醒脑静制剂对脑卒中模型大鼠的保护作用及机制研究

【作者】 刘洋；

【导师】 孙建宁；

【作者基本信息】 北京中医药大学 ， 中药药理学， 2012， 博士

【摘要】 缺血性脑卒中是一种严重威胁人类健康和生命的疾病,尤其在老年人中多发。由于缺血性脑卒中具有高致残率和致死率,给个人、家庭及社会带来沉重负担。在既往报道中,绝大多数脑卒中患者即使脱离生命危险,也会伴有不可恢复的神经功能缺损,表现为口眼歪斜、肢体功能障碍、思维迟钝、记忆力减退、情绪不稳定、精神抑郁等。这些症状可能一直困扰患者,如不采用适当措施加以治疗和干预,可能会随着时间的推移而持续加重,给病人带来极大痛苦。中药对于缺血性脑卒中的疗效已得到广泛认可,其作用靶点广泛,作用持久、温和,毒副作用小,在对症治疗的同时,也可对机体整体状态进行调节。我国医药资源丰富,经典名方、验方,经现代工艺制剂后,在确保临床疗效的同时,也扩大药物的应用范围,得到广泛认可。醒脑静注射液由麝香、冰片、栀子、郁金等药物组成,能够清热泻火,凉血解毒,开窍醒脑。临床上用于常用于治疗神经系统损伤性疾病,如脑卒中、一氧化碳中毒、肝昏迷等急重症,尤其是对于急性缺血性脑病具有良好疗效,得到临床工作者的认可。然而,由于其剂型原因,醒脑静注射液不便于病人自行给药,使其不能长期应用于脑缺血恢复期的治疗,为其临床应用带来一定局限性。醒脑静口服制剂是注射液原方提取物,在确保疗效的基础上,可延长药物临床应用时间,且便于服用。经过前期筛选,目前制剂工艺已确定。并其对缺血性脑卒中模型大鼠具有一定作用保护作用,其对抗缺血性脑损伤的作用得到初步肯定。本研究采用大脑中动脉血栓(MCAT)模型,通过比较两种醒脑静复方制剂(醒脑静注射液和醒脑静口服制剂)对局灶性脑缺血模型动物神经功能缺损评分和脑梗死体积的影响,明确口服制剂对大鼠缺血性脑损伤模型的保护作用。并采用永久性局灶性中动脉阻断脑缺血(pMCAO)大鼠模型,观察醒脑静口服制剂在脑缺血恢复早期(14 d)对缺血模型动物肢体运动、平衡能力相关指标,评价药物在缺血性脑损伤恢复早期的神经保护作用。另外,本研究中还采用转基因脑卒中动物模型——自发性脑卒中大鼠(SHRSP),评价醒脑静口服制剂对SHRSP血压、心率及一般体征的影响,并从对抗氧化性损伤的角度评价了醒脑静口服制剂对SHRSP的保护作用。已有研究结果表明,醒脑静注射液对缺血性脑损伤具有明确保护作用,其作用机制与抗炎、抗氧化、改善能量代谢障碍等作用有关,但其对突触结构、功能的影响未见报道。本研究采用永久性局灶性中动脉阻断脑缺血(pMCAO)大鼠模型,从神经元病理改变、突触结构、功能角度,观察醒脑静注射液对脑缺血急性期神经元病理改变、突触结构改变、及突触功能相关蛋白表达的影响,探讨醒脑静注射液对局灶性脑缺血模型大鼠的保护作用机制。本研究主要内容如下：第一部分醒脑静口服制剂对局灶性脑缺血模型大鼠和自发性脑卒中大鼠的影响第一节醒脑静口服制剂对局灶性脑缺血模型大鼠的保护作用实验一两种醒脑静制剂对三氯化铁所致局灶性脑缺血模型大鼠脑损伤的保护作用目的：观察两种醒脑静复方制剂对局灶性脑缺血模型大鼠的神经功能、脑梗死范围的影响,明确口服制剂在脑缺血急性期的脑保护作用。方法：实验动物随机分为5组,即模型组、醒脑静注射液3、9mL/kg组(相当于临床人用量9倍、27倍)、醒脑静口服制剂40mg/kg、120mg/kg(每10 mL注射液生药量相当于口服制剂0.1289g,分别相当于临床人用量9倍、27倍)组,另设假手术组。造模前,醒脑静口服制剂组灌胃给药两天2 d,醒脑静注射液组腹腔注射给药,1次/d；第3 d给药1 h后,采用三氯化铁凝闭大脑中动脉,造成大鼠大脑中动脉血栓形成(MCAT)模型,造模6 h后给药1次。于造模后6h、24 h分别对大鼠进行神经功能缺损症状评分。并于造模24 h后,取脑,冠状切片,进行TTC染色,对白色梗死组织进行称重,以梗塞组织重量占总脑重量的百分比作为脑梗塞范围。结果：实验结果显示,缺血后模型动物均表现出一定程度的神经功能缺损症状,表现为：身体蜷缩,身体平衡能力下降,向左侧旋转、倾倒,不能行走等。术后6 h,与模型组相比,醒脑静注射液9 mL/kg组神经功能缺损症状评分明显降低(P<0.05),醒脑静口服制剂也能够明显减轻缺血后神经功能损伤(P<0.05)；术后24h,与模型组相比,醒脑静3 mL/kg、9 mL/kg组神经功能缺损均明显减轻(P<0.05,P<0.01),醒脑静口服制剂120mg/kg组大鼠神经功能缺损症状明显减轻(P<0.05)。手术24 h后,缺血组动物脑组织可见不同程度的白色梗死灶。其中,模型组脑梗死组织可达14.9 g,经给药治疗,给药组脑梗死范围较模型组明显降低,醒脑静注射液3 mL/kg、9 mL/kg组均能够明显减小梗死范围(P<0.05,P<0.01)；醒脑静口服制剂40mg/kg、120mg/kg组能够显著减少梗死体积(P<0.01)。结论：醒脑静口服制剂在缺血性脑损伤发生后24 h内能够改善缺血模型动物的神经功能缺损症状,减少缺血后脑组织梗死范围,且与醒脑静注射液组相比,效果无显著性差异。结果表明,醒脑静口服制剂对三氯化铁所致局灶性脑缺血模型大鼠具有保护作用。实验二醒脑静口服制剂对永久性局灶性大脑中动脉阻断脑缺血(pMCAO)模型大鼠缺血损伤恢复早期神经功能的保护作用目的：考察醒脑静口服制剂在脑缺血损伤恢复早期对pMCAO模型大鼠神经功能缺损的改善作用。方法：采用线栓法制作pMCAO大鼠模型。术后3 d,对缺血动物进行神经功能缺损评分,根据评分结果将动物随机分为5组：模型组、醒脑静口服制剂12mg/kg组(相当于临床人用量的3.5倍)、醒脑静口服制剂25 mg/kg组(相当于临床人用量的7倍)、醒脑静口服制剂50 mg/kg (相当于临床人用量的14倍),以丁苯酞70 mg/kg组为阳性对照组,另设假手术组。给药组灌胃给服相应药物,假手术组与模型组灌胃给予等体积橄榄油。脑缺血后3 d至14 d灌胃给药,1次/d。分别于pMCAO后3d、6d、9d、12 d. 14 d,通过神经功能评分、横木行走实验、前肢触觉刺激实验、前肢抓握力量测试评价药物对缺血模型动物神经功能的保护作用。于14 d末次给药后,取动物脑组织,生化法测定脑组织中MDA含量及SOD活力。结果：与假手术组相比,缺血模型大鼠表现出明显神经功能缺损症状。缺血后14 d,与模型组相比,醒脑静12 mg/kg,25 mg/kg和50 mg/kg组神经功能缺损症状明显改善,神经功能缺损症状评分降低(P<0.01),横木行走实验评分明显增加(P<0.05,P<0.01),撕除胶布潜伏期明显缩短(P<0.05,P<0.01),前肢抓握力量明显增加(P<0.01)。缺血14 d后,与假手术组相比,模型组大鼠脑组织中SOD活力明显下降,MDA含量明显增加,且与模型组相比,醒脑静12 mg/kg,25 mg/kg和50 mg/kg组脑组织MDA含量均明显降低(P<0.01)。结论：醒脑静口服制剂在脑缺血恢复早期(14 d)对pMCAO模型大鼠具有神经保护作用,能够改善缺血动物神经功能缺损症状,减轻缺血后脑组织脂质过氧化损伤。第二节醒脑静口服制剂对自发性脑卒中大鼠(SHRSP/Izm)的影响及抗氧化性损伤作用的研究实验一醒脑静口服制剂对SHRSP机体状态的影响目的：采用自发性脑卒中大鼠(SHRSP)模型,观察醒脑静口服制剂对于SHRSP血压、心率及一般体征的影响。方法：SHRSP给予食盐负荷饮食(40 g/kg饮食),按体重随机分为4组：SHRSP组、维生素C组(ascorbic acid, AA)、醒脑静25 mg/kg组、醒脑静50 mg/kg组。AA组每日给予维生素C水溶液(1000 mg/d),醒脑静组灌胃给服相应剂量药物,SHRSP组灌胃等体积饮用水,1次/d,连续给药6周。