【作者】 秦文熠；

【导师】 罗勇；

【作者基本信息】 重庆医科大学 ， 神经病学， 2013， 博士

【摘要】 背景：缺血性脑血管病属一类严重危害人类健康和严重影响生活质量的神经系统危、急、重症。其中，局灶脑缺血/再灌注是主要的发病类型，具有“高发病率、高致残率及高死亡率”的特点。局灶脑缺血/再灌注后会产生一系列复杂的病理变化过程，其缺血缺氧损害影响脑内较广的范围，不仅仅局限于缺血病灶局部及灶周组织，其远隔损害也可破坏脑内其他重要的功能结构，如海马、黑质等。研究证实，大脑重要组织结构中以大脑皮质及海马结构是对缺血缺氧损害较为敏感的区域。海马的CA1区是其重要的功能结构，是对短暂性脑缺血最为敏感的区域。对于大脑中动脉梗塞/再灌注引起的病理损害，非缺血缺氧局部的海马仍受其损伤而引起相应的病理变化即为脑缺血/再灌注后产生的远隔损害。而在远隔损害的病理机制中，炎症反应起着非常重要的作用，即局灶脑缺血/再灌注后诱发的炎症反应，对非缺血病灶内的海马同样产生严重的病理损害，是对海马区域的远隔炎性效应。在局灶脑缺血/再灌注发生的起始阶段，各种复杂的病理过程共同作用于机体，导致脑功能受到严重的损伤。在其复杂的病理损伤机制中炎症反应是极其重要的组成部分，不仅参与局部缺血灶内的病理损伤，同时也会引起非缺血区内海马部位的远隔损伤效应。在大量实验性脑缺血/再灌注动物模型中发现，脑缺血缺氧损伤后缺血病灶局部及灶周等均有炎性细胞因子的表达和炎性细胞的浸润，说明脑内炎症反应被激活，加重机体组织的损伤，促进继发性脑损害发生，成为局灶脑缺血/再灌注损伤的主要过程之一。核因子-κB（Nuclear factor-κB,NF-κB)信号通路为炎症反应病理环节中主要的组成部分，属于调节炎症反应的关键节点，且活化的NF-κB可单独或协同其他与炎症相关的转录因子共同作用参与多种炎症介质基因的诱导表达。由于NF-κB信号通路能启动下游多个炎症介质，具有级联放大的特点，因此NF-κB信号通路应视为炎症反应调控的中心和关键起始步骤。同时，NF-κB信号通路本身就是一个复杂的细胞转导通路，由多个起关键作用的重要蛋白相互作用、相互协同激活或抑制NF-κB信号通路，调节其在生理病理状态下的反应平衡。在NF-κB信号通路中，由发挥最重要且最基本作用的抑制蛋白IκB家族及激活蛋白IKKs家族共同调节NF-κB信号通路的活性。因此，以抑制NF-κB信号通路过度激活为主要靶向，调节抑制蛋白与激活蛋白之间的平衡为有效抑制局灶脑缺血/再灌注炎症反应的损害的关键节点。在调节NF-κB信号通路的多种干预手段中，电针是目前较为常用的“非药物”治疗手段，正被越来越多地运用于脑血管病的临床治疗与基础研究中。同时，电针在局灶脑缺血/再灌注后的抗炎作用也在大量研究中得到证实。但电针的抗炎作用机制中是否通过干预NF-κB信号通路而发挥作用仍未有较深入的研究。同时，作为NF-κB信号通路的特异性抑制多肽（nemo binding domain, NBD）NBD多肽，针对局灶脑缺血/再灌注后的抗炎干预作用的研究也未有更深入的报道。目的：观察局灶脑缺血/再灌注后炎症反应的激活变化及探讨电针与NF-κB特异性抑制剂NBD多肽对于NF-κB信号通路中关键蛋白活性的调节作用及其主要的作用靶点，以探索电针及NBD多肽调节信号通路激活作用的可能机制；将电针干预NF-κB信号通路激活的作用机制与NBD多肽的作用机制作对照，以深入探讨电针调节NF-κB信号通路的激活或抑制的平衡反应，从而得出电针抑制局灶脑缺血/再灌注后炎症损伤可能的作用靶点和作用机制。另外，以右侧海马CA1区为研究区域，以NBD多肽为干预手段，取再灌注后24h及7d为观察时相点，以NF-κB信号通路中关键蛋白NF-κB p65及IκBα作为研究对象，研究探讨局灶脑缺血/再灌注后给予该药抑制海马CA1区炎症反应进而减轻远隔损害的神经保护作用及其可能的作用机制。方法：1.