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The Mechanism of Electroacupuncture and NBD Peptide Regulating the Activation of NF-ΚB Signaling Pathway in Cortex and Hippocampus to Ameliorate the Inflammatory Injury after Focal Cerebral Ischemia/Reperfusion

Author: QinWenZuo
Tutor: LuoYong
School: Chongqing Medical University
Course: Neurology
Keywords: focal cerebral ischemia/reperfusion EA NBD peptide NF-κBsignaling pathway inflammatory injury
CLC: R743.31
Type: PhD thesis
Year: 2013
Downloads: 166
Quote: 0
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Abstract


Background: Ischemic cerebrovascular disease belongs to a kind ofnervous system diseases caused serious damage to human health, the focalcerebral ischemia/reperfusion is one of the main disease types. It exists aserious of brain tissue damage cell death process and complex pathologicalmechanism and so on. Meanwhile, the ischemia anoxic pathological damagewidely exists in the brain, not just confined to the ischemic lesions in thecortex and nearly area, the damage can affect other functional importantbrain structures, such as the hippocampus, nigra and so on. In the focalcerebral ischemia/reperfusion complex pathological mechanism,inflammation is an important part of that, it was not only involved in focalischemia in the pathological injury, at the same time, it was also participatedin the non-ischemic area of hippocampus. In the early stage of focal cerebral ischemia/reperfusion, the pathological mechanism is complicated, theinflammatory injury is important part which was induced by focal cerebralischemia/reperfusion. The inflammatory cascade amplification reaction willaggravate ischemic hypoxic damage, cause severe pathologcial damage tothe body. At the same time, a large number of experimental animal models ofcerebral ischemia/reperfusion also showed that the inflammatory cytokinesand inflammatory cells infiltration were all expressed in the cerebralischemia lesion area and focal approach area after focal cerebralischemia/reperfusion injury. Exacerbated the body tissues damage andenhanced the secondary injury by activation of the inflammatory responseafter focal cerebral ischemia/reperfusion is one of the main reasons.Therefore, to inhibit the overeaction of inflammation is considered to be theeffective and important way to relieve the damage of ischemic. In thecomplex web of cytokines in inflammatory response, the activation of thenuclear factor-κB signaling pathway is considered to be the key link and thecentre reaction, which was closely associated with the inflammatoryresponse, the many kinds of mediators of inflammation gene promoter andenhancer predominated the κB sequence. The activated NF-κB induced theexpression of inflammatory mediators gene alone or with othertranslocation factors. Due to the NF-κB signaling pathway can launchmultiple downstream inflammatory mediators, has the characteristic ofcascade amplification and is the center and key of the inflammatory response control. Meanwhle, the NF-κB signaling pathway itself is acomplex cell transcription signaling pathways plays a key role by multipleimportant protein interaction with activation and inhibiton, regulating thereaction equilibrium in physiological and pathological conditions. Therewere important inhibiting protein IκB family and predominate activatedprotein IKKs family working together to regulate the NF-κB signalingpathway activity. Therefore, to achieve effective inhibition of inflammatoryreaction after focal cerebral ischemia/reperfusion, suppressing theactivation of NF-κB pathway was considered to be the main target andcritical nodes for regulating the balance between inhibition of protein andactivation of protein. The electroacupuncture (EA) is as the moreconmmonly way of non-drug therapy among the variety of interventionsfor regualting the NF-κB signaling pathways, are being increasingly used inclinical treatment and basic research of cerebrovascular disease. And theanti-inflammatory effects of EA for the inflammation after focal cerebralischemia/reperfusion in a number of studies have also been confirmed. Butwhether or not the mechanism and anti-inflammatory effect of EA rely onintervening the NF-κB signaling pathway is still not more in-depth research.Meanwhile, as the NF-κB signaling pathway specific inhibitor peptide(nemo binding domain, NBD), which against the focal cerebralischemia/reperfusion also no further reports. Meanwhile, the hippocampusrepresents