每周监测大鼠体重变化情况,并于给药前(0周),给药第2、4、6周进行收缩压(SBP)和心率(HR)测定,及一般体征(包括进食量、饮水量、24小时尿量、24小时排便量)的监测。结果：药前(第0周)各组动物体重、一般体征无差异。自给药第2周开始,给予食盐负荷后大鼠饮水量、排尿量、排便量明显增加,但与SHRSP组相比,AA组进食量明显减少(P<0.01),饮水量、排尿量、均显著降低(P<0.01),体重减轻(P<0.01)。给药第6周,各组进食量无差异,但AA组体重仍明显低于SHRSP组(P<0.01)。醒脑静组体重、进食量与SHRSP组相比均无显著性差异(P>0.05)。给药前,各组大鼠SBP无显著性差异；给予食盐负荷后,第2周,各组大鼠SBP均出现明显上升趋势；第4周,AA组、醒脑静高剂量组SBP较SHRSP组有下降趋势,但未表现出显著性差异；第6周此种趋势消失,除AA组SBP仍低于SHRSP组以外,其他各组SBP较模型组有所升高,但结果尚无统计学意义。给药第2周,各组HR有下降趋势,但从第4周至第6周实验结束,HR呈现出上升趋势,且各组间无显著性差异。结论：醒脑静口服制剂对SHRSP SBP及HR无显著影响,对动物体重增长、进食量、饮水量等一般体征无明显影响。实验二醒脑静口服制剂对SHRSP氧化性损伤的影响目的：观察醒脑静口服制剂对SHRSP氧化性损伤状态的影响,评价醒脑静口服制剂对SHRSP的保护作用。方法：SHRSP给予盐负荷饮食(40 g/kg饮食),按体重随机分为4组：SHRSP组、维生素C组(ascorbic acid, AA)、醒脑静25 mg/kg组、醒脑静50 mg/kg组。AA组每日给予维生素c水溶液(1000 mg/d),醒脑静组灌胃给服相应剂量药物,SHRSP组灌胃给予等体积饮用水,1次/d,连续给药6周。大鼠于给药第2、4、6周采集血浆样本,生化法测定血中总体抗氧化状态(TAS, total antioxidant status);并于第6周给药结束后TBARS法测定脑组织、血浆中丙二醛(MDA)含量,ELISA法测定尿液8-羟基脱氧鸟苷(8-OHdG)含量,RT-PCR (?)去测定脑组织中SOD、TNF-αmRNA表达水平。结果：给药第2周,与SHRSP组相比,AA组及醒脑静给药各组动物表现出自身抗氧化能力增加。醒脑静50 mg/kg组血浆TAS明显提高,抑制过氧化物能力增强(P<0.01)：第4周,AA组血浆TAS明显增加(P<0.05),醒脑静各剂量组TAS也较SHRSP组有所增加,但未表现出统计学差异；第6周,与SHRSP组相比,醒脑静50 mg/kg组血浆TAS均显著增加(P<0.01)。给药第6周,与SHRSP组相比,AA组尿液中8-OHdG含量明显降低(P<0.05),其他给药组末见统计学差异；各组大鼠脑组织中MDA含量无显著性差异；SHRSP组血浆MDA含量较高,各给药组血浆MDA含量均有不同程度降低,其中醒脑静50 mg/kg组血浆MDA含量明显降低(P<0.05,P<0.01)。与SHRSP组比较,给药第6周,给药组脑组织中SOD mRNA表达均增加,但至实验结束,尚未显示出统计学差异；醒脑静25 mg/kg组、50 mg/kg组TNF-a mRNA表达出现下降趋势,但尚无统计学意义。结论：醒脑静口服制剂能够降低SHPSP体内脂质过氧化物含量,提高动物体内总体抗氧化能力,这种抗氧化能力的改善或许对SHPSP起保护作用。第二部分醒脑静注射液的作用机制研究——醒脑静注射液对pMCAO模型大鼠突触可塑性的影响实验一醒脑静注射液对pMCAO模型大鼠缺血神经元的保护作用目的：观察醒脑静注射液对pMCAO模型大鼠在脑缺血急性期神经元的保护作用。方法：采用线栓法建立pMCAO大鼠模型,缺血6 h后,模型动物随机分成5组：模型组、醒脑静1.2 mL/kg组(相当于临床人用量的3.5倍)、醒脑静2.5 mL/kg组(相当于临床人用量的7倍)、醒脑静5 mL/kg(当于临床人用量的14倍),以金纳多2.5 mL/kg组为阳性对照组,另设假手术组。给药组腹腔注射相应药物,假手术组与模型组腹腔注射等体积生理盐水。脑缺血后6 h至7 d给药,1次/d。采用苏木素-伊红染色(H&E staining)和尼氏染色(Nissl staining)(?)去观察醒脑静注射液对缺血后神经元病理改变的影响,采用透射电镜观察醒脑静注射液对脑缺血后神经元超微结构改变的影响。结果：缺血2 d后,光镜下可见假手术组皮层神经元细胞形态圆整,排列整齐,神经元胞浆丰富,尼氏小体分布均匀；模型组脑组织皮层神经元大量变性坏死,核固缩深染,尼氏小体含量明显减少；与模型组相比,给药组皮层完整神经元数目增加,海马区神经元排列较为整齐,死亡神经元数目减少,神经元损伤程度明显减轻,尼氏小体脱失减轻。电镜下可见假手术组神经元细胞核形态规整,核膜光滑完整,核仁明显,染色质分布均匀,胞浆内细胞器丰富,线粒体成椭圆或圆形,脊清晰；模型组神经元核固缩,细胞周围呈现空泡状,细胞器少见；给药组细胞形态基本正常,核膜核仁清晰可见,且细胞器较为丰富。缺血7 d后,模型组缺血周围区域仍可见大量神经元缺失,神经元变性死亡,尼氏小体脱失；与模型组相比,醒脑静注射液组神经元损失数目减少,结构完整、可辨认的神经元数量较多,尼氏体表达增加。电镜下可见缺血至7 d,模型组神经元固缩,核膜形态不规则,核内染色质浓缩、边集；醒脑静注射液组可见神经元形态基本正常,核膜光滑,核仁明显,且染色质均匀分布。结论：醒脑静注射液在脑缺血急性期对神经元起保护作用,减轻因缺血造成的神经元死亡、尼氏小体脱失及神经元超微结构破坏。实验二醒脑静注射液对pMCAO模型大鼠缺血突触结构的影响目的：观察醒脑静注射液对pMCAO模型大鼠在脑缺血急性期突触结构,及突触结构相关蛋白表达的影响。方法：采用线栓法建立pMCAO大鼠模型,缺血6 h后,模型动物随机分成5组：模型组、醒脑静1.2 mL/kg组、醒脑静2.5 mL/kg组、醒脑静5 mL/kg,以金纳多2.5 mL/kg组为阳性对照组,另设假手术组。给药组腹腔注射给予相应药物,假手术组与模型组腹腔注射等体积生理盐水。脑缺血后6 h至7 d给药,1次/d。采用透射电镜观察pMCAO模型大鼠突触结构,免疫组织化学染色法观察缺血后突触结构相关蛋白的表达情况以及醒脑静注射液的干预作用。结果：缺血2 d后,电镜下可见假手术组突触结构正常,突触前后膜结构清晰；模型组动物突触数量稀少,突触结构不清；给药组可见较多突触结构,形态丰富。缺血7 d后,模型组突触结构少见；给药组可见较为清晰突触结构,突触间隙可见,突触前膜靠近突触间隙一侧偶见突触小泡聚集。与假手术组相比,缺血2 d后,模型组动物synapsin-Ⅰ、PSD-95在海马CA1区、CA3区、皮层均明显下降(P<0.01),α-synuclein在海马CA1区、CA3区、皮层表达均明显增加(P<0.01)。与模型组相比,synapsin-Ⅰ在醒脑静1.2 mL/kg组CA3区,2.5 mL/kg组CA3区和皮层,5 mL/kg组CA1区、CA3区、皮层表达明显升高(P<0.05,P<0.01);PSD-95在醒脑静1.2mL/kg组皮层,2.5 mL/kg组CA1区,5 mL/kg组CA1区和皮层表达明显升高(P<0.05,P<0.01);α-synuclein在醒脑静1.2 mL/kg组CA3区,2.5 mL/kg组皮层,醒脑静5 mL/kg组CA1区、CA3区和皮层表达均显著减少(P<0.05,P<0.01)。缺血7d后,与假手术组相比,模型组synapsin-Ⅰ在海马CA1区、皮层表达仍显著降低(P<0.05,P<0.01), PSD-95在CA1区、CA3区和皮层表达仍显著降低(P<0.01),其中在CA1、CA3区表达略有上升,但与2d表达水平相比无显著性差异,α-synuclein在海马CA1区、CA3区和皮层表达仍显著增加(P<0.01)。