【作者】 周庆博；

【导师】 迟兆富；

【作者基本信息】 山东大学 ， 神经病学， 2011， 博士

【摘要】 脑出血(Intracerebral Hemmorhage, ICH)以高发病率、致残率和病死率严重危害人民的身心健康。ICH后损伤机制十分复杂,尚未完全阐明。已有研究普遍认为,脑出血后血肿凝固产生的凝血酶在继发性脑损伤中占有非常重要的地位,与出血后脑水肿、炎症反应、细胞凋亡及兴奋性氨基酸毒性等损伤因素关系密切。ICH一直缺乏循证医学确切有效的治疗手段,疗效至今不令人满意,寻找切实有效的神经保护药物一直是脑出血研究领域的重要方向。ICH后血肿产生的凝血酶通过作用于脑组织内蛋白酶激活受体-1(PAR-1)而发挥细胞毒性作用。我们既往的研究发现黄芩等中药对实验性大鼠脑出血后PAR-1的表达具有抑制作用。黄芩苷(baicalin)是从传统中药黄芩根部提取的主要活性成分,对脑缺血性神经损伤具有保护作用,其对脑出血后神经损伤的干预作用报道很少。目的：本研究以胶原酶Ⅶ诱导大鼠脑出血后,给予不同剂量的黄芩苷药物干预,观察脑出血后的脑组织病理生理动态变化的特点,观察黄芩苷对实验性大鼠脑出血后的神经功能缺损、脑水肿及神经元病理形态学变化的影响,观察黄芩苷对实验性大鼠脑出血后的凝血酶毒性、兴奋性氨基酸毒性、炎症因子、细胞凋亡和基质金属蛋白酶-9(MMP-9)等继发损伤因素的影响；探讨黄芩苷对脑出血后脑组织损伤的神经保护机制,探索脑出血的有效治疗药物。方法：健康雄性Wistar大鼠,10～12月龄,体重320-350g,随机分为5组,即假手术组(Group A),脑出血模型组(Group B),脑出血+黄芩苷小剂量组(25mg/kg) (Group C),脑出血+黄芩苷中剂量组(50mg/kg) (Group D),脑出血+黄芩苷大剂量组(100mg/kg) (Group E).采用立体定向技术将1μL的胶原酶Ⅶ(0.5U/1μL)注入大鼠尾状核区制备大鼠大脑内出血模型。脑出血模型组和黄芩苷各剂量组：大鼠右侧尾状核区注射1μL的胶原酶Ⅶ；假手术组：大鼠右尾状核区注射等量的1μL生理盐水。模型制备成功后2h开始给药,黄芩苷各剂量组腹腔注射给予相应剂量的黄芩苷混悬液,假手术组和脑出血模型组均分别腹腔注射给予等容量生理盐水,连续用药,均每日1次。各组在脑出血后不同时间点(1、3、5、10天)进行神经行为缺损评分,每个时间点从各组中随机取6只大鼠处死后,取脑组织,采用干-湿比重法检测脑组织水含量,RT-PCR和’Western blot法检测脑出血周围脑组织PAR-1 mRNA和PAR-1的表达,RT-PCR法检测Caspase-3mRNA和MMP-9mRNA的表达,Western blot法检测兴奋性氨基酸受体(NMDA-NR)的表达,免疫组织化学法检测核转录因子-κB (NF-κB)、Caspase-3和MMP-9的表达,TUNEL原位标记法检测阳性凋亡细胞数量,HE染色法观察脑出血周围脑组织病理形态变化。应用SPSS13.0统计软件包进行数据统计分析。结果：(1)黄芩苷小中大剂量组各时间点神经功能评分均低于脑出血模型组各时间点的评分,有明显统计学差异(P<0.05)；各时间点的黄芩苷中大剂量组的神经功能评分明显低于黄芩苷小剂量组(P<0.05)。(2)脑出血模型组较假手术组脑组织水含量明显升高(P<0.01),出血后3d时达到高峰,之后缓慢下降,10d仍高于正常；黄芩苷小中大剂量组各时间点脑组织水含量均较脑出血模型组的脑组织水含量明显减少(P<0.01),出血后3d和5d,黄芩苷中大剂量组的脑组织水含量明显低于黄芩苷小剂量组(P<0.05)。(3)脑出血后脑组织PAR-1 mRNA、Caspase-3 mRNA、MMP-9 mRNA和PAR-1、Caspase-3、MMP-9表达及TUNEL阳性凋亡细胞数量,出血后1d均已明显升高,3d时达到高峰,5-10d逐渐降低,在各时间点与假手术组间比较,表达均明显增多(P<0.01)；黄芩苷小中大剂量组各时间点的脑组织PAR-1 mRNA、Caspase-3mRNA、MMP-9mRNA和PAR-1、Caspase-3、MMP-9的表达及TUNEL阳性凋亡细胞数量均明显低于脑出血模型组(P<0.01),出血后3d和5d,黄芩苷中大剂量组的表达明显低于黄芩苷小剂量组(P<0.05)。(4)脑出血模型组脑组织NF-κB和NMDA-NR的表达出血后1d时明显增多、达到高峰,3d时仍很多,5-10d逐渐减少,与假手术组间比较有显著差异(P<0.01)；黄芩苷小中大剂量组各时间点的脑组织NF-κB和NMDA-NR的表达均较脑出血模型组明显降低(P<0.01),出血后1d和3d,黄芩苷中大剂量组的脑组织NF-κB和NMDA-NR的表达明显低于黄芩苷小剂量组(P<0.05)。(5)病理形态学改变：肉眼观察：ICH后1d和3d,ICH模型组右侧大脑半球肿胀,脑回增宽,脑沟变浅,冠状切面可见血肿位于基底节区,呈不规则球形或椭圆形,与ICH模型组比较,黄芩苷小中大剂量组上述改变有减轻；假手术组两侧大脑半球基本对称,未见脑组织肿胀。HE染色观察：脑出血模型组血肿周围组织疏松,血管周围间隙增宽,神经元数目明显减少,排列紊乱,细胞形态不完整,可见大量炎性细胞浸润并可见被吞噬的核碎片和核溶解现象,神经细胞明显肿胀呈空泡样、细胞质透亮,Nissled体消失,胞核深染、偏位,脑出血后3d最明显,之后渐减轻。黄芩苷各剂量组与同时间点的ICH组比较,血肿周围水肿带缩小,炎症细胞浸润数量减少,神经细胞损伤减轻。假手术组大鼠右侧基底节区细胞形态结构完整,胞核及核仁清晰可见。结论：(1)大鼠脑出血后脑水肿、脑组织PAR-1mRNA和PAR-1表达、Caspase-3mRNA、MMP-9mRNA、Caspase-3、MMP-9、NF-κB和NMDA-NR表达、及TUNEL阳性凋亡细胞数量均呈现先上升后渐降的动态变化过程,从变化曲态看,脑出血后神经功能缺损、脑水肿与脑组织PAR-1mRNA、PAR-1表达、血脑屏障通透性增加、炎症反应、细胞凋亡等损伤因素有不同程度的相关性；(2)黄芩苷能有效减低脑出血后脑水肿,对实验性大鼠脑出血后的神经功能缺损有一定程度的促恢复进作用；(3)黄芩苷能有效抑制脑出血后脑组织PAR-1mRNA和PAR-1表达、Caspase-3mRNA、MMP-9mRNA、Caspase-3、MMP-9、NF-κB和NMDA-NR表达,减少TUNEL,阳性细胞数量,这可能是黄芩苷减轻脑水肿、保护神经细胞及改善神经功能缺损的机制之一,可能会成为有效治疗急性脑出血疾病的手段之一。更多还原

【Abstract】 Intracerebral hemorrhage (ICH) is a devastating neurological disorder with high mortality rate and poor prognosis. ICH-associated brain damage involves numerous mediators, such as thrombin, inflammatory injury, apotosis, matrix metalloproteinase-9, neurotoxicity of excitabilitical amine acid, etc. Thrombin may play an essential role in this process and thrombin-induced extravascular neurotoxicity has been shown to be mediated by protease activated receptors (PARs).To date, no curative therapy is available for the treatment of ICH, and supportive therapy is