运用大鼠右侧大脑中动脉闭塞/再通模型（middle cerebralartery occlusion/reperfusion, MCAO/R），将355只SD雄性健康清洁级大鼠随机分为假手术组（n=70）、模型组（n=95）、电针组（n=95）及NBD药物组（n=95），每组将按照再灌注后6h、12h、24h及48h四个时相点分为4个亚组，每组每个时间点5只大鼠。利用电针刺激模型大鼠的“百会”穴（GV20）及“四关”穴（“合谷”LI4/“太冲”LR3）进行电针干预；同时利用侧脑室注射NBD多肽进行药物干预。运用TTC染色观察局灶脑缺血/再灌注后24h时脑梗死体积的变化；HE及FJB染色观察局灶脑缺血/再灌注后24h脑缺血再灌注、电针及NBD多肽干预后病灶内神经细胞变性损伤及丢失情况；ELISA检测缺血病灶区内脑组织及外周血血清中6h、12h、24h及48h四个时间点细胞因子（IL-1β/IL-13）的含量变化；免疫组化定位观察再灌注后24h时间点NF-κB p65蛋白胞浆/胞核的表达情况；免疫荧光检测再灌注后24h时间点缺血灶内NF-κB p65/IκBα胞浆/胞核蛋白的表达及IKKα及IKKβ的蛋白变化；Western blot检测各组NF-κB p65/IκBα胞浆或胞核蛋白的表达变化；Western blot、Q-PCR检测各组内IKKα及IKKβ的蛋白及mRNA表达变化；EMSA实验检测各组内NF-κB与DNA结合能力的变化。2.将138只雄性健康清洁级SD大鼠随机分为假手术组（n=24）、模型组（局灶脑缺血/再灌注组，n=38）、NBD多肽药物组（局灶脑缺血/再灌注+NBD多肽, n=38）及NBD对照药物组（局灶脑缺血/再灌注+NBD多肽转化型, n=38）。运用苏木素-伊红染色及FJB染色观测局灶脑缺血/再灌注及NBD多肽干预后大鼠海马CA1区内神经细胞变性损伤、丢失情况；运用ELISA检测海马区组织内IL-1β及IL-1Ra的含量变化；免疫荧光双标、免疫蛋白印迹检测NF-κB p65及IκBα蛋白在胞核表达的变化情况探讨各组别海马CA1区内NF-κB的核转位活化过程。结果：1. TTC染色显示局灶脑缺血/再灌注24h后模型组脑梗死体积约为40%，电针组及NBD组脑梗死体积明显减小（P<0.05）（电针组约为30%、NBD组约为22%）。HE及FJB染色显示，局灶脑缺血/再灌注24h后模型组内神经细胞受损及丢失明显；电针组及NBD组缺血区内神经细胞受损及丢失情况明显减轻（P<0.05）；与模型组、电针组比较，NBD组减轻效应更显著（P<0.05）。ELISA检测显示，与假手术组比较，模型组中促炎因子IL-1β含量在缺血脑组织及外周血血清中明显增高，电针组及NBD组IL-1β含量水平明显低于模型组（P<0.05），NBD组比电针组降低更明显（P<0.05）；抑炎因子IL-13含量在模型组、电针组及NBD组的脑组织及外周血血清中均升高，但比模型组比较，电针组、NBD组升高更明显（P<0.05），同时模型组中IL-13含量高峰时间点为48h，电针组将IL-13含量高峰时间点提前至24h及NBD组内的提前至12h。免疫组化显示模型组NF-κB p65在胞浆及胞核均表达，特别在胞核内高表达（P<0.05），电针组及NBD组结果与模型组比较，以胞浆表达为主，胞核表达明显减少（P<0.05），电针组与NBD组结果比较无明显差别（P>0.05）。免疫荧光、Western blot检测显示，与假手术组比较，模型组胞核内NF-κB p65蛋白高表达（P<0.05），而IκBα蛋白呈低表达（P>0.05）；电针组及NBD组与模型组比较，均能明显增加胞浆及胞核内IκBα的表达、降低NF-κB p65在胞核内的高表达（P<0.05），抑制NF-κB的核转位。