背景:黄芪可通过减轻兴奋性氨基酸的释放及在细胞间的堆积、缓解Ca2+超载、抗氧化等途径抑制凋亡的发生.目的:将黄芪用于未成熟脑缺氧缺血脑损伤的治疗,一方面检测其对海马缺氧缺血后半胱氨酸天冬氨酸蛋白酶-3 mRNA表达水平的影响,另一方面通过迷宫实验观察黄芪对缺氧缺血脑损伤的成熟鼠学习记忆能力的干预.设计:随机对照实验.单位:东南大学临床医学院/附属中大医院儿科,基础医学院病理科.材料:实验于2002-10/2003-06在东南大学临床医学院实验中心完成.选取出生7 d的同窝SD大鼠114只,随机分成3组:假手术组18只,模型组48只,黄芪治疗组48只.黄芪注射液由成都地奥九泓制药厂生产,规格为10 mL/支,含生药20 g.方法:模型组与黄芪治疗组建立缺氧缺血脑损伤模型,假手术组不造模.黄芪治疗组于造模后即刻及每天同一时间腹腔注射0.08 mL黄芪注射液,7 d后停药,模型组于同时间腹腔注射等量生理盐水,假手术组不给药.黄芪治疗组及模型组在缺氧缺血后24 h,5 d断头取脑,假手术组于假手术后24 h断头取脑.各组海马区脑损伤行组织病理学检测,半胱氨酸天冬氨酸蛋白酶-3 mRNA的表达采用半定量反转录-聚合酶反应方法进行检查,成年90 d龄的大鼠进行三等分迷宫测试其学习记忆能力,3个实验各自独立.主要观察指标:①各组海马区脑损伤组织病理学检测.②各组结扎侧海马半胱氨酸天冬氨酸蛋白酶-3 mRNA的表达.③三等分迷宫试验结果.结果:实验纳入大鼠114只,全部进入结果分析.①各组海马区脑损伤组织病理学检测:假手术组双侧海马区组织无水肿、坏死,神经细胞形态正常,神经细胞数为(87.7±0.6)×103/高倍视野.模型组24 h时结扎侧海马区水肿,细胞周围间隙增宽,神经细胞数减少为(68.8±3.0)×103/高倍视野,与假手术组比较差异显著(P＜0.01);5 d时结扎侧海马体积缩小,锥状细胞层紊乱,神经细胞稀少至(48.7±2.2)×103/高倍视野,与假手术组及同侧24 h时比较均有显著差异(P＜0.01).黄芪治疗组24 h时结扎侧海马区组织水肿较模型组明显减轻,5 d时可观察到完整的海马形态,此两时间点神经细胞死亡率均较模型组明显减低(P＜0.01).②各组结扎侧海马半胱氨酸天冬氨酸蛋白酶-3 mRNA的表达:假手术组以低水平表达,吸光度值为0.220±0.009.模型组于缺氧缺血后逐渐升高,6 h时比假手术组升高11%,至24 h时mRNA水平达高峰,较假手术组约升高260%(P＜0.01),高峰持续至48 h后下降,5 d和7 d时恢复基础水平.黄芪治疗组变化趋势与模型组相似,但在24,48 h两时间点时峰值降低了44%～46%,与模型组比较差异显著(P＜0.01).③三等分迷宫试验结果:与模型组比较,黄芪治疗组达到学会标准所需训练次数明显减少[(45.7±2.7),(16.1±2.5)次,P＜0.01],缺氧缺血24 h后记忆保持率显著提高[(48.3±11.7),(80.0±9.0)%,P＜0.01].结论:黄芪可以有效抑制未成熟脑缺氧缺血损伤后海马区神经细胞的凋亡,提高神经细胞存活率,此种保护作用与抑制半胱氨酸天冬氨酸蛋白酶-3的表达有关.同时黄芪能够明显改善未成熟脑缺氧缺血损伤后的学习记忆能力.%BACKGROUND: Astragalus root can inhibit apoptosis through reducing the release and interstitial accumulation of excitatory amino acids, alleviating calcium overloading and antioxidative effect.OBJECTIVE: Astragalus root was used to treat anoxic-ischemic brain injury in immature brain. We evaluated the effect of astragalus root on caspase-3 mRNA expression, and meanwhile, labyrinth test was employed to investigate the intervention of astragalus root on learning and memory function of mature rats after anoxic-ischemic brain injury.DESIGN: Randomized and controlled study.SETTING: Pediatric Department, Zhongda Hospital Affiliated to the Medical College of Southeast University; Pathological Department, the Basic Medical Sciences Institute of Southeast University.MATERIALS: From October 2002 to June 2003, this study was conducted at the Experiment Center of the Medical College, Southeast University.A batch of 114 seven-day-old SD rats were selected from the same brood and divided into 3 groups, namely, sham-operation group (n=18), model group (n=48) and astragalus root group (n=48). Astragalus injection was produced by Chengdu DIAO Pharmaceutical Factory, with 10 mL astragalus injection corresponding to 20 g raw material.METHODS: Animal model of anoxic-ischemic brain injury was established in model group and astragalus root group, but was not established in sham-operation group. In astragalus root group, immediately after establishing anoxic-ischemic model and at the same time point each day, 0.08 mL astragalus injection was administered intraperitoneally until the 7th postoperative day. In model group, 0.08 mL normal saline was administered at the same time points. In sham-operation group, no treatment was given. In astragalus root group and model group, animals were decollatedat 24 hours and 5 days postoperatively to take out the brains. In sham-operation group,animals were decollated and their brains were taken out at 24 hours postoperatively. In all the groups, hippocampal brain injury was detected using histopathological method combined with semi-quantified RT-PCR methods for detecting caspase-3 mRNA. Adult rats aged 90 days were used in modified y maze to examine their learning and memory functions. All these three experiments were independent.MAIN OUTCOME MEASURES:① Hippocampal brain injury in each group was evaluated using pathological method.