Experimental Study In The Correlation Among Expression Changes Of Mitogen-and Stress-activated Protein Kinase And Traumatic Brain Injury

BackgroundTraumatic brain injury (TBI) is still the major problems affecting human s health. The research about pathogenesis of TBI is always a hot spot in the field of neurosurgery. In recent years, as the development of molecular biology, neurological surgery, nerve imaging and nerve monitoring system’s, the research on craniocerebral injury improved more deeply.Now most people think that the traumatic brain injury only cause impairment partly at first, but after several hours to several days there will be secondary damage caused by excess extracellular glutamate, derangements in intracellular calcium, and caspases.The primary insult to the CNS that initiates a major pathological process known as reactive gliosis which was called secondary injury produces a significant inflammatory reaction and a complex cellular response ensues. The typical features of neuro-trauma include neuronal death, microglial activation, and reactive astrogliosis, and eventually, a dense astrocytic scar formed. All these alterations may serve to impair recovery and meanwhile influence clinical outcome in TBI patients by impeding proper regeneration and neuronal growth. To-date, few pharmacological or treatment options are available to reduce these TBI-induced disabilities. True advances in clinical treatment depend on further understanding of the underlying molecular and cellular mechanisms of TBI.Mitogen-and stress-activated protein kinases (MSKs) land2are nuclear serine/threonine protein kinases that are activated in vivo downstream of either the ERK1/2or p38mitogen-activated protein kinase (MAPK) cascades. MSKs contain two kinase domains:an N-terminal kinase domain related to the AGC kinase family and a C-terminal kinase domain related to the CaMK family. The upstream MAPK phosphorylates the C-terminal domain, which then phosphorylates and activates the N-erminal domain. Once activated, the N-terminal domain phosphorylates substrates. MSKs do not have a precisely defined substrate consensus sequence; however, they do have a preference for a basic cluster prior to the phosphorylated residue. In cells, MSKs phosphorylate several substrates including cAMP response element binding protein (CREB),nuclear factor kappa B (NFcB), HMGN1, and histone H3. The major role of MSKs appears to be in the regulation of immediate early (IE) genes, and consistent with this, the transcription of several CRE-dependent IE genes is compromised in MSK knockouts. The physiological roles of MSKs still remain to be completely determined; however, recent work has suggested a role for MSKs in neuronal synaptic plasticity and in regulating cytokine production in the innate immune system. The number of MSKs targets keeps on growing. In addition to factors regulating gene transcription, MSKs have also been suggested to regulate cell-death-associated proteins and translation initiation.Although MSK1is known to be expressed at relatively high levels in the nervous system, its function has not been well understood. Our research is conducted to gain a better insight into the physiologic functions of MSK1in the normal and traumatized brain and its association with the cellular and molecular mechanisms underlying nervous system lesion and repair.Objective:To investigate the effects of mitogen-and stress-activated protein kinase (MSK1) in rats suffered with a improved traumatic brain injury, and further study the role and mechanism of MSK1after rat traumatic brain injury at the cellular level.Methods:Healthy male Sprague-Dawley rats were randomly divided into3groups: operation group, sham operation group, and control group. In operation group, We use homemade instrument to simulate the weight-drop TBI models. A hammer fall freely and hit the right parietal lobe of rats directly, resulting in mild-to-moderate cerebral contusion.The procedure to establish the sham operation group was similar to the operation group but without brian injury. The expression of MSK1, caspase-3, PCNA in rat brain were measured by immunoblotting. The localization of MSK1in rat brain was measured by immunofluorescence assay. In model of neuronal apoptosis and astrocyte proliferation response, through over-expressing MSK and transfection of siRNA against MSK, the effects of MSK1in cell apoptosis and proliferation were respectively studied by using the CCK-8method and immunoblotting.Results:1, Homemade improved ability to simulate brain injury model of physiological and pathological changes after traumatic brain injury.Homemade improved combat equipment to simulate gravity accelerated damage model to combat weight free fall, directly impacting a hard film, rat brain damage on the right side.Injury in rat brain and cell levels changed significantly after injury.2.The expression profiles of MSK1after traumatic brain injury in the operation group. Western blot and immunohistochemistry were performed to investigate MSK1expression after TBI. In the cortex surrounding the wound, the MSK1protein level was relatively higher in normal cortex, then progressively decreased from12h after TBI, peaked at day3(P<0.01), and then gradually increased to normal level. Besides, we also detected the expression of MSK1in the contralateral, unoperated