与模型组相比,醒脑静1.2mL/kg、2.5 mL/kg、5 mL/kg组synapsin-Ⅰ在CA3、皮层表达仍有明显增加(P<0.05, P<0.01), PSD-95在醒脑静1.2 mL/kg组CA3、皮层表达有所上升(P<0.05,P<0.01), 2.5 mL/kg组CA1表达增加(P<0.05),5 mL/kg组CA1、CA3、皮层表达均有明显增加(P<0.05,P<0.01);α-synuclein在醒脑静1.2 mL/kg、2.5 mL/kg、5 mL/kg组CA1区、CA3区、皮层表达均有所下降(P<0.05,P<0.01)。结论：脑缺血急性期,pMCAO模型大鼠突触结构破坏,与突触功能密切相关蛋白synapsin-Ⅰ、PSD-95、α-synuclein表达发生改变,表明突触结构、功能受到损伤。醒脑静注射液在缺血后2 d即能增加皮层、海马部位的PSD-95、synapsin-Ⅰ表达,并减少α-synuclein的积聚,这种作用持续到缺血后7 d仍然存在,表明醒脑静注射液在脑缺血急性期、亚急性期对神经元突触结构、功能具有保护作用。实验三醒脑静注射液对pMCAO大鼠突触功能相关蛋白表达的影响目的：观察醒脑静注射液对pMCAO模型大鼠突触功能相关蛋白表达的影响。方法：采用线栓法建立pMCAO大鼠模型,缺血6 h后,模型动物随机分成4组：模型组、醒脑静2.5 mL/kg组、醒脑静5 mL/kg,以金纳多2.5 mL/kg组为阳性对照组,另设假手术组。给药组腹腔注射给予相应药物,假手术组与模型组腹腔注射等体积生理盐水。脑缺血后6 h至7 d给药,1次/d。采用Western Blot (?)去观察缺血2 d、7 d后,pMCAO模型大鼠皮层、海马区域PSD-95、p-CaMKⅡ、NR2B蛋白的表达情况,以及醒脑静注射液对蛋白表达的影响。结果：缺血后2 d,与假手术组相比,模型组PSD-95蛋白在皮层、海马表达均明显降低(P<0.01)；与模型组相比,给药组PSD-95蛋白明显增加(P<0.05,P<0.01)。缺血后7 d,与假手术相比,PSD-95蛋白的表达仍显著降低(P<0.05,P<0.01)；与模型组相比,醒脑静给药组皮层PSD-95蛋白的表达均有不同程度增加(P<0.05,P<0.01),金纳多组、醒脑静5.0 mL/kg组海马PSD-95蛋白表达也有增加,但未见统计学差异。缺血后2 d,与假手术组相比,模型组p-CaMKⅡ蛋白在皮层、海马表达均明显降低(P<0.05)；与模型组相比,金纳多组p-CaMKⅡ蛋白表达明显增加(P<0.05)。缺血后7 d,模型组p-CaMKⅡ蛋白的表达与假手术组无显著性差异；与模型组相比,给药组p-CaMKⅡ蛋白的表达未见统计学差异。缺血后2 d,与假手术组相比,模型组NR2B蛋白表达有所下降,其中在海马的表达显著降低(P<0.01)；与模型组相比,醒脑静给药组NR2B蛋白表达明显增加(P<0.05,P<0.01)。缺血后7 d,与假手术组相比,模型组NR2B蛋白在皮层、海马表达无显著性差异；但与模型组相比,醒脑静给药组表达水平仍有明显增加(P<0.05,P<0.01)。结论：醒脑静注射液在缺血损伤急性期能够增加PSD-95、p-CaMKⅡ、NR2B蛋白的表达,对抗缺血引起的突触功能损伤。醒脑静注射液对突触的保护作用在脑缺血急性期(2d)作用较强,这或许是醒脑静注射液对抗急性缺血性脑损伤的机制之一。更多还原

【Abstract】 Cerebral ischemic stroke is one of serious diseases that threaten human being, which with high incidence, especially in the senile. Due to its high morbidity and mortality, cerebral ischemic stroke imposes a heavy burden on individuals, families and society. In previous reports, the vast majority of patients with stroke suffer from unrecoverable neurological defects even when they are treated, such as limb dysfunction, mental retardation, memory loss, depression, and so on. These symptoms may have troubled the patients all the time. With no appropriate measures of treatment and intervention, they may continue and aggravate over time. Therefore, treatment in sub-acute phase is essential.Traditional Chinese medicines are effective for cerebral ischemia, which always act through various pathways. They are usually mild with lasting effect, with few side effects. They can treat both symptoms and causes of diseases, with regulation on generalized condition. In China, there are many famous classic formulas in ancient times, which can be prepared into Chinese patents by modern techniques. These patents are effective as ancient formulas in clinic, and can be applied for more symptoms because of their convenience for administration.Xingnaojing Injection is commonly used in clinic, which is composed of she xiang (Moschus), bing pian (Borneolum Synthcticum), yu jin (Radix Curcumae Wenyujin), and zhi zi(Fructus Gradeniae). It is commonly applied to nervous system diseases, such as carbon monoxide poisoning, coma hepaticum, and so on. It is effective significantly for acute cerebral ischemia. However, injection is usually applied for acute symptoms. Because of its dosage form, it can not be administered out of hospitals. Development of peroral preparation of Xingnaojing injection will ensure its wide application. Peroral preparation