【摘要】 背景:已知脑缺血后再恢复血流可使脑损伤加重,也有一些研究结果证实中药的活血化瘀成分能对抗脑缺血再灌注损伤。目的:观察活血化瘀4个最基本中药(红花、桃仁、川芎、赤芍)作为施加因素对脑缺血再灌注大鼠血清、脑组织匀浆的免疫球蛋白、C 反应蛋白及补体等免疫指标变化的影响,并验证其量效和时效关系。设计:随机对照实验,单盲法评估。单位:广州市红十字会医院暨南大学附属第四医院神经内科。材料:实验于2001-01/2002-12在广州市红十字会医院创伤研究所实验室完成。实验动物选择成年雌性SD 大鼠138只,体质量280～300g,由广州中医药大学实验动物中心提供。红花、川芎、桃仁、赤芍为单味中药饮片浓缩颗粒,按1∶1∶1∶2比例制成含2.5g/m L 生药汤剂(活血化瘀药)。干预:采用线栓法制备大鼠大脑中动脉阻塞模型(经2h 大脑中动脉阻断后再灌注24h)。138只大鼠随机分6组,每组23只。假手术组:结扎各血管,不阻塞大脑中动脉。灌药1组:术前30m in 灌胃给予2g/kg 活血化瘀药。灌药2组:术前30m in 灌胃给予2.5g/kg 活血化瘀药。灌药3组:术前连续7d 灌胃给予2g/kg 活血化瘀药。灌药4组:术前连续7d 灌胃给予2.5g/kg 活血化瘀药。对照组给予等容积生理盐水。①全部大鼠清醒后于缺血2h 再灌注24h 进行神经功能缺损评分(5分制,0～1分为神经功能轻度缺损,2～4分为神经功能重度缺损)。②再灌注24h 后从各组大鼠中随机选取10只,采用速率散射比浊法检测脑匀浆、血清C 反应蛋白,补体C3,C4水平及血清IgG 含量。③再灌注24h后从各组大鼠中随机选取10只,麻醉后迅速断头取脑,然后在110℃烘箱中烘干至恒重,计算脑组织含水量。④光学显微镜下观察各组大鼠脑组织细胞形态。⑤再灌注24h 后从各组大鼠中随机选取3只,透射电镜观察各组大鼠脑组织超微结构。主要观察指标:①各组大鼠脑组织神经功能缺损评分结果。②各组大鼠脑匀浆、血清C 反应蛋白及补体C3和C4水平及血清IgG 含量。③各组大鼠脑组织含水量及脑组织细胞形态。④各组大鼠脑组织超微结构变化。结果:138只大鼠全部进入结果分析。①灌药各组大鼠缺血再灌注24h后重度神经功能缺损所占比例明显低于对照组(P <0.01),灌药4组低于灌药2组(13%,48%,χ2=6.571,P <0.05)。②脑组织含水量:假手术组和灌药各组大鼠脑组织含水量低于对照组(P <0.05)。灌药3组低于灌药1组(P <0.01)。③血清C 反应蛋白含量:假手术组和灌药各组低于对照组(P <0.01)。④血清补体C3含量:假手术组和灌药各组低于对照组(P <0.01)。⑤血清补体C4含量:假手术组和灌药各组低于对照组(P <0.01)。灌药3组低于灌药1组(P <0.01),灌药4组低于灌药2组和灌药3组(P <0.05)。⑥血清IgG 含量:假手术组和灌药各组低于对照组(P <0.01)。灌药4组低于灌药2组(P <0.05)。⑦脑匀浆C反应蛋白含量:假手术组和灌药各组低于对照组(t=5.626～17.929,P <0.01),灌药3组低于灌药1组(P <0.01),灌药4组低于灌药2组(P <0.05)。⑧脑匀浆补体C3水平:假手术组和灌药各组低于对照组(P <0.05～0.01)。灌药3组低于灌药1组(P <0.01)。灌药4组低于灌药2组(P <0.01)。⑨脑匀浆补体C4含量:假手术组和灌药各组低于对照组(P <0.05～0.01)。灌药3组低于灌药1组(P <0.01)。灌药4组低于灌药2组和灌药3组(P <0.01)。⑩脑组织充血水肿情况:对照组炎症反应明显,灌药组脑水肿、病理损害较对照组轻,⒒假手术组超微结构正常;对照组皮质坏死边缘区的细胞、毛细血管、髓鞘水肿明显,神经元细胞器减少,灌药3,4组细胞膜界限清楚,结构完整,线粒体丰富,大小均匀,有髓纤维形态正常,灌药1,2组介于两者之间。结论:①给予活血化瘀药使大鼠神经功能缺损评分降低,用药时间长的大鼠神经功能缺损评分低,提示用药时间长能更好地改善大鼠神经功能缺损程度。②给予活血化瘀中药可使大鼠脑匀浆和血清中补体C3和C4水平明显降低,血清IgG 含量降低,说明该药可通过启动补体系统而减轻对脑组织的损伤,C 反应蛋白也明显降低,更进一步提示该药可抑制炎症反应。③用药时间越长脑损伤越轻,且用药剂量2.5g/L 效果较好。更多还原

【Abstract】 BACKGROUND It has been known that re-recovery of blood flow after cerebral ischema may aggravate brain injury. It has been verified in some researches that the components for activating blood circulation and removing stasis in Chinese herbs can resist cerebral ischemic reperfusion injury.OBJECTIVE To observe 4 most basic Chinese herbs for activating blood circulation and resolving stasis honghua Flos Carthami taoren Semen Persicase chuanxiong Rhizoma ligustici Chuanxiong and chishao Radix Paeoniae Rubra as the exertive factors on their influences on immune index changes in IgG C-reactive protein CRP and complements of serum and homogenate in brain tissue so as to verify their graded and time effects.DESIGN Randomized control experiment and single blind method.SETTING Guangzhou Red Cross Hospital and Department of Neurology of the Fourth Hospital Affiliated to Jina University.MATERIALS The experiment was performed from January 2001 to December 2002 in Experimental Room of Traumatic Institute of Guangzhou Red Cross Hospital. Totally 138 SD female adult rats were employed mass weighted varied from 280 to 300 g provided from Experimental Animal Center of Guangzhou University of Chinese Medicine. Honghua Flos Carthami taoren Semen Persicase chuanxiong Rhizoma ligustici Chuanxiong and chishao Radix Paeoniae Rubra were the concentrated granules individually. At the ratio of 1∶1∶1∶2 they were prepared into 2.5 g/mL raw herbal decoction huoxue huayu tang.INTERVENTIONS Thread embolic method was used to prepare cerebral medium sized artery obstruction model in rats reperfusion for 24 hours after cerebral medium sized artery obstruction for 2 hours. Totally 138 rats were randomized into 6 groups 23 rats in each. Sham operation group ligature was done in each vessel without blocking medium sized artery of cerebrum. First perfusion group 30 minutes before operation gastric perfusion was done with 2 g/kg huoxue huayu tang. Second perfusion group 30 minutes before operation gastric perfusion was done with 2.5 g/kg huoxue huayu tang. Third perfusion group before operation gastric perfusion was done with 2 g/kg huoxue huayu tang continuously for 7 days. Fourth perfusion group before operation gastric perfusion was done with 2.5 g/kg huoxue huayu tang continuously for 7 days. In the control physiological saline of same dose was given at the same time points continuously for 7 days. ① In all of the groups when rats woke up after 24 hours reperfusion and 2 hours ischemia neural functional deficits were evaluated 5-score system 0-1 mild neural functional deficits 2-4 severe