cortex. MSK1protein level had no alterations, regardless of injury. In order to identify the changes of MSK1immunoreactivity in the brain cortex3days after traumatic brain injury, we then performed immunohistochemistry with anti-MSK1mouse monoclonal antibody on verse cryosections of brain tissues,1mm distal to the lesion site. Notably, MSK1staining was extensively low expressed in the ipsilateral brain at3days (120/mm2to30/mm2). But in the contralateral brain, there were high levels of MSK1staining. In addition, MSK1expression was evaluated by cell counting between contralateral and ipsilateral brains; the quantitative changes were consistent with the Western blot results.3, The colocalization of MSK1with different cellular markers in the adult rat brain cortex after TBI. To determine which kind of cell types may be associated with the peak expression of MSK1after injury, we used double immunofluorescent microscopy studies in transverse cryosections of brain tissues within1-mm distance from the lesion site by co-labeling with different cellular markers.MSK1was expressed in neurons and astrocytes.4. Detection of cellular proliferation in the adult rat brain cortex after TBI. To certify the relationship between MSK1and proliferative cells, we examined the expression changes of PCNA, which has been used as a general marker of dividing cells. Its expression was markedly increased at day3post-injury and remained until day14. To our surprise, the expression of PCNA had a negative correlation with MSK1. To identify the proliferative cell types present after brain injury, we performed double immunofluorescence staining with PCNA-specific antibody and GFAP staining (an astrocyte marker). At day3post-injury, most reactive astrocytes were PCNA positive. In addition, cell proliferation evaluated by PCNA appeared in many MSK1-express-ing cells at day3after injury.5, Detection of the neuronal apoptosis after TBI. The apoptosis of neurons with MSK1in CNS injury remains unclear. Therefore, we examined the expression of active caspase3by Western blot, which was known as a marker of apoptosis. To our surprise, the expression of active caspase3also had a negative correlation with MSK1. We found that the protein level of active caspase3increased significantly from day3to day7after injury. To examine the distribution and co-localization of MSK1and activated caspase-3in the injured brain at3days post-injury, we next performed double-labeling immunofluorescent staining of active caspase3and NeuN in the injured brains. In addition, the co-localization of activated caspase3and MSK1was observed at3days after TBI.6, Analyses of hippocampus neuron vitality in newborn rats. Detece the vitality of hippocampus neuron cell of newborn rats using the CCK-8method at6h,12h,18,24h after glutamate injury, and result show that the decrease of the vitality depend on the action time.7. The expression profiles of MSK1in hippocampus neuron cell after glutamate injury. Western blot was performed to investigate MSK1expression after the hippocampus neuron cells of newborn rats were treated with glutamate. the MSK1protein level was decreased while in the normal brain, there were high levels of MSK1staining.8. Detection the relationship of alteration between the express of MSK1and the the vitality of hippocampus neuron cells after glutamate injury. To certify the relationship between MSK1and the vitality of hippocampus neuron cell in response to glutamate, we build the small interfering RNA (siRNA) plasmid and the expression plasmid (EGFP-N2-MSK), which can interfere or over-expresse the expression of MSK1. Apoptosis decreased, when MSK1was over-expressed; while apoptosis increased, when the expression of MSK was interfered. These results suggest that MSK1may play a regulatory role in impairment and restoration of hippocampal neuron, and may has an anti-apoptosis effect on neurons.9. The astrocyte proliferation in response to LPS. The expression of PCNA and Ki67were significantly increased after astrocytes were treated with LPS, which depended on the concentration of LPS and action time10. The expression changes of MSK1affect the proliferation induced by LPS. We also use the small interfering RNA (siRNA) plasmid and the expression plasmid (EGFP-N2-MSK1), which can interfere or over-expresse the expression of MSK1in astrocyte. The result suggest that proliferation may decreased, when MSK1was over-expressed; while proliferation increased, when the expression of MSK1was interfered.Conclusions:1, In operation group, We use homemade instrument to simulate the weight-drop TBI models. The crackdown is controlled and accurate, the maneuverability and repeat-ability is good.2, The expression profiles of MSK1after traumatic brain injury in the operation group. The MSK1protein level was relatively higher in normal cortex, then progressively decreased from12h after TBI, peaked at day3, and then gradually increased to normal level.3, MSK1was expressed in neurons and astrocytes.4, The expression changes of caspase-3were in the opposite trend with respect to MSK1, which indicates that MSK1plays an important role in rats with traumatic brain injury.5, MSK has an anti-apoptosis effect on neurons.6, MSK1may inhibit proliferation of astrocytes.7, The molecular biology of rats after TBI associated with the biological activities of MSK1.