a very prominent structure in the rat brain, the focal ischemic lesions of the cortex and/or striatum which was induced by the temporarydeprivation of brain blood flow, always causes severe hippocampal regiondamage, secondary changes have been observed in the hippocampus,especially in the CA1region, results in the insidious degeneration of specificvulnerable neurons such as pyramidal cells in the hippocampus. Focalcerebral ischemia lesions could lead to the secondary remote damage in thehippocampus and the inflammatory changes in remote areas might beinvolved in the pathogenesis of secondary neuronal damage. Therefore, toinhibit the inflammatory reation after focal cerebral ischemia/reperfusioneffectively is the important access for relieving the remote damage in thehippocampus CA1region, even for the remote injury in the other regions.During the inflammation which was induced by focal cerebralischemia/reperfusion, the NF-κB signaling pathway is the centre and keystep, it is meaningful to restrain the activation of NF-κB to reduce theinflammatory injury after cerebral ischemia/reperfusion.Objective: Our purpose of the present study is to figure out themechanism of EA and NBD peptide regulating the activation of theimportant protein in the NF-κB signaling pathway to explore the effecttargets and mechanism of EA and NBD peptide inhibiting the inflammatoryinjury after focal cerebral ischemia/reperfusion. We will compare with themechansim of EA and NBD peptide for the inhibition of NF-κB signaling pathway, in order to explore the mechanism of activiation and inhbitionbalance in the NF-κB signaling pathway, to make clear the effect targets andmechanism of EA for the focal cerebral ischemia/reperfusion. Furthermore,we used the NBD peptide (NF-кB inhibitor) to inhibit the activation ofNF-кB signaling pathway, and the NF-κB p65and IκBα were considered tobe the key proteins in the NF-κB signaling pathway to explore its mechanismand function for restraining inflammation in hippocampus CA1region afterfocal cerebral ischemia/reperfusion at24h and7d.Methods:1. We adopts the middle cerebral artery occlusion andreperfusion models, the355SD male rats were randomly divided into shamgroup (n=70), I/R group (n=95), EA group (n=95) and NBD group (n=95).And they were further divided respectively into4subgroups according to thedifferent duration of reperfusion as6,12,24and48h, there were5rats in eachgroup of each time point. We used the TTC staining to evaluate the infarctvolume at24h after reperfusion; HE and FJB staining to observe the neuroncells damage change after ischemic stimulation24h, EA and NBD peptidetreatment which were located in the ischemic region. ELISA detected thelevel of cytokines (IL-1β/IL-13) in the ischemic brain tissue and peripheralblood serum. The location of NF-κB p65protein expression in the cytoplasmand nucleus was detected by immunohistochemistry at24h after reperfusion.The change of NF-κB p65/IκBα feedback loop protein expression was testedin the cytoplasm and nucleus by immunofluorescence and Western blot. The expression of IKKα and IKKβ protein and mRNA were tested byimmunofluorescence, Western blot and Q-PCR. The ability of NF-κBbinding DNA was tested by EMSA.2. The138male Sprague-Dawley (SD) rats were randomly divided intoa sham group, an I/R (ischemia/reperfusion) group, a NBD group and aMT-NBD (modified type) group. The H&E and Flour-Jade B staining wereperformed in the hippocampus CA1region to observe the degenerativeinjury and loss of the neuron cells. The level of IL-1β and IL-1Ra was testedby ELISA analysis to explicit the inflammatory reaction in the hippocampusCA1region. The protein of NF-κB p65and IκBα were analyzed byimmunofluorescence and Western blot to detect the nuclear translocationprocess of NF-κB in the nucleus.Results:1. The infarct volume was about40%in the right brain tissueafter I/R damage, which was significantly reduced in EA group and NBDgroup (P<0.05)(about30%in EA group and22%in NBD group). The HEand FJB staining showed that neuron cells damage was remarkable in thefocal ischemia area after focal cerebral ischemia/reperfusion, and it wasalleviated in the EA group and NBD group, especially in the NBD group(P<0.05). The level of IL-1β in the brain and serum was reduced by EA andNBD peptide treatment which was high in I/R group (P<0.05). The level ofIL-13was increased among the three groups, but it was higher in the EAgroup and NBD group than that in I/R group, meanwhile