of Xingnaojing is made from extract of identical Chinese medicinals of Xingnaojing injection. Based on previous study, effect of peroral preparation of Xingnaojing has been proved.In this study, neuroprotective effect of Xingnaojing peroral preparation was evaluated in MCAT (Middle Cerebral Artery Thrombosis) model rats. We observed improvements in neuro-functions and the size of cerebral infarction to make sure both injection and peroral preparations have neuroprotective effect in acute phase of cerebral ischemia. In addition, we further explored action of peroral preparation in convalescence in pMCAO (permanent middle artery occlusion) model rats, and evaluated its effect as improvement on neurological defects and relief on oxidative stress. In addition, in this study, SHRSP was used to evaluate antioxidant action of Xingnaojing peroral preparation.Studies have shown that the protection of Xingnaojing injection for ischemic injury is related to its anti-inflammatory, sedative, anti-oxidatant and other effects. However, whether its neuroprotective action is related to synaptic function has not been explored yet. We evaluated protective effect of Xingnaojing injection on neurons and synapse in acute and sub-acute phases in rat model of pMCAO. Post-ischemia synaptic structure and function was observed, and action of Xingnaojing Injection on synaptic structure and function was evaluated.This study is composed of the two following parts:1 Effect of Xingnaojing peroral preparation on rat model of focal cerebral ischemia and stroke-prone spontaneously hypertension rat1.1 Neuroprotection of Xingnaojing peroral preparationin on rat model of focal cerebral ischemia1.1.1 Protection from two preparations of on Xingnaojing on rat model of FeCl3-induced focal cerebral ischemiaObjective:it is to evaluate effects of preparations of Xingnaojing in acute phase of cerebral ischemia, and further demonstrate neuroprotection of Xingnaojing peroral preparation.Methods:SD rats were randomly divided into model group, Xingnaojing injection 3 mL/kg group and 9 mL/kg group (equal to 9- and 27-time clinical dosage of adult),40 mg/kg group and 120 mg/kg group of peroral preparation, and the sham group. Rats were administered with peroral preparation two days earlier before the establishment of model, or intraperitoneally administered with Xingnaojing injection, once per day. One hour later administration on the third day, MCAT model in rats was established with FeCl3 Rats were administered with medicines 6 hours later after establishment of model. On 6 h and 24 h after establishment of model, scores on neurological defects were measured. And 24 hours after establishment of model, coronal sections of brain tissue was taken and stained with TTC. Weight of white infarction and whole brain tissues was measured, taking percentage weight of infarction to the total weight of brain as degree of cerebral infarct.Results:after MCAT, rats got some neurological defects, such as curled body, poor balance, rotation and falling to the left side, inability to walk, and so on. Compared with model group, 9 mL/kg group of Xingnaojing Injection and 120 mg/kg group of Xingnaojing peroral preparation got lower score on neurological defects at 6 h post-MCAT (P<0.05); 24 h post-MCAT, Xingnaojing Injection 