【作者】 白雪；

【导师】 张祥建；

【作者基本信息】 河北医科大学 ， 神经病学， 2013， 硕士

【摘要】 目的：脑血管病被认为是危害人类健康的三大疾病之一，尤其是缺血性脑血管病以其较高的发病率、致残率、死亡率、复发率给人类生命造成了严重威胁。脑缺血后继发性脑损伤是病情加重及影响预后的重要原因，包括炎症反应、氧化应激、钙超载、谷氨酸的兴奋毒性、细胞凋亡及线粒体功能异常等因素。其中炎症反应在脑血管病中的作用机制日益得到重视，如何减轻梗死后炎症反应成为治疗的重要方法。近年来，随着对免疫炎症研究的深入， nucleotide oligomerization domain-like receptors2(NOD2)/receptor-interacting protein2(RIP2)诱导的nuclear factor kappaB(NF-κB)信号通路被证实参与脑缺血后炎症反应，而下调该通路可以减轻缺血后的炎症反应，发挥脑保护作用。大量的药理和临床研究发现柚皮素（Naringenin，NG）具有抗炎、抗氧化、抗凋亡、抗病毒、抗肿瘤等多种药理作用。由于柚皮素是脂溶性的，容易透过血脑屏障，已有实验研究证明柚皮素可以对帕金森病，阿尔茨海默氏病等中枢神经系统疾病发挥脑保护作用。但有关柚皮素在脑梗死急性期的保护作用及机制的研究甚少。本实验在大鼠永久性大脑中动脉闭塞（Permanent Middle Cerebral ArteryOcclusion，pMCAO）所致脑缺血模型上观察局部脑缺血后柚皮素的神经保护作用及其可能的机制。方法：采用健康成年雄性Sprague-Dawley大鼠，应用改良的Longa线栓法建立大鼠右侧pMCAO模型。120只实验动物随机分为假手术组（Sham）、单梗组（pMCAO）、对照组（Vehicle）、柚皮素小剂量组（naringenin50mg/kg per day，NG-L）、柚皮素大剂量组（naringenin100mg/kg per day，NG-H）。术后24h对各组大鼠进行神经功能评分。评分完毕后将动物断头处死，用干湿重法检测脑组织含水量，用2%2，3，5-三苯基四唑氮红（Triphenyltetrazolium chloride，TTC）染色法测量脑梗死体积，用免疫组织化学（Immunohistochemistry）、蛋白印记（Western blot）和实时荧光定量逆转录多聚酶链反应（Real-time reverse transcription-quantitative PCR，RT-qPCR）测定缺血大脑皮层NOD2、RIP2、NF-κB、MMP-9和claudin-5的表达。结果：1Sham组无神经功能缺损，而pMCAO、Vehicle组、柚皮素小剂量组和柚皮素大剂量组大鼠均出现不同程度的左侧肢体偏瘫。柚皮素大剂量组的神经功能评分在24h低于pMCAO组和Vehicle组，差异有统计学意义（pMCAO group vs. NG-H group:3.38±0.16vs.2.67±0.20, P <0.05;Vehicle group vs. NG-H group:3.42±0.13vs.2.67±0.20, P <0.05）。柚皮素小剂量组与pMCAO组和Vehicle组相比，神经功能评分有所改善，但差异无统计学意义（pMCAO group vs. NG-L group:3.38±0.16±vs.3.04±0.13, P>0.05; Vehicle group vs. NG-H group:3.42±0.13vs.3.04±0.13, P>0.05）。2柚皮素大剂量组病变侧脑组织含水量在24h时低于pMCAO组和Vehicle组，差异有统计学意义（pMCAO group vs. NG-H group:85.46±0.40%vs.83.17±0.55%, P <0.05; Vehicle group vs. NG-H group:85.15±0.32%vs.83.17±0.55%, P <0.05）。柚皮素小剂量组与pMCAO组和Vehicle组相比，脑组织含水量有所改善，但差异无统计学意义（pMCAOgroup vs. NG-L group:85.46±0.40%vs.84.36±0.38%, P>0.05; Vehiclegroup vs. NG-H group:85.15±0.32%vs.84.36±0.38%, P>0.05）。3与pMCAO组和Vehicle组相比，柚皮素大剂量组梗塞体积在24h明显减小，差异有统计学意义（pMCAO group vs. NG-H group:44.35±2.23vs.34.44±1.65, P <0.05; Vehicle group vs. NG-H group:44.42±1.53vs.34.44±1.65, P <0.05）。柚皮素小剂量组与pMCAO组和Vehicle组相比，梗塞体积有所减小，但差异无统计学意义（pMCAO group vs. NG-L group:44.35±2.23vs.38.99±2.54, P>0.05; Vehicle group vs. NG-H group:44.42±1.53vs.38.99±2.54, P>0.05）。4在脑梗塞后24h，柚皮素大剂量组NOD2、RIP2、NF-κB和MMP-9表达均降低，而claudin-5的表达增加（P <0.05）。柚皮素小剂量组也可以下调NOD2、RIP2、NF-κB和MMP-9的表达且上调claudin-5的表达，但差异无统计学意义（P>0.05）。结论：脑缺血后脑组织中NOD2、RIP2、NF-κB和MMP-9的表达显著升高，claudin-5的表达显著降低。大剂量柚皮素可以改善神经症状，减轻脑梗死体积，减轻脑水肿，对局灶性脑缺血具有神经保护的作用。其保护作用可能与下调NOD2、RIP2、NF-κB和MMP-9的表达，上调claudin-5的表达有关。更多还原