② Caspase-3 mRNA in the ligated side of hippocampus was detected.③ Results of modified Y maze test were analyzed.RESULTS:All of the 114 rats entered the statistical analysis.① Assessment ofhippocampal brain injury in each group with pathological method:In sham-operation group, the bilateral hippocampus showed no swelling or necrosis, and neural cells in this area had normal morphological features with a density of (87.7±0.6) × 103 per high amplification field. In model group, the ligated side of hippocampus was swollen with a widened spatium and the cell density decreased to (68.8±3.0) × 103 per high amplification field, which significantly differed from that in sham-operation group (P ＜ 0.01). At the fifth day, the volume of ligated side of hippocampus reduced with pyramid layer disorganized and neural cells sparse at a density of (48.7±2.2) × 103 per high amplification field. These changes were significantly different from those of sham-operation group and the same side at 24 hours (P ＜ 0.01). At 24 hours the ligated side of hippocampus was less swollen in astragalus root group than in model group.At day 5, the whole hippocampus was observed. At these two time points,cell death rate in astragalus root group was significant lower than that in model group (P ＜ 0.01).②Caspase-3 mRNA in the ligated side of hippocampus in all the groups: In sham-operation group, the expression of caspase-3 was low, with an absorbency value of 0.220±0.009. In model group, after ischemia and anoxia its expression increased. At 6 hours, it was 11% higher than that in sham-operation group. In astragalus root group, mRNA level reached its peak, which was 260% higher than that in sham-operation group (P ＜ 0.01). The peak of mRNA continued, decreased after 48 hours and returned to baseline at 5 days and 7 days. The fluctuation of mRNA was similar between astragalus root group and model group,but the peak value at 24 hours and 48 hours in astragalus root group was 44％-46％ lower than that in model group (P ＜ 0.01). ③ Results of modified Y maze test: As compared to model group, in astragalus root group, the number of training times for meeting the standard made by the Association was significantly smaller [(45.7±2.7), (16.1±2.5) times, P ＜ 0.01] and at 24 hours after anoxia and ischemia, memory retention was significantly higher [(48.3±11.7), (80.0±9.0)%, P ＜ 0.01].CONCLUSION: Astragalus root can effectively inhibit the apoptosis of neural cells in hippocampus in immature brain after anoxia and ischemia and enhance the survival rate of them. This protective effect may be related to its inhibitory effect on the expression of caspase-3. Meanwhile, astragalus root can dramatically improve learning and memory function of the immature brain after anoxia and ischemia.
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