活血化瘀药对大鼠脑缺血再灌注损伤的保护作用时效量效关系对照验证

背景脑缺血后再灌注性血脑屏障损伤是脑缺血和再灌注损伤的重要病理生理基础.目的观察以活血化瘀的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和脑组织匀浆中的一氧化氮、免疫球蛋白、C反应蛋白、补体等免疫指标及脑组织细胞形态和超微结构产生的变化,并验证其时效和量效关系.设计随机对照实验,单盲法评估.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.红花、川芎、桃仁、赤芍为单味中药饮片浓缩颗粒,按1112比例制成含2.5 g/mL生药汤剂(活血化瘀药).实验动物选择成年雌性SD大鼠138只,体质量280～300 g,由广州中医药大学实验动物中心提供.干预采用线栓法制备大鼠大脑中动脉阻塞模型(经2 h大脑中动脉阻断后再灌注24h).138只大鼠随机分为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30 min灌胃给予2 g/kg活血化瘀药.灌药2组术前30 min灌胃给予2.5 g/kg活血化瘀药.灌药3组术前连续7 d灌胃给予2 g/kg活血化瘀药.灌药4组术前连续7 d灌胃给予2.5 g/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2 h再灌注24 h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24 h后从各组大鼠中随机选取10只,检测脑匀浆、血清C反应蛋白及补体C3和C4水平采用速率散射比浊法,检测脑匀浆和血清中一氧化氮浓度采用硝酸还原酶法.③再灌注24 h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24 h后从各组中随机选取3只大鼠,制备脑组织冠状切片,采用透射电镜观察各组大鼠脑组织超微结构.主要观察指标①各组大鼠神经功能缺损评分结果.②各组大鼠脑匀浆及血清中C反应蛋白及补体C3和C4水平,一氧化氮浓度.③各组大鼠脑组织含水量.④各组大鼠脑组织细胞形态和脑组织超微结构.结果138只大鼠全部进入结果分析.①各组大鼠神经功能缺损程度比较灌药各组大鼠缺血再灌注24 h后重度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(P＜0.05).②各组大鼠脑组织含水量比较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).③各组大鼠脑匀浆一氧化氮浓度比较假手术组和灌药各组大鼠低于对照组(P＜0.01).灌药3组低于灌药1组(P＜0.05).灌药4组低于灌药2,3组(P＜0.01).④各组大鼠血清一氧化氮浓度比较假手术组和灌药各组血清一氧化氮浓度均高于对照组(P＜0.01).灌药3组高于灌药l组(P＜0.05).灌药4组高于灌药2,3组(P＜0.05).⑤各组大鼠脑匀浆和血清C反应蛋白水平比较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2,3组(P＜0.05～0.01).⑥各组大鼠脑匀浆和血清补体C3水平比较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＜0.05).灌药4组低于灌药2,3组(P＜0.01).⑦各组大鼠脑匀浆和血清血清补体C4水平比较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l组(P＜0.05).灌药4组低于灌药2,3组(P＜0.01).⑧各组大鼠脑水肿情况比较对照组脑组织明显充血水肿,表明炎症反应明显.灌药组脑水肿、病理损害较对照组轻.⑨各组大鼠脑组织超微结构的改变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,提示大鼠神经功能缺损程度改善与用药时间延长有关.②给予活血化瘀中药大鼠脑组织一氧化氮浓度降低,血清中一氧化氮浓度增高,说明活血化瘀中药可以逆转缺血再灌注后一氧化氮浓度在不同组织中的异常改变来减轻脑损伤.③给予活血化瘀中药大鼠脑组织和血清中补体C3和C4水平明显降低,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.④用药时间越长脑损伤越轻,且用药剂量以2.5 g/L效果较好.

Protective effect of blood-activating and stasis-resolving medicines against cerebral ischemia-reperfusion injury in rats: A controlled trial to verify the time-effect and dose-effect relation