the major option for most cases. Targeting PAR-1 might offer a potential therapeutic option for patients with ICH.Baicalin (7-glucuronic acid,5,6-dihydroxyflavone) is one of the major flavonoid compounds isolated from the dry roots of Scutellaria baicalensis Georgi. Numerous studies have shown that baicalin has neuroprotective effects on ischemic cerebral injury. However, little is known about the effects of baicalin on hemorrhagic brain injury. Previous studies have shown that complex prescriptions containing Scutellaria baicalensis Georgi were able to inhibit PAR-1 expression in rat brain after ICH. The aim of this study was to evaluate the effects of baicalin on ICH-induced brain injury using an ICH rat model. In addition, the mechanisms underlying were also investigated.Objectives:To observe pathophysiological changes in experimental ICH model of rats. To investigate the effect and mechanisms of baicalin on neurologic impairment, brain edema, neurotic pathomorphology, expression and significance of PAR-1mRNA and PAR-1, Caspase-3 mRNA and Caspase-3, MMP-9 mRNA and MMP-9, NMDA-NR, NF-κB, and number of TUNEL-positive cells in the brain issue of rat with experimental intracerebral hemorrhage, and to explore its possible protective mechanism, to find a effective therapeutic drug.Methods:ICH was induced by intracerebral administration of 0.5 U collagenaseⅦin 1μl saline into the right caudate nucleus of rats, sham operation rats were administered with equal volume of saline without collagenaseⅦ. Rats with ICH were randomly divided into five groups: sham-operated group (Group A), ICH group (Group B) and ICH with baicalin small dose (25 mg/kg, Group C), moderate dose (50 mg/kg, Group D), large dose (100 mg/kg, Group E) treatment group. Rats with sham operation were used as controls. Two hours after the ICH induction, saline was intraperitoneally injected into the animals both in sham-operated group and ICH group, while baicalin into the baicalin treatment Group at the indicated doses. The same treatments were conducted once a day thereafter.On day 1, day 3, day 5 and day 10, rat neurologic defecit scores were observed, six brain tissue samples were collected from the perihematoma areas. The water content of the brain tissue was quantitated with wet/dry weight measurement. Brain tissue pathomorphology was observed in electron microscope by dyeing the brain tissue slice with HE. The perihematomal brain tissue were extracted to determine the differential expression of PAR-1mRNA, Caspase-3 mRNA and MMP-9 mRNA, and protein levels of PAR-1, NMDA-NR, Caspase-3, MMP-9, and NF-κB by RT-PCR, Western Blot and immunohistochemical methods. Cell apoptosis was evaluated by terminal transferase dUTP nick end labeling (TUNEL) staining. All data were presented as means±SD and analyzed with one-way analysis of variance (ANOVA) and Student’s t-test by SPSS 13.0 soft ware package.Results:(1) Neurologic deficit scores were higher in ICH group than those in sham-operated group at each time point, peaking on day 1. Baicalin group got lower neurologic defecit scores than that in ICH group at every time points (P<0.05), there was difference between Group C and Group D, E (P<0.05). (2) After ICH, the brain water content was much higher than that in sham-operated group (P<0.01), it increased on day1, peaked on days 3 and then declined