3 mL/kg and 9 mL/kg groups got significantly improved on neurological defects (P<0.05,P<0.01),120 mg/kg group of peroral preparation of Xingnaojing also got milder defects (P<0.01) than control group. In the sham group, there was no infarction on brain tissue, while white infracted area in the model group. In the medicated group, infarction size was more reduced than the model group. In the 3 mL/kg and 9 mL/kg groups of Xingnaojing Injection,120 mg/kg group of peroral preparation of Xingnaojing, infarction size was significantly smaller (P<0.05,P<0.01).Conclusion:both Xingnaojing Injection and peroral preparation of Xingnaojing had significant neuroprotective effects within 24 hour after cerebral ischemia.1.1.2 Protective effects of peroral preparation of Xingnaojing on neurological defects and oxidative damage in pMCAO model rats in early convalescenceObjective:it is to evaluate peroral preparation of Xingnaojing in pMCAO model rats in early convalescenceMethod:pMCAO model was established with suture. Rats were randomly divided into model group, sham group, peroral preparation of Xingnaojing 12 mg/kg group,25 mg/kg group, and 50 mg/kg group, taking Butylphthalide 70 mg/kg group as positive control group. All animals were administered medicines 3-14 d post-ischemia, rats in the sham group were administered with same volume of oleum olivae. Neurological deficit scores, tactile stimulation experiments, beam walking test, strength test were operated to observe protective effects of peroral preparation of Xingnaojing on neural function in pMCAO model rats on day 3,6,9, 12, and 14; and measure SOD activity and MDA content in ischemic brain tissue.Results:compared with the sham group, rats in the model group got obvious symptoms of neurological defects. After treatment, Xingnaojing 12 mg/kg group,25 mg/kg group, and 50 mg/kg group got significantly improvement on their neurological functions, manifesting improvement on scores of neurological defect (P<0.01), beam walking ability (P<0.05, P<0.01), and recovery of forelimbs strength (P<0.05,P<0.01), and enhancement of tactile sensitivity (P<0.01). Peroral preparation of Xingnaojing was able to significantly reduce the MDA content of ischemic brain tissue.Conclusion:peroral preparation of Xingnaojing had protective action on pMCAO rat, by reducing oxidative damage.1.2 Study on protection of Xingnaojing peroral preparation on SHRSP and related mechanism1.2.1 Action of of Xingnaojing peroral preparation on general status in SHRSPObjective:it is to explore impacts of peroral preparation of Xingnaojing on body weight, blood pressure, heart rate, and general condition in SHRSP.Method:all SHRSP were given salt-loaded diet, and were randomly divided into SHRSP group, the AA group (vitamin C,1000 mg/d), Xingnaojing 25 mg/kg group and 50 mg/kg group. All rats were intragastrically administered with medicines for 6 weeks. Food intake, water intake, urine output, and defecation output within 24-hour were measured on the 2nd,4th and 6th weeks respectively.Results:there were no significant differences on body weight and general condition between groups pre-medication (0 week). Since second week, given the salt-loaded food, water intake, urine output, the amount of defecation of SHRSP increased significantly. Compared with the