在IKKα、IKKβ指标的观测中，与假手术组比较，模型组内IKKα、IKKβ蛋白及mRNA表达明显增高，特别是再灌注后24h及48h，IKKβ蛋白及mRNA升高明显（P<0.05）；与模型组比较，电针组IKKα、IKKβ蛋白及mRNA表达显著下降（P<0.05），而NBD组与模型组及电针组比较，IKKβ蛋白及mRNA表达下降最明显（P<0.05），但对IKKα无明显抑制作用（P>0.05）。在NF-κB与DNA结合能力变化的检测中，与假手术组比较，模型组内NF-κB与DNA结合能力明显升高（P<0.05），电针组及NBD组内NF-κB与DNA结合能力与模型组比较明显下降，其中电针组与NBD组比较，电针组下降更显着（P<0.05）。显示电针及NBD多肽对于NF-κB信号通路中其关键抑制蛋白及关键激活蛋白均有调节作用。2.海马CA1区检测结果示：HE及FJB染色显示，与假手术组比较，局灶脑缺血/再灌注后模型组24h及7d海马CA1区出现明显的神经细胞变性坏死损伤（P<0.05），神经细胞丢失明显（P<0.05），MT-NBD药物对照组显示相同结果（P<0.05），模型组内24h与7d之间比较神经细胞受损数目无显著差异（P>0.05），MT-NBD组结果与模型组结果比较无差异（P>0.05）；同时，与假手术组比价，模型组及MT-NBD组内1L-1β及IL-1Ra含量显著增高，特别是再灌注后24h时间点最显著（P<0.05）。与模型组及MT-NBD组比较，NBD组内24h及7d变性受损及丢失的神经细胞数目明显减少（P<0.05），特别是再灌注后7d改善最显著（P<0.05）；同时，NBD组内IL-1β含量显著降低（P<0.05），IL-1Ra含量明显增高（P<0.05）。免疫荧光及western blot检测结果显示，与假手术组比较，模型组及MT-NBD组于再灌注后24h及7d海马组织CA1区细胞核内p65蛋白表达显著增加（P<0.05），而IκBα呈低表达（P>0.05）；NBD组与模型组及MT-NBD组比较，胞核内p65蛋白表达显著降低（P<0.05），而IκBα蛋白在胞核内大量表达（P<0.05），表明NBD多肽能有效调节NF-κB p65及IκBα在胞核的表达，从而有效干预NF-κB的核转位过程。结果表明局灶脑缺血/再灌注后，海马CA1区内神经细胞受损明显，炎症反应被激活；NBD多肽干预后促炎因子水平下降，神经细胞受损减轻，证实海马区内炎症反应被抑制，能有效缓解脑缺血/再灌注对海马产生的远隔损害。结论：1.局灶脑缺血/再灌注后炎症反应被激活，局部缺血大脑皮质内神经细胞受损明显；电针刺激及NBD多肽干预均能抑制NF-κB信号通路的激活：二者均能通过增加NF-κB/IκBα反馈环路中IκBα的表达在局灶脑缺血/再灌注早期有效抑制NF-κB核转位过程；但二者对于IκB激酶的作用却不相同：电针极有可能是通过有效抑制IKKα、IKKβ的表达，阻止IκBα降解的程度，调节NF-κB/IκBα反馈环路的平衡而抑制NF-κB核转位，同时抑制NF-κB增高的活性而达到有效抑制NF-κB信号通路激活的目的；而NBD多肽能显著抑制IKKβ的表达调节NF-κB/IκBα反馈环路的平衡而抑制NF-κB信号通路的激活，对于IKKα作用却不显著，其抑制NF-κB活性的能力也弱于电针，因此，NBD多肽的作用靶点精确与电针多靶点的特点有所不同。综上，电针及NBD多肽通过有效调节NF-κB信号通路在病理状态下过度活化的平衡，达到减轻局灶脑缺血/再灌注后炎性反应损害的目的。2.局灶脑缺血/再灌注后，缺血缺氧远隔损害部位的大鼠海马CA1区内NF-κB核转位过程启动，NF-κB信号通路被活化，海马CA1区内炎症反应被激活，加剧了局灶脑缺血/再灌注产生的远隔损害对脑组织的破坏；NBD多肽干预能抑制NF-κB的核转位过程，阻止NF-κB信号通路的活化从而有效抑制炎症反应的级联放大，达到减轻局灶脑缺血/再灌注后海马CA1区内远隔损害的目的。更多还原

【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.更多还原