BACKGROUND: The injury of blood-brain barrier following cerebral ischemia reperfusion is a considerate pathological basis for injury caused by cerebral ischemia and reperfusion.OBJECTIVE: To investigate the effects of four most basic Chinese medicinal herbs, or safflower, peach seed, ligusticum and red peony with actions of activating blood and resolving the stasis on the contents of nitric oxide, immunoglobulins, C-reactive protein (CRP) and complements (immunological indices) of serum and cerebral homogenate, as well as the morphological and structural changes of cerebral tissue cells in rats with ischemia and reperfusion to vertify relationship between time effectiveness and quantitative effectiveness.DESIGN: A randomized controlled study, and evaluation by single blind.MATERIALS: The experiment was completed in the Laboratory of Traumatology Institute of Guangzhou Red Cross Hospital from January 2001 to De cember 2002. Safflower, peach seed, szechwan lovge rhizome and red peony are concentrated granules of single decocting pieces, the blood activating and stasis resolving decoction was prepared at 2.5 g/L according to 1:1:1:2 ratio.Totally 138 adult female SD rats were selected for the experiment, weighing 280-300 g, provided by Animal Center of Guangzhou University of TCM.INTERVENTIONS: The models rats with middle cerebral artery occlusion were set up by thread ligation (24 hours reperfusion after 2 hours middle cerebral artery occlusion). All 138 rats were randomly divided into 6 groups with 23 in each group. Sham operation group: The vessels were ligated but the middle cerebral artery was not occluded. No.1 medicated group: The BASR was by gavage given in a dose of 2 g/kg 30 minutes before operation. No. 2 medicated group: The BASR was by garage given in a dose of 2.5 g/kg 30 minutes before operation. No. 3 medicated group: The BASR was by garage given in a dose of 2 g/kg for consecutive 7 days be fore operation. No. 4 medicated group: The BASR was by gavage given in a dose of 2.5 g/kg for consecutive 7 days before operation. Control group:the same volume of saline was by gavage given for consecutive 7 days before operation. ① Scoring of dysneuria (5-score system: 0-1 score meant mild dysneuria, 2-4 scores meant severe dysneuria) for all rats were performed after consciousness following 2 hours ischemia and 24 hours reperfusion. ② After 24 hours reperfusion, 10 rats in each group were at random selected for assay of levels of CRP, complement 3 (C3) and complement 4 (C4) (rate nephelometry), and concentration of nitric oxide (nitrate reductase method) in both cerebral homogenate and serum. ③ After 24hours reperfusion, 10 rats in each group were at random selected, and after anesthesia was completed, the brain was quickly collected through decapitation, put into a 110 ℃ drying oven till its constant weight, then the water content in brain was calculated. ④ The cerebral cytomorphology in every group was observed under light microscope. ⑤ After reperfusion, 3 rats in each group were randomly selected for preparation of coronal section of cerebral tissue, the cerebral ultrastructure in each group was observed under transmission electronic microscope.MAIN OUTCOME MEASURES: ① The results of dysneuria scoring in each group. ② The levels of CRP, C3 and C4, and concentration of nitric oxide in both cerebral homogenate and serum. ③ The water content in brain. ④ The cerebral cytomorphology and the cerebral ultrastructure. RESULTS: All 138 rats entered into the result analysis. ① Comparison of the extents of dysneuria of rats in each group: The ratio of severe dysneuria after 24 hours ischemic reperfusion in all medicated groups was obviously lower than that in control group (P ＜ 0.01), and the ratio in No.4 medicated group was lower than that in No.2 medicated group (P ＜ 0.05).② Comparison of water contents in brain of rats in each group: The water contents in sham operation group and all medicated groups were obviously lower than that in control group (P ＜ 0.05). ③ Comparison of the nitric oxide concentration in cerebral homogenate of rats in each group: The concentration in sham operation group and all medicated groups were obviously lower than that in control group (P ＜ 0.01). The concentration in No. 3medicated group was obviously lower than that in No. 1 medicated group (P ＜ 0.05). The concentration in No. 4 medicated group was obviously lower than that in No. 2 medicated group (P ＜ 0.01). ④ Comparison of the nitric oxide concentration in serum of rats in each group: The concentrations in sham operation group and all medicated groups were obviously lower than that in control group (P ＜ 0.01). The concentration in No. 3 medicated group was higher than that in No. 1 medicated group (P ＜ 0.05). The concentration in No. 4 medicated group was higher than those in No. 2 and in No. 3 medicated groups (P ＜ 0.05). ⑤ Comparison of the levels of CRP in cerebral homogenate and serum of rats in each group: The levels in sham operation group and all medicated groups were lower than that in control group (P ＜ 0.05-0.01). The level in No. 3 medicated group was lower than that in No. 1 medicated group (P ＜ 0.01). The level in No. 4 medicated group was lower than those in No. 2 and in No. 3 medicated groups (P ＜0.05-0.01). ⑥ Comparison of the levels of C3 in cerebral homogenate and serum of rats in each group: The levels in sham operation group and all medicated groups were lower than that in control group (P ＜ 0.05-0.01). The level in No. 3 medicated group was lower than that in No. 1 medicated group (P ＜ 0.05). The level in No. 4 medicated group was lower than those in No. 2 and in No. 3 medicated groups (P ＜ 0.01). ⑦ Comparison of the levels of C 4 in cerebral homogenate and serum of rats in each group: The levels in sham operation group and all medicated groups were lower than that in control group (P ＜ 0.05-0.01). The level in No. 3 medicated group was lower than that in No. 1 medicated group (P ＜ 0.05). The level in No.4 medicated group was lower than those in No. 2 and in No. 3 medicated groups (P ＜ 0.01). ⑧ Comparison of the condition of cerebral edema of rats in each group: In control group there was obvious cerebral congestive edema, indicating an obvious infection; while in medicated groups the extent of cerebral edema was milder than that in control group. ⑨ Changes of cerebral ultrastructure of rats in each group: The ultrastructure in sham group was normal. In control group, there obvious edema of cells, capillaries and sheaths in the marginal zone of cortex necrosis, and reduction of organelles of neuron. As well. In No. 3 and No. 4 medicated groups, the limits of cell membranes were clear, the structure was integral, the chondriosomes were rich and even in size, the medullated fibers were morphologically normal. And in No. 1 and No. 2 medicated groups the changes were between the twoCONCLUSION: ① The scoring of dysneuria in rats was decreased after the blood-activating and stasis-resolving medicine was given, and it was lower in rats that were given a longer period of medication, indicating that the improved extent for dysneuria is related to prolonged medication. ②The nitric oxide concentration of cerebral tissue in rats that recevied the blood-activating and stasis-resolving medicine was decreased, and the nitric oxide concentration of serum in the rats was increased, indicating that the blood-activating and stasis-resolving medicine can reverse the anomalies of nitric concentration in different tissues after ischemic reperfusion so as to reduce cerebralinjury. ③ The levels of C3 and C4 of cerebral tissue and serum in rats that received the blood-activating and stasis-resolving medicine were obviously decreased, indicating that the medicine may reduce the cerebral injury through triggering complement system; and the CRP was also get decreased, further suggesting that the medicine can inhibiting infective reaction. ④ The longer the period of medication, the milder the cerebral injury, and the dose of 2.5 g/L was better in effect.