SHRSP group, food intake, water intake, and urine output in AA group was significantly reduced (P<0.01), with great body weight loss (P<0.01). In the 6th week of medication, there were no significant differences on food intake between groups, but the body weight in the AA group was still significantly less than others’(P<0.01). There was no significant difference on body weight, food intake Xingnaojing-treated groups (P>0.05). Given salt-loaded food, SBP of rats significantly increased since 2nd week of treatment; in the 4th week, SBP in the AA group, and Xingnaojing 50 mg/kg group slightly reduced, but with no significant differences compared with SHRSP group. This tendency did not vanish unitl the 6th week, in which, SBP in AA group was lower than in the SHRSP group, while was slightly higher in the Xingnaoj ing-treated groups. However, there was no significant difference between groups. Since 2nd week of medication, HR decreased in every group slightly, while increased until 6th week, with no significant difference between groups.Conclusion:peroral preparation of Xingnaojing had no significant impact on body weight, general condition, blood pressure, and heart rate.2.2 Antioxidant action of Xingnaojing peroral preparation of in SHRSPObjective:to investigate antioxidant action and related mechanism of Xingnaojing peroral preparation in SHRSP.Method:all SHRSP given the high salt load diet were randomly divided into SHRSP group, the AA group (1000 mg/d), Xingnaojing 25 mg/kg, and 50 mg/kg group. Rats were administered with medicine for 6 weeks. Plasma TAS was measured respectively in 2nd,4th and 6th week, and MDA content in the brain tissue and plasma was measured on 6th week, and expression of SOD and TNF-αmRNA was measured by RT-PCR.Results:in 2nd week, Xingnaojing peroral preparation was able to increase level of plasmaTAS. It had no significant action in 4th week. However, it was able to increase level of plasma TAS. It was able to reduce content of MDA in the plasma. There was only tendency of increased level of SOD and decreased TNF-αmRNA expression in medicated group, but no significant difference.Conclusion:Xingnaojing peroral preparation of had protective action on SHRSP, which might relate to reduction on oxidative damage.2 Study on protection of Xingnaojing injection on synapse and mechanism2.1 Protection of Xingnaojing injection on neurons in pMCAO model rat Objective:to investigate neuronal protection of XNJ injection in pMCAO model rats in acute and sub-acute phase.Methods:pMCAO model was established with suture. Rats were randomly divided into sham group, model group, XNJ Injection 1mL/kg,2.5 mL/kg, the 5 mL/kg groups, and taking Ginkgo injection 2.5 mL/kg group as control. Postoperative 6 h-7 d rats were administered respectively, once per day. H&E staining and Nissl staining was applied to observe morphology changes on neurons on 2 d and 7 d after ischemia. Microstructure of neurons was observed by transmission electron microscope.Results:on 2nd d post-ischemia, in visual fields of optical microscope, cortical neurons in the sham group were round with complete form. It was rich in cytoplasm in neurons with plenty of Nissl bodies evenly distributed. In the model group, there were a great number of degenerated and necrotic neurons in the