gradually, still higher on day5. The brain water content in baicalin group was obviously lower than that in ICH group at each time point (P<0.01), there was difference between Group C and Group D, E at 3d and 5d post-ICH (P<0.05). (3) After ICH, the expression of brain tissue PAR-1mRNA, Caspase-3 mRNA, MMP-9 mRNA and the protein levels of brain issue PAR-1, Caspase-3, MMP-9, and the number of TUNEL-positive cells were much higher than those in sham-operated group (P<0.01), it was higher on day 1, peaked on days 3 and then declined gradually on day5 and day 10. At each time point, baicalin at all doses significantly reduced PAR-1mRNA, Caspase-3 mRNA and MMP-9 mRNA expression, PAR-1, Caspase-3, MMP-9 protein levels and the number of TUNEL-positive cells in comparison with those of the vehicle treated ICH group (P<0.01, respectively). On day 3 and 5, there was significant difference between Group C and Group D, E. (4) After ICH, the brain tissue NF-κB and NMDA-NR expression was significantly higher than that in sham-operated group (P<0.01), it rose on day1 and then reduced gradually on day3 to day 10. The NF-κB and NMDA-NR expression in baicalin group was obviously lower than that in ICH group at each time point (P<0.01), there was difference between Group C and Group D, E at 1d and 3d post-ICH (P<0.05). (5) Observe the gestalt form of the brain:in ICH groups, the right cerebral hemisphere got swollen, the gyri extended and the sulci shallow; the changes in baicalin treatment groups were slighter; the bilateral hemispheres were approximately symmetric in sham-operated group. The hematoma, which was irregularly spheroid or oval, could been viewed at the area of basal nucleus in the coronal slice. Observe the HE-stained tissue with a light-microscope:in ICH groups, the red cells were of integrity, the brain edema and neural injury surrounding the hematoma were observed, inflammatory cells transmigrated, the tissues were loose, nerve cells and cotloidal cells swollen, and vacuoles of different sizes were formed in cell space and the cell space increased, the cell ballooning degeneration and necrosis were observed, that peaked on days 3 and then reduced gradually; in baicalin treatment groups, the brain tissues were swollen and loose around the injected area, inflammatory cells transmigrated, the changes in baicalin treatment groups were slighter. There were not obviously pathological changes in sham-operated groups.Conclusions:(1) The results demonstrated that the upregulation expression of PAR-1mRNA, Caspase-3 mRNA, MMP-9 mRNA, PAR-1, Caspase-3, MMP-9, NF-κB, NMDA-NR and the increased number of TUNEL-positive cells after ICH were in a time-dependent manner, and the neurologic deficit and brain edema formation were correlated with brain tissue PAR-1, MMP-9, NMDA-NR, NF-κB expression and cell apoptosis in rats. (2) Baicalin effectively reduced the brain edema, inhibited cell apoptosis and inflammatory injury and improved the neurologic deficit following ICH in a dose- and time-dependent manner, with concomitant suppression of brain tissue PAR-1, Caspase-3 and MMP-9, NF-κB, NMDA-NR expression at both mRNA and (or) protein levels, which may be one of the mechanisms of neuroprotective effects of baicalin on ICH-induced brain injury. (3) The study raised the possibility that baicalin may be a potential agent for ICH therapy.更多还原