Regulated expression of pancreatic triglyceride lipase after rat traumatic brain injury

Pancreatic triglyceride lipase (PTL), an enzyme of digestive system, plays very important roles in the digestion and absorption of lipids. However, its distribution and function in the central nervous system (CNS) remains unclear. In the present study, we mainly investigated the expression and cellular localization of PTL during traumatic brain injury (TBI). Western blot and RT–PCR analysis revealed that PTL was present in normal rat brain cortex. It gradually increased, reached a peak at the 3rd day after TBI, and then decreased. Double immunofluorescence staining showed that PTL was co-expressed with neuron, but had a few colocalizations in astrocytes. When TBI occurred in the rat cortex, the expression of PTL gradually increased, reached the peak at the 3rd day after TBI, and then decreased. Importantly, more PTL was colocalized with astrocytes, which is positive for proliferating cell nuclear antigen (PCNA). In addition, Western blot detection showed that the 3rd day post injury was not only the proliferation peak indicated by the elevated expression of PCNA, glial fibrillary acidic protein (GFAP) and cyclin D1, but also the apoptotic peak implied by the alteration of caspase-3 and bcl-2. These data suggested that PTL may be involved in the pathophysiology of TBI and PTL may be complicated after injury, more PTL was colocalized with astrocytes. Importantly, injury-induced expression of PTL was colabelled by proliferating cell nuclear antigen (proliferating cells marker), and the western blot for GFAP, PCNA and cyclin D1, showed that 3 days post injury was the proliferation peak, in coincidence to it, the protein level change of caspase-3 and bcl-2 revealed that the stage was peak of apoptotic too. These data suggested that PTL may be involved in the pathophysiology of TBI and that PTL may be implicated in the proliferation of astrocytes and the recovery of neurological outcomes. But the inherent mechanisms remained unknown. Further studies are needed to confirm the exact role of PTL after brain injury.     

Upregulation of p21-activated Kinase 6 in rat brain cortex after traumatic brain injury

p21-activated Kinase 6 (PAK6) is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family. PAK kinases are well-known regulators of a wide variety of cellular functions, including regulation of cytoskeleton rearrangement, cell survival, apoptosis and the mitogen-activated protein kinase signaling pathway. To elucidate the expressions and possible functions of PAK6 in central nervous system (CNS) lesion and repair, we performed a traumatic brain injury (TBI) model in adult rats. Western blot analysis revealed that PAK6 level significantly increased at day 3 after damage, and then declined during the following days. Besides, double immunofluorescence staining showed PAK6 was primarily expressed in the neurons and a few of glial cells in the normal group. While after injury, the expression of PAK6 was increased significantly in the astrocytes and neurons, and the astrocytes had largely proliferated. We also examined the expression of proliferating cell nuclear antigen (PCNA) whose change was correlated with the expression of PAK6. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many PAK6-expressing cells at day 3 after injury. In addition, injury-induced expression of PAK6 was co-labeled by active caspase-3 during neuronal apoptosis after injury. Collectively, we hypothesized PAK6 may play important roles in CNS pathophysiology after TBI and further research is needed to have a good understanding of its function and mechanism.     

Increased expression of BAG-1 in rat brain cortex after traumatic brain injury

BAG-1 protein was initially identified as a Bcl-2-binding protein. It was reported to enhance Bcl-2 protection from cell death, suggesting that BAG-1 represents a new type of anti-cell death gene. Moreover, recent study has shown that BAG-1 can enhance the proliferation of neuronal precursor cells, attenuate the growth inhibition induced by siah1. However, its function and expression in the central nervous system lesion are not been understood very well. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of BAG-1 expression in the brain cortex. Double immunofluorescence staining revealed that BAG-1 was co-expressed with NEURON and glial fibrillary acidic protein (GFAP). In addition, we detected that proliferating cell nuclear antigen had the co-localization with GFAP, and BAG-1. All our findings suggested that BAG-1 might involve in the pathophysiology of brain after TBI.     

SCY1-like 1 binding protein 1 (SCYL1-bp1) interacts with p53-induced RING H2 protein (Pirh2) after traumatic brain injury in rats

Traumatic brain injury (TBI) triggers a complex series of neurochemical and signaling changes that lead to neuronal dysfunction and overreactive astrocytes. In the current study, we showed that interactions between SCYL1-bp1 and Pirh2 are involved in central nervous system (CNS) injury and repair. Western blot and immunohistochemical analysis of an acute traumatic brain injury model in adult rats revealed significantly increased levels of SCYL1-bp1 and Pirh2 in the ipsilateral brain cortex, compared to contralateral cerebral cortex. Immunofluorescence double-labeling analyses further revealed that SCYL1-bp1 is mainly co-expressed with NeuN. Terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling staining data supported the involvement of SCYL1-bp1 and Pirh2 in neuronal apoptosis after brain injury. We additionally examined the expression profiles of active caspase-3, which were altered in correlation with the levels of SCYL1-bp1 and Pirh2. Notably, both SCYL1-bp1 and Pirh2 were colocalized with active caspase-3, and all three proteins participated in neuronal apoptosis. Immunoprecipitation experiments further revealed interactions of these proteins with each other in the pathophysiology process. To our knowledge, this is the first study to report interactions between SCYL1-bp1 and Pirh2 in traumatic brain. Our data collectively indicate that SCYL1-bp1 and Pirh2 play important roles in CNS pathophysiology after TBI.     

