM KC1,0.1mM EDTA,0.5mM PMSF, and1mM DTT, and incubated on ice for30min with intermittent mixing and centrifuged at 12,000×g at4℃for15min. The supernatant containing nuclear proteins was collected and stored at-80℃for EMSA analysis. Protein concentration was determined using a bicinchoninic acid (BCA) assay kit with bovine serum albumin as the standard.3. Western blot analysis for Myd88protein expression.50μg cytosolic protein extracts were treated with SDS-PAGE sample buffe, and boiled5min at95℃. Equal amount of proteins were loaded onto a10%SDS-polyacrylamide gel electrophoresis, and then electrotransferred onto a PVDF membrane. Following transfer, the membranes were blocked with5%nonfat milk in Tween-TBS (TBST) overnight at4℃. The membranes were then washed and incubated with primary rabbit anti-Myd88antibody (1:1000, Santa Cruz, CA, USA), rabbit anti-β-actin monoclonal antibody (1:1000; Sigma, MO, USA) for2hr at room. The membranes were washed in TBST for10min each, and then incubated with HRP-linked secondary antibody (1:1000, Jackson ImmunoResearch, PA, USA) for1hr at room temperature followed by washing in TBST for10min each, three times. Positive signals were developed using enhanced chemiluminescence (ECL; Amersham Biosciences, United Kingdom), and serial exposures were made to radiographic films (Fuji Hyperfilm, Tokyo, Japan). Optical densities were obtained using the image analysis program Image J.4. Electrophoretic mobility shift assay (EMSA) for NF-κB binding activity.EMSA was performed using a commercial kit (Gel Shift Assay System; Promega, WI, USA). Consensus oligonucleotide probe (5’-AGT TGA GGG GAC TTT CCC AGG C-3’) was end-labeled with [y-32P] ATP (Free Biotech., Beijing, China) and T4-polynucleotide kinase. Nuclear protein (40μg) was pre-incubated in9μl of binding buffer for10min at room temperature. After adding of the32P-labled oligonucleotide probes, the incubation was continued for20min at room temperature. Reaction was stopped by adding1μl of gel loading buffer and the mixture was subjected to nondenaturing4%polyacrylamide gel electrophoresis in0.5×TBE buffer (Tris-borate-EDTA). After electrophoresis was conducted at350V for1h, the gel was vacuum-dried and exposed to X ray film (Fuji Hyperfilm, Tokyo, Japan) at-80℃with an intensifying screed.5. Quantity real-time polymerase chain reaction (PCR) to detect the transcription of TNF-α、IL-1β、ICAM-1.Total RNA was extracted from frozen tissues via a single step method using TRIZOL reagent (Invitrogen Life Technologies, CA, USA), following a standard protocol. The concentration and quality of the RNA in each sample were determined by gel visualization and spectrophotometric analysis (OD260/280). RNA was reversely transcribed to complementary DNA (cDNA) using the Reverse transcription system (Bio-Rad, CA, USA). Quantitative real-time PCR analysis was performed by the Stratagene Mx3000P real-time PCR system (Stratagene, CA, USA), applying real-time SYBR Green PCR technology. The reaction mixtures contained2μl cDNA,0.4μl ROX Reference Dye and10μl SYBR Green I (Takara Bio Inc, Shiga, Japan),1μl of forward and reverse primer (1μM) and nuclease-free water to a final volume of20μl. After denaturation at95℃for30sec,40PCR cycles were performed, each consisted of a denaturation step (95℃,5sec) and an annealing step (62℃,34sec). Total RNA concentrations from each sample were normalized by the quantity of β-actin mRNA, and the expression levels of target genes were evaluated by the number of target mRNA to β-actin mRNA. All samples were analyzed in triplicate. 6. Immunohistochemistry for Myd88in brain subjected to TBI.The4%buffered formalin-fixed brain tissue was embedded in paraffin, sectioned in4μm thickness with a microtome. The anti-Myd88antibody (same as that used in Western blot) was diluted at1:100with1%bovine serum albumin/phosphate-buffered solution (BSA/PBS)(1g BSA/100ml PBS, pH7.4,0.01mol/L). The sections were incubated with the diluted antibodies overnight at4℃respectively, washed, and blocked with1.6%H2O2in phosphate-buffered saline (PBS) for10min. After washing with PBS, sections were incubated with HRP-conjugated goat anti-rabbit IgG (diluted at1:500) for60min at room temperature. DAB was used as chromogen and counterstaining was done with hematoxylin.Six views were selected randomly for each section and observed under a light microscope (×400). Then mean number of positive cells in the six views was used for statistical analysis.7. MPO activity assay.Samples of surrounding brain tissue of the injured cortex were homogenized at room temperature in50mmol/L of PPB (potassium phosphate buffer), pH6.0, and centrifuged for20min at60,000×g4℃. After centrifugation, the pallet was washed in PPB to remove the inhibitors of MPO activity and was resuspended in0.5%hexadecyltrimethylammonium bromide in PPB to liberate MPO from neutrophilic granules. Samples were then centrifuged for15min at15,000×g, and a portion of supernatant was added to PPB containing o-dianisidine dihydrochloride (0.2mg/ml) and0.001%H2O2. Changes in absorbance at460nm were assayed spectrophotometrically. One unit of MPO activity was defined as the degradation of1mmol of H2O2per minute. Results are expressed as units of MPO activity per gram of tissue. 