neural functional deficits. ② After 24 hours reperfusion 10 rats were randomized from each group for determination of the contents of CRP in homogenate and serum complement C3 and C4 as well as serum IgG with rate nephelometer. ③ After 24 hours reperfusion 10 rats were randomized from each group. After anesthetized they were sacrificed rapidly to collect brains. Dried over heat in oven at 110°C till constant weight the water content of brain tissue was calculated. ④ Cytomorphology of brain tissue observed under optic microscope in rats of each group. ⑤After 24 hours reperfusion 3 rats were randomized from each group to observe ultrastructure of brain tissue under transmission electron microscope TEM of rats in each group. MAIN OUTCOME MEASURES ① Evaluation of neural functional deficits of brain tissue in rats of each group. ② Contents of CRP in serum and homogenate complement C3 and C4 as well as serum IgG in rats of each group. ③ Water content and cytomorphology of brain tissue in rats of each group. ④ Ultrastructure of brain tissue in rats of each groupRESULTS Totally 138 rats all entered result analysis. ① The percentage of severe neural functional deficits after 24 hours ischemic reperfusion in rats of each perfusion group was lower remarkably than the control P < 0.01 in which that in fourth perfusion group was lower than second group 13% 48% χ2=6.571 P < 0.05. ② Content of brain tissue That in sham operation group and each perfusion group was lower than the control P < 0.05 in which that in third perfusion group was lower than first groupP < 0.01. ③ Content of serum CRP That in sham operation group and each perfusion group was lower than the control  P < 0.01. ④ Content of serum complement C3 That in sham operation group and each perfusion group was lower than the control P < 0.01. ⑤ Content of serum complement C4 That in sham operation group and each perfusion group was lower than the control  P < 0.01 in which that in third perfusion group was lower than first group P < 0.01 That in fourth perfusion group was lower than second and third group  P < 0.05. ⑥ Content of serum IgG That in sham operation group and each perfusion group was lower than the control P < 0.01 in which that in fourth perfusion group was lower than second groupP < 0.05. ⑦ Content of CRP in brain homogenate that in sham operation group and each perfusion group was lower than the control t=5.626-17.929 P < 0.01 in which that in third perfusion group was lower than first group P < 0.01 that in fourth group was lower than second group P < 0.05. ⑧ Level of complement C3 in brain homogenate That in sham operation group and each perfusion group was lower than the control P < 0.05-0.01 in which third perfusion group was lower than first group P < 0.01 that in fourth group was lower than second group  P < 0.01. ⑨ Level of complement C4 in brain homogenate That in sham operation group and each perfusion group was lower than the control  P < 0.05-0.01 in which that in third perfusion group was lower than first group P < 0.01 that in fourth group was lower than second and third groups P < 0.01. ⑩ Congestive edema in brain tissue The inflammatory reaction in the control was obvious. Brain edema and pathological damage in perfusion groups were milder than the control. Ultrastructure was normal in sham operation group. The edema was obvious in cells capillaries and myelin sheath in cortical necrosis margin area and neuronal cell organs were decreased in the control. In third and fourth perfusion groups cellular membrane was clear in boundary line integrative in structure rich in mitochondria and even in size and the morphology of medullated fibers was normal. The situation of first and second perfusion was between two of them.CONCLUSION ① Huoxue huayu tang decreases neural functional deficits in the evaluation of rats and the score with long medication was lower suggesting that long medication can even better improve neural functional deficits. ② Huoxue huayu tang reduces remarkably the level of complement C3 and C4 in brain homogenate and serum and decreases serum IgG content explaining that the medicine alleviates brain tissue injury by startup complement system. The remarkable decrease of C-reactive protein further suggests the inhibition of such medicine on inflammatory reaction. ③ It is concluded that the longer the medication is the milder the brain injury is and the better result is achieved at dosage of 2.5 g/L.更多还原