【Abstract】 Objective: Cerebrovascular disease is considered to be one of the threeleading causes of death in the world, especially ischemic cerebrovasculardisease, with high rate of morbidity, disability, mortality and recurrence,posing a serious threat to human life. Brain tissue injuries secondary toischemia, which include inflammation, oxidative stress, calcium overload,glutamate excitotoxicity, apoptosis and mitochondrial dysfunction, aggravatethe illness and inhibit the recovery of patients. Thereinto, the role ofinflammatory response in cerebrovascular disease is gaining increasingattention, how to reduce the post-infarcted inflammatory response becomes animportant therapy. In recent years, with further study on immune inflammation,the nucleotide oligomerization domain-like receptors2(NOD2)/receptor-interacting protein2(RIP2) induced nuclear factor kappa B(NF-κB) activation pathway is supposed to participate in the inflammatoryresponse secondary to cerebral ischemia, while down-regulating this pathwaycould reduce the inflammatory response after ischemia and offerneuroprotection. Ample pharmacological and clinical studies have shownnaringenin possesses a vanity of pharmacological effects, such asanti-inflammatory, anti-oxidative, anti-apoptotic, anti-viral, anti-tumor and soon. As naringenin is liposoluble, it exhibits high permeability across theblood-brain barrier, the neuroprotective effects of naringenin on the centralnervous system have been well documented in Parkinson’s disease andAlzheimer’s disease. However, there is still a paucity of data regarding theeffect of naringenin in the acute phase of ischemic stroke. The purpose of thisstudy is to evaluate the potential neuroprotective effect of naringenin andunderlying mechanisms after cerebral ischemia induced in male adultSprague-Dawly rats by permanent middle cerebral artery occlusion (pMCAO). Methods: Healthy, male Sprague-Dawley rats were subjected tomodified permanent MCAO as described by Longa previously.120rats wererandomly assigned into Sham operated group (Sham), pMCAO group(pMCAO), pMCAO with vehicle group (Vehicle), pMCAO with low-dose (50mg/kg per day) naringenin group (NG-L) and pMCAO with high-dose (100mg/kg per day) naringenin group (NG-H). At24h after ischemia, neurologicaldeficit was evaluated, brain water content was measured by wet-dry method,infarct size were analyzed with2,3,5-triphenyltetrazolium chloride (TTC)staining; immunohistochemistry, western blot and real-time reversetranscription-quantitative PCR (RT-qPCR) were used to detect the variation ofNOD2, RIP2, NF-κB, MMP-9and claudin-5in ischemic cerebral cortex.Results:1Rats in Sham group had a neurological score of zero at all time points.Rats in pMCAO group, Vehicle group, low dose group and high dose groupperformed a right palsy. Neurological deficit score in high dose group wassignificantly decreased compared with pMCAO and Vehicle groups (pMCAOgroup vs. NG-H group:3.38±0.16vs.2.67±0.20, P <0.05; Vehicle group vs.NG-H group:3.42±0.13vs.2.67±0.20, P <0.05). There was a decrease inthe neurological deficit score following low dose NG administration, but therewas no significant difference in low dose group compared with pMCAO andVehicle groups (pMCAO group vs. NG-L group:3.38±0.16±vs.3.04±0.13,P>0.05; Vehicle group vs. NG-H group:3.42±0.13vs.3.04±0.13, P>0.05).2The brain water content in high dose group was significantly decreasedcompared with that in pMCAO and Vehicle groups at24h (pMCAO group vs.NG-H group:85.46±0.40%vs.83.17±0.55%, P <0.05; Vehicle groupvs. NG-H group:85.15±0.32%vs.83.17±0.55%, P <0.05). There was adecrease in the brain water content following low dose NG administration, butthere was no significant effect in NG-L group compared with pMCAO andVehicle groups at24h (pMCAO group vs. NG-L group:85.46±0.40%vs.84.36±0.38%, P>0.05; Vehicle group vs. NG-H group:85.15±0.32%vs. 84.36±0.38%, P>0.05).3Infarct size was significantly decreased in NG-H group compared withthat in pMCAO group and Vehicle group at24h (pMCAO group vs. NG-Hgroup:44.35±2.23vs.34.44±1.65, P <0.05; Vehicle group vs. NG-H group:44.42±1.53vs.34.44±1.65, P <0.05). There was a decrease in the infarctsize following low dose NG administration, but no statistical significance inNG-L group compared with pMCAO and Vehicle groups at24h (pMCAOgroup vs. NG-L group:44.35±2.23vs.38.99±2.54, P>0.05; Vehicle groupvs. NG-H group:44.42±1.53vs.38.99±2.54, P>0.05).4High dose of naringenin significantly down-regulated the expression ofNOD2, RIP2, NF-κB and MMP-9, up-regulated the expression of claudin-5at24h after ischemia (P <0.05). Low dose of naringenin can alsodown-regulated the expression of NOD2, RIP2, NF-κ B and MMP-9,up-regulated the expression of claudin-5, but the trend is unstable (P>0.05).Conclusions: This work shows that the expression of NOD2, RIP2, NF-κB and MMP-9is significantly up-regulated and the expression of claudin-5has a markedly shrinkage after ischemia. Systemic administration ofnaringenin is effective which can ameliorate the neurological deficit, decreasethe infarct size and improve the brain edema caused by pMCAO. These effectsmay be through down-regulation of NOD2, RIP2, NF-κB and MMP-9andup-regulation claudin-5expression.更多还原