Expression of Dixdc1 and its Role in Astrocyte Proliferation after Traumatic Brain Injury

DIX domain containing 1 (Dixdc1), a positive regulator of Wnt signaling pathway, is recently reported to play a role in the neurogenesis. However, the distribution and function of Dixdc1 in the central nervous system (CNS) after brain injury are still unclear. We used an acute traumatic brain injury (TBI) model in adult rats to investigate whether Dixdc1 is involved in CNS injury and repair. Western blot analysis and immunohistochemistry showed a time-dependent up-regulation of Dixdc1 expression in ipsilateral cortex after TBI. Double immunofluorescent staining indicated a colocalization of Dixdc1 with astrocytes and neurons. Moreover, we detected a colocalization of Ki-67, a cell proliferation marker with GFAP and Dixdc1 after TBI. In primary cultured astrocytes stimulated with lipopolysaccharide, we found enhanced expression of Dixdc1 in parallel with up-regulation of Ki-67 and cyclin A, another cell proliferation marker. In addition, knockdown of Dixdc1 expression in primary astrocytes with Dixdc1-specific siRNA transfection induced G0/G1 arrest of cell cycle and significantly decreased cell proliferation. In conclusion, all these data suggest that up-regulation of Dixdc1 protein expression is potentially involved in astrocyte proliferation after traumatic brain injury in the rat.     

TNF‐α–induced p53 activation induces apoptosis in neurological injury

It was previously confirmed that the apoptotic and necrotic neurons are found during the acute post‐traumatic period, suggesting the induction of apoptosis after traumatic brain injury (TBI). To further explore the involvement of apoptotic factors in TBI, an apoptosis antibody array was conducted to measure the alterations of apoptotic factors in rat brain cortex after TBI. As a result, the Neurological Severity Scale (NSS) scores after TBI were increased, and the cell morphology of the brain cortex was destructed with increased neuronal apoptosis. Furthermore, the caspase‐3 activity was increased, and the apoptotic‐related factors TNF‐α and p53 were up‐regulated in the brain cortex. More importantly, in vitro experiments demonstrated that down‐regulation of TNF‐α in oxygen‐glucose deprivation/reoxygenation (OGD/R) cells increased cell viability and decreased apoptosis and the p53 expression. These results suggested the involvement of TNF‐α–induced apoptotic signalling pathway by activating p53 in the molecular mechanism of neurological injury.     

Cloning, expression, and characterization of TNFSF14 (LIGHT) gene in mefugu, Takifugu obscurus

Tumor necrosis factor receptor-associated factor 6 (TRAF6), which plays an important role in inflammation and immune response, is an essential adaptor protein for the NF-κB (nuclear factor κB) signaling pathway. Recent studies have shown that TRAF6 played an important role in tumorigenesis and invasion by suppressing NF-κB activation. However, up to now, the biologic role of TRAF6 in glioma has still remained unknown. To address the expression of TRAF6 in glioma cells, four glioma cell lines (U251, U-87MG, LN-18, and U373) and a non-cancerous human glial cell line SVG p12 were used to explore the protein expression of TRAF6 by Western blot. Our results indicated that TRAF6 expression was upregulated in human glioma cell lines, especially in metastatic cell lines. To investigate the role of TRAF6 in cell proliferation, apoptosis, invasion, and migration of glioma, we generated human glioma U-87MG cell lines in which TRAF6 was either overexpressed or depleted. Subsequently, the effects of TRAF6 on cell viability, cell cycle distribution, apoptosis, invasion, and migration in U-87MG cells were determined with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry analysis, transwell invasion assay, and wound-healing assay. The results showed that knockdown of TRAF6 could decrease cell viability, suppress cell proliferation, invasion and migration, and promote cell apoptosis, whereas overexpression of TRAF6 displayed the opposite effects. In addition, the effects of TRAF6 on the expression of phosphor-NF-κB (p-p65), cyclin D1, caspase 3, and MMP-9 were also probed. Knockdown of TRAF6 could lower the expression of p-p65, cyclin D1, and MMP-9, and raise the expression of caspase 3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, invasion, and migration of U-87MG cell, as well as inhibition of apoptosis of U-87MG cell by abrogating activation of NF-κB.     

