肌苷对脑缺血再灌注后神经细胞凋亡和细胞色素C基因表达的影响

背景细胞色素C(chromosome C,CytC)的释放是细胞凋亡的重要环节之一.肌苷对脑缺血损伤神经细胞凋亡可能具有一定的抑制作用,但其机制尚不十分清楚.目的研究肌苷对大鼠局灶性脑缺血再灌注后神经细胞凋亡和CytC基因表达的影响.设计随机对照的实验研究.地点和材料本实验在青岛大学医学院脑血管病研究所和山东省脑血管病防治重点实验室完成.成年健康雌性SD大鼠68只,体质量230～280 g,清洁级,由中国科学院上海实验动物中心提供.干预应用线栓法建立大鼠大脑中动脉阻塞再灌注模型,随机分为治疗组(腹腔注射肌苷,100mg/kg)32只和对照组(腹腔注射生理盐水)32只,每组再随机分为缺血1.5 h再灌注2,6,12,24 h,2,3,7,14 d组(n=4),另外4只作假手术组.原位末端标记和原位杂交技术分别观察神经细胞凋亡和CytC mRNA表达.主要观察指标脑缺血再灌注后神经细胞凋亡和CytC mRNA表达.结果脑缺血再灌注后2 h皮质区与纹状体区即出现凋亡细胞并逐渐增加,分别于1 d和2 d达高峰,之后逐渐减少,至14 d接近于假手术组水平;经肌苷治疗后凋亡神经细胞减少,其中再灌注12 h～7 d较对照组有显著性差异.CytC mRNA于脑缺血再灌注2h开始表达,皮质区12 h达高峰,纹状体区1 d达高峰,以后逐渐下降;肌苷治疗组CytC mRNA表达于再灌注12 h～7 d在皮质区、12 h～14d在纹状体区较对照组显著降低.结论肌苷可能通过抑制细胞凋亡及相关基因表达而发挥其神经保护作用,对治疗脑血管病有潜在的应用价值.

Effects of inosine on apoptosis and expression of cytochrome CmRNA in neurons after cerebral ischemia-reperfusion in rats

BACKGROUND: The release of cytochrome C (Cyt C) from mitochondria is a critical step in the apoptosis process. Inosine could play a certain inhibiting role in neuronal apoptosis after cerebral ischemic injury, but its mechanism is not known thoroughly.OBJECTIVE: To investigate the effects of inosine on neuronal apoptosis and expression of cytochrome C mRNA in rats after focal cerebral ischemic reperfusion.DESIGN: A randomized controlled experimental research.SETTING and MATERIALS: This experiment was carried out in the Institute of Cerebrovascular Diseases, Medical College of Qingdao University and the Key Laboratory of the Prevention and Cure of Cerebrovascular Diseases of Shandong Province. Sixty-eight adult healthy female SD rats,weighting 230 - 270 g, clearing grade, were purchased from Shanghai Experimental Animal Center of Chinese Academy of Science.INTERVENTION: The model of ischemic reperfusion in SD rats was established by middle cerebral artery occlusion with a nylon monofilament suture. The rats were randomly divided into treatment group(32 rats injected with inosine intraperisoneally, 100 mg/kg) and control group(32 rats injected with saline solution intraperisoneally). Each group was then randomly divided into eight subgroups with 4 rats in each at 2 hours, 6, 12, 24 hours,and 2, 3, 7, 14 days of reperfusion after 1.5 hours' ischemia. The other 4rats served as sham-operation group. In situ hybridization was performed to examine the expression of cytochrome C mRNA while terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-label ing(TUNEL) staining was made to characterize apoptosis.MAIN OUTCOME MEASURES: Neuronal apoptosis and expression of cytochrome C mRNA in brain tissues after cerebral ischemic reperfusion.RESULTS: TUNEL-positive cells were observed at 2 hours of reperfusion, the number increased gradually and peaked at 1 day and 2 days of reperfusion in the cortex and striatum, respectively, then reduced to the level of sham-operation group at 14 days. In inosine group, the number of TUNEL-positive cells decreased during 12 hours-7 days of reperfusion compared to that of the control group. Cytochrome C mRNA was expressed in the cortex and striatum of ischemic hemisphere as early as 2 hours of reperfusion, reached a peak at 12hours and 1 day in the cortex and striatum, respectively. Inosine treatment could diminish the cytochrome C mRNA expression at 12 hours to 7 days in the cortex and 12 hours to 14 days in the striatum, respectively.CONCLUSION: Inosine can exert a protective effect on nerves by inhibiting apoptosis and related mRNA expression. Therefore, it may be a potential compound in treating cerebrovascular diseases.