cortex, manifesting as shrunken and darkly stained nucleus, and great loss of Nissl bodies. In the treatment groups, there were more complete neurons in the cortex, with clearly arranged neurons in the hippocampus areas. There were less dead neurons, less damage to neurons, and less loss of Nissl bodies. In the field of transmission electron microscope, nuclear morphology in the sham group was clear with complete nuclear membrane, unabroken karyosome, and evenly distributed chromatin. There were plenty of organelles in the cytoplasm, of which itochondria was oval or round. However, in the model group, there were pycnotic neurons, around which, there were vacuolars. There was less organelle in the cytoplasm. In the treatment groups, morphology of neurons was normal with clear nuclear membrane and plenty of organelles.Conclusion:in acute phase of cerebral ischemia, Xingnaojing Injection has protective effect on neurons by preventing neuronal death, loss of Nissl bodies, and ultrastructure damage of neurons induced by ischemia.2.2 Effect of Xingnaojing injection on synaptic structure in pMCAO model ratsObjective:to investigate the effect of Xingnaojing injection on synaptic structure after cerebral ischemia at different time points.Methods:pMCAO model was established by suture. Rats were randomly divided into model group, Xingnaojing Injection 1.2 mL/kg group,2.5 mL/kg group, and 5mL/kg group, taking Gingko injection 2.5mL/kg group as the control group, and the sham group. Rats were administered with medicines 6 h-7 d post-operation. On 2 d and 7 d after ischemia, synapse-related proteins, synapsin-Ⅰ, PSD-95, andα-synuclein were detected with immunohistochemical staining.Results:compared with the sham group, on 2nd day post-ischemia, in the model group, expression of synapsin-Ⅰ, PSD-95 in hippocampal in CA1 and CA3 area, and cerebral cortex were significantly decreased (P<0.01);α-synuclein in hippocampal CA1 and CA3 area, and cortical expression were significantly increased (P<0.01). Compared with model group, expression of synapsin-Ⅰon CA3 area in Xingnaojing 1.2 mL/kg group, in CA3 and cortex of 2.5 mL/kg group, in CA1 area, CA3 area, and the cortex of 5 mL/kg group were significantly increased (P<0.05,P<0.01); PSD-95 expression was significantly increased in cortex and CA1 area in Xingnaojing 1.2 mL/kg group, CA1 area in 2.5 mL/kg group, and the cortex in 5 mL/kg group (P<0.05,P<0.01); expression of a-synuclein was decreased in the CA3 area in 1.2 mL/kg group, cortex in 2.5 mL/kg group, and CA1 and CA3 area in 5 mL/kg group (P<0.05.P<0.01).Compared with the sham group, on 7th d post-ischemia, expression of synapsin-Ⅰin the model group in the CA1 area and cerebral cortex was still significantly lower (P<0.05, P<0.01), expression PSD-95 in CA1, CA3 areas and cortex remained significantly lower (P<0.01) than the sham group; expression ofα-synuclein in CA1 area, CA3 area, and cerebral cortex was still significantly increased (P<0.01). Compared with model group, expression of synapsin-Ⅰon CA3 area, and cerebral cortex in Xingnaojing 1.2 mL/kg,2.5 mL/kg,5 mL/kg groups were significantly increased (P<0.05,P<0.01); expression of PSD-95 on CA3 and cerebral cortex in Xingnaojing 1.2 mL/kg group was increased (P<0.05, P<0.01), in CA1 area in 2.5 mL/kg group was increased (P<0.05), in CA1, CA3 area, and cerebral cortex in 5 mL/kg group was increased significantly (P<0.05,P<0.01); expression ofα-synuclein in CA1, CA3, and cerebral cortex in Xingnaojing 1.2 mL/kg,2.5 mL/kg,5 mL/kg groups were decreased (P<0.05,P<0.01).Conclusion:after pMCAO, expression of synaptic function related proteins, synapsin-Ⅰ, PSD-95,α-synuclein were significantly changed, indicating damage of synaptic function. On 2nd d after ischemia, Xingnaojing Injection was able to increase expression PSD-95 and synapsin-Ⅰ, and to reduce the accumulation ofα-synuclein. Part of this action lasted until 7th d post ischemia, indicating that Xingnaojing injection had a certain effect on neuronal synaptic function.2.3 Effect of Xingnaojing injection on synaptic function in pMCAO model ratsObjective:it is to investigate the effect of Xingnaojing injection on synaptic structure after cerebral ischemia at different time points.Methods:pMCAO model was established by suture. Rats were randomly divided into model group, Xingnaojing Injection 2.5 mL/kg group and 5mL/kg group, taking Gingko injection 2.5 mL/kg group as the control group, taking normal SD rats as the sham group. Rats were administered with medicines 6 h-7 d post-operation. At 2 d and 7 d after ischemia, expression PSD-95, p-CaMKⅡ, NR2B proteins were detected with western blotting.Results:on 2nd d post-ischemia, in model group, PSD-95 protein expression in the cortex and hippocampus was significantly lower (P<0.01); compared with model group, in treatment group, protein expression of PSD-95 was significantly increased (P<0.05,P<0.01). On 7th day post-ischemia, compared with the sham group, protein expression of PSD-95 was still significantly lower (P<0.05,P<0.01); PSD-95 protein expression in cortex in treatment groups increased (P<0.05,P<0.01), PSD-95 protein expression also increased in Gingko group, Xingnaojing Injection 5 mL/kg group in hippocampal area, but there was no significant difference.On 2nd d post ischemia, compared with the sham group, in model group, protein expression of p-CaMKII in the cortex and hippocampus areas significantly decreased (P<0.05); compared with model group, protein expression of p-CaMKⅡwas significantly increased in Gingko group (P<0.05). On 7th day after ischemia, in model group, there was no significant difference on protein expression p-CaMKII compared with the sham group. There was no significant difference on protein expression of p-CaMKII between the mdoel group and treatment groups.At 2nd d post ischemia, in model group, protein expression of NR2B was significantly lower (P<0.01) than in the control group; compared with model group, NR2B protein expression in Xingnaojing treatment groups significantly increased (P<0.05,P<0.01). On 7th d post ischemia, there was no significant difference on NR2B protein expression between the sham group and model group. Compared with model group, the expression level of Xingnaojing treatment group has increased (P<0.05,P<0.01).Conculsion:Xingnaojing injection was able to affect protein expression in both cortex and hippocampus areas in rat model of pMCAO in the acute and sub-acute phase. It was able to prevent protein loss related to ischemia-induced synaptic dysfunction; this might be one of the mechanisms for its neuroprotective effect.更多还原