8. Statistical analysis.The software SPSS13.0was used in the statistical analysis. Each parameter was expressed as the mean±SD and compared using One-way ANOVA followed by LSD and Dunnett’s T3analysis for multiple groups analysis. The relation between variables was analyzed using bivariate correlation with two-tailed test. The level of significance was set as P≤0.05.RESULT:1. General observation and NSS score:There was no significantly difference between all groups in body weight. The procedure trauma model-making and drugs administration were same in each mouse. The animals survived in both TBI and treatment group. There was no significantly difference between TBI and treatment group in NSS score.2. Myd88mRNA expression in brain tissue:The real-time PCR result showed that very low level of Myd88mRNA was found in control brain and it was significantly increased in TBI groups. The Myd88mRNA level was obviously decreased in mice brain after administrated with ONO-5046(P<0.05, respectively).3. Myd88protein expression in brain tissue:The western blot result showed that low level of Myd88protein expression was found in the control group. While in the TBI group, the Myd88protein was expressed at a high level. Compared to the TBI group, level of Myd88protein expression was significantly decreased in ONO-5046administration group, and this result was coincidence with real-time PCR. There was a statistically significant difference between the control group and each TBI groups (P<0.05, respectively). 4. Immunohistochemistry for Myd88in brain subjected to TBI: Immunohistochemistry for Myd88was performed to assess the localization of Myd88expression. A few Myd88-positive cells were observed in the control group, which indicates the constitutional expression of Myd88in the normal brain of mice (10.00±2.23/visual field). Increased Myd88-positive cells (38.83±3.77/visual field) in the TBI group could be found in the brain. Myd88-positive cells (25.33±6.15/visual field) was significantly decreased in TBI+ONO-5046group (P<0.05, respectively).5. NF-κB DNA-binding activity in brain tissue after TBI:NF-κB activation in the nuclear extracts was determined by EMSA. The result showed that low NF-κB binding activity was found in the control group. Compared with that of the control group, NF-κB binding activity in the part of the brain increased significantly after cortical contusion trauma (P<0.05), In the TBI+ONO-5046group, the NF-κB binding activity was significantly down-regulated (P<0.05) in the brain area surrounding the injury site after TBI.6. TNF-a, IL-1β and ICAM-1transcription in brain tissue after TBI:We used quantitative real-time PCR to analyze brain expression of pro-inflammatory cytokines (TNF-α, IL-1β and ICAM-1). Our data demonstrated that the mRNA level of TNF-a, IL-1β and ICAM-1were low in the sham-operated mice brain. Compared with those of the control group, mRNA level of TNF-a, IL-1β and ICAM-1in the brain tissue were significantly increased after TBI (P<0.05). ONO-5046administration after TBI leads to decreased TNF-α, IL-1β and ICAM-1expression.7. Neutrophil infiltration in brain tissue after TBI:We analyzed MPO activity, an index of neutrophils infiltration in brain. Low MPO activity (U/g)(0.33±0.14) was found in the control group, and it was increased significantly after cortical contusion trauma (1.36±0.22). The MPO activity (U/g)(0.97±0.09) remained markedly suppressed after treatment with ONO-5046.CONCLUSION:Immune inflammatory response plays an important role in secondary brain injury followed the TBI. After TBI, large number of neutrophils infiltrated into the brain parenchyma and released sufficient amount of NE by activated in several ways, and excessive NE release into the brain tissue can exacerbate secondary brain injury. NF-κB in the center of inflammation response, and has an important role in the progress of the inflammatory response followed the TBI. Myd88, the up-stream mediator factor of NF-κB, has an important role in the regulation of NF-κB binding activity. After administration of ONO-5046, specific inhibition of NE activity can significantly suppressed the expression of Myd88and NF-κB binding activity, down-regulated the transcription of pro-inflammatory cytokines, decreased neutrophils infiltration into injured mice brain followed the TBI. ONO-5046attenuated the Myd88/NF-KB signaling pathway, decreased neutrophils infiltration in injured mice brain, and this showed that may be NE has play an important role in promoting inflammatory responses by activating Myd88/NF-KB signaling pathway following TBI. |
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