Tetramethylpyrazine Nitrone Improves Neurobehavioral Functions and Confers Neuroprotection on Rats with Traumatic Brain Injury

Oxidative stress is one of the major secondary injury mechanisms after traumatic brain injury (TBI). 2-[[(1,1-Dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a derivative of the clinically used anti-stroke drug tetramethylpyrazine armed with a powerful free radical-scavenging nitrone moiety, has been demonstrated promising therapeutic efficacy in ischemic stroke and Parkinson’s models. The present study aims to investigate the effects of TBN on behavioral function and neuroprotection in rats subjected to TBI. TBN (90 mg/kg) was administered twice daily for 7 days by intravenous injection following TBI. TBN improved neuronal behavior functions after brain injury, including rotarod test and adhesive paper removal test. Compared with the TBI model group, TBN treatment significantly protected NeuN-positive neurons, while decreased glial fibrillary acidic protein (GFAP)-positive cells. The number of 4-hydroxynonenal (4-HNE)-positive and 8-hydroxy-2′-deoxyguanosine (8-OHdG)-positive cells around the damaged area after TBI were significantly decreased in the TBN treatment group. In addition, TBN effectively reversed the altered expression of Bcl-2, Bax and caspase 3, and the down-regulation of nuclear factor erythroid-derived 2-like 2 (Nrf-2) and hemeoxygenase-1 (HO-1) proteins expression stimulated by TBI. In conclusion, TBN improves neurobehavioral functions and protects neurons against TBI. This protective effect may be achieved by anti-neuronal apoptosis, alleviating oxidative stress damage and up-regulating Nrf-2 and HO-1 expression.     

Caspase 7: increased expression and activation after traumatic brain injury in rats

Caspases, a cysteine proteinase family, are required for the initiation and execution phases of apoptosis. It has been suggested that caspase 7, an apoptosis executioner implicated in cell death proteolysis, is redundant to the main executioner caspase 3 and it is generally believed that it is not present in the brain or present in only minute amounts with highly restricted activity. Here we report evidence that caspase 7 is up-regulated and activated after traumatic brain injury (TBI) in rats. TBI disrupts homeostasis resulting in pathological apoptotic activation. After controlled cortical impact TBI of adult male rats we observed, by semiquantitative real-time PCR, increased mRNA levels within the traumatized cortex and hippocampus peaking in the former about 5 days post-injury and in the latter within 6-24 h of trauma. The activation of caspase 7 protein after TBI, demonstrated by immunoblot by the increase of the active form of caspase 7 peaking 5 days post-injury in the cortex and hippocampus, was found to be up-regulated in both neurons and astrocytes by immunohistochemistry. These findings, the first to document the up-regulation of caspase 7 in the brain after acute brain injury in rats, suggest that caspase 7 activation could contribute to neuronal cell death on a scale not previously recognized.     

TRAF6 regulates cell fate decisions by inducing caspase 8-dependent apoptosis and the activation of NF-kappaB

Tumor necrosis factor receptor-associated factor 6 (TRAF6) functions as an adaptor, positively regulating the NF-kappaB pathway. Here we report a new function of human TRAF6, the direct stimulation of apoptosis. The mechanism of apoptosis induction results from the capacity of human TRAF6 to interact and activate caspase 8. Both the C-terminal TRAF domain of human TRAF6, which directly interacts with the death effector domain of pro-caspase 8, and the N-terminal RING domain, which is required for activation of caspase 8, are necessary for the induction of apoptosis. The role of endogenous TRAF6 in regulating apoptosis was confirmed by extinguishing TRAF6 expression with specific small-hairpin RNA that resulted in diminished spontaneous apoptosis and resistance to induced apoptosis. In contrast to the human molecule, murine TRAF6 displayed less ability to induce apoptosis and a greater capacity to stimulate NF-kappaB activity. Human and murine TRAF6 are similar except in the region between zinc finger 5 and the TRAF domains. Reciprocal transfer of this connecting region completely exchanged the ability of human and murine TRAF6 to induce apoptosis and activate NF-kappaB. Unique regions of TRAF6 therefore play an important role in determining cell fate.     

Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury

Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and in vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and cleaved caspase 8 p20 from 1 h to 14 days after cortical impact-induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT-PCR, were elevated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8- and caspase 3-immunopositive cells with apoptotic-like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2-5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.     

Spatiotemporal Pattern of RNA-Binding Motif Protein 3 Expression After Spinal Cord Injury in Rats

RNA-binding motif protein 3 (RBM3) belongs to a very small group of cold inducible proteins with anti-apoptotic and proliferative functions. To elucidate the expression and possible function of RBM3 in central nervous system (CNS) lesion and repair, we performed a spinal cord injury (SCI) model in adult rats. Western blot analysis revealed that RBM3 level significantly increased at 1 day after damage, and then declined during the following days. Immunohistochemistry further confirmed that RBM3 immunoactivity was expressed at low levels in gray and white matters in normal condition and increased at 1 day after SCI. Besides, double immunofluorescence staining showed RBM3 was primarily expressed in the neurons and a few of astrocytes in the normal group. While after injury, the expression of RBM3 increased both in neurons and astrocytes at 1 day. We also examined the expression profiles of proliferating cell nuclear antigen (PCNA) and active caspase-3 in injured spinal cords by western blot. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many RBM3-expressing cells and rare caspase-3 was observed in RBM3-expressing cells at 1 day after injury. Our data suggested that RBM3 might play important roles in CNS pathophysiology after SCI.