brought forward the peak time points from48h to12h and24h (P<0.05). The NF-κB p65proteinexpressed mainly in the cytoplasm rather than in the nucleus in EA groupand NBD group (P<0.05), which expressed both in the cytoplasm andnucleus in I/R group (P<0.05). The IκBα protein expression was increasedboth in the cytoplasm and nucleus in EA group and NBD group than that inI/R group, which could maintain the NF-κB p65in the cytoplasm to inhibitthe nuclear translocation (P<0.05). The EA stimulation decreased theexpression of IKKα and IKKβ protein and mRNA, which increased afterreperfusion, especially at24h and48h (P<0.05). Meanwhile, the NBDpeptide restrained the expression of IKKβ protein and mRNA significantly(P<0.05), but there were no obvious inhibitory effect for IKKα. The abilityof NF-κB binding DNA was decreased in EA group and NBD group whichwas high in I/R group (P<0.05), especially it was decreased significantly inthe EA group (P<0.05).2. Compared with the sham group, the degenerative injury of neuroncells in the hippocampus CA1region was significantly increased (P<0.05),meanwhile the loss of neuron cells was also increased (P<0.05), and the levelof IL-1β was increased in the hippocampus in the I/R group and MT-NBDgroup (P<0.05). On the contrary, the degenerative injury and loss of neuroncells in the hippocampus CA1region were significantly relieved after NBDpeptide treatment (P<0.05). At the same time, the level of IL-1β wasdecreased and the level of IL-1Ra was increased obviously than that in I/R group and MT-NBD group (P<0.05). The results showed that theinflammation was activated and the remote damage was involved in thehippocapmus after focal cerebral ischemia/reperfusion. After the NBDpeptide treatment, the inflammatory injury and remote damage wererelieved. Furthermore, compared with the sham group, the protein ofNF-κB p65expression was increased significantly in the nucleus in I/Rgroup and MT-NBD group(P<0.05), and the protein of IκBα expression wasnot remarkable (P>0.05). Compared with the I/R group and MT-NBD group,the protein expression of p65was obviously decreased (P<0.05) and theIκBα was significantly increased in the nucleus in NBD group (P<0.05). Theresults demonstrated that the NBD peptide could regulate the proteinexpression of p65and IκBα in the nucleus to inhibit the nuclear translocationof NF-κB.Conclusion:1. The EA and NBD peptide could reduce the infarctvolume, ameliorate the damage of neuron cells in the ischemic region anddown-regulate the level of proinflammatory cytokines in ischemic brain areaand peripheral blood serum. The EA and NBD peptide both could regulatethe activation of NF-κB signaling pathway: they both increase the IκBαexpression in the NF-κB/IκBα feedback loop at the early stage of focalcerebral ischemia/reperfusion to remain the nuclear translocation of NF-κB.Moreover, the EA highly possible inhibited the expression of IKKα andIKKβ to restrain the phosphorylation of the IκBα to stop the nuclear translocation and inhibit the activation of NF-κB in order to regulate theactivation of NF-κB signaling pathway to restrain the inflammatory injuryafter focal cerebral ischemia/reperfusion. The mechanism of NBD peptidewas different from the mechanism of EA, NBD peptide could significantlyinhibit the expression of IKKβ achieves the goal of inhibiting the NF-κBactivation, and its ability of inhibiting the NF-κB activity is weaker than EA.Therefore, the mechanisms and effect targets of EA regulating the NF-κBsignaling pathway are various which is different from NBD peptide2. We conclude that NF-κB nuclear translocation process and theNF-κB signaling pathway was activated in the hippocampus CA1regionafter focal cerebral ischemia/reperfusion, therefore,the inflammatory injurywas irritated and enhanced the remote damage for the hippocampus afterfocal cerebral ischemia/reperfusion. The NBD peptide treatment contributesto alter the NF-κB p65/IκBα expression in nucleus effectively to regulate thenuclear translocation to stop the activation of NF-κB signaling pathway andinhibit the cascade amplification of inflammation, then relieve effectivelythe remote damage in the hippocampus CA1region after focal cerebralischemia/reperfuson.

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CLC: > Medicine, health > Neurology and psychiatry > Neurology > Cerebrovascular disease > Acute cerebrovascular disease ( stroke) > Transient ischemic
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