葛根素对大鼠脑复苏后海马CA1区神经细胞凋亡相关基因的影响(英文)

Effect of puerarin on apoptosis-related gene of nerve cell in hippocampus CA1 region of rats after cerebral resuscitation

背景:Fas及 P53是重要的促进凋亡的调控基因,属于肿瘤坏死因子 /神经生长因子受体家族,其表达产物具有传递凋亡信号的作用,可调控脑缺血再灌注损伤时细胞的凋亡,而葛根素则能够减轻细胞的凋亡程度。目的:观察葛根素对大鼠脑复苏后海马 CA1区神经细胞凋亡相关基因Fas及 P53的影响。设计:随机对照实验。单位:华中科技大学同济医学院附属同济医院急诊科。材料:实验于 2001-09/2002-02在华中科技大学同济医学院附属同济医院急诊科实验室完成。选取清洁级 3个月龄 W istar大鼠 45只,随机分为假手术组、模型对照组、葛根素治疗组,15只 /组。方法:葛根素治疗组及模型对照组建立大鼠急性全脑缺血再灌注模型。假手术组只分离第一颈椎两侧翼小孔,但不电凝两侧椎动脉,只分离两侧颈总动脉,但不夹闭之。葛根素治疗组于缺血前 1h 给予葛根素注射液 100m g/kg,模型对照组给予等量生理盐水,假手术组不给药。各组大鼠分别于脑缺血再灌注后 3,6,12,24,48h 即速处死,每次每组 3只。分离海马组织,制备组织切片,利用免疫组化法、原位末端标记法检测各组大鼠脑缺血再灌注后不同时间点 Fas及 P53蛋白表达的水平及凋亡细胞数的变化。主要观察指标:①各组脑缺血再灌注后不同时间点海马 CA1区 Fas及P53蛋白表达的阳性细胞数。②各组脑缺血再灌注后不同时间点海马CA1区凋亡细胞数的比较。结果:实验纳入大鼠 45只,全部进入结果分析。①各组脑缺血再灌注后不同时间点海马 CA1区 Fas蛋白表达的阳性细胞数:假手术组未见明显Fas 基因表达。与模型对照组比较,葛根素治疗组于脑缺血再灌注后各时间点均明显降低,6,12,24,48h 时差异显著 [(15.0±4.3),(13.5±4.9);(40.7±3.4),(27.2±3.1);(37.0±4.8),(22.0±2.1);(24.7±4.1),(18.9±5.3)个 /m m ;P <0.05,P <0.01]。②各组脑缺血再灌注后不同时间点海马 CA1区P53蛋白表达的阳性细胞数:假手术组未见明显 P53基因表达。与模型对照组比较,葛根素治疗组于脑缺血再灌注后 24,48h 均明显降低[(25.3±4.4),(12.8±2.7);(24.3±3.6),(10.9±3.0)个 /m m ;P <0.01]。③各组脑缺血再灌注后不同时间点海马 CA1区凋亡细胞数的比较:与模型对照组比较,葛根素治疗组于脑缺血再灌注后 12,24,48h 均明显降低[(34.0±3.7),(21.0±3.7);(41.0±4.2),(33.0±4.8);(71.0±5.5),(41.0±3.4)个 /m m ;P <0.01]。结论:给予葛根素治疗的大鼠缺血再灌注 6～48h 时 Fas的表达显著降低,缺血再灌注 24～48h 时 P53的表达亦明显减少,且凋亡细胞数也呈下降趋势,进一步证实葛根素的脑保护作用,提示葛根素抑制脑复苏后的细胞凋亡可能与其减少促凋亡基因 Fas及 P53蛋白表达有关。

BACKGROUND： Fas and P53 are important regulator and control gene which can promote apoptosis. They belong to the receptor family part of tumor necrotic factor/nerve growth factor. Their expression products have effects on apoptosis signal transmission, and can regulate and control cell apoptosis in cerebral ischemia-reperfusion injury. And puerarin can alleviate the level of cell apoptosis. OBJECTIVE： To observe the effect of puerarin on Fas and P53, the apoptosis-related gene of nerve cell in hippocampus CA1 region of rats after cerebral resuscitation. DESIGN＂ Randomized controlled trial. SETTING： Department of Emergency, Tongji Hospital, Tongji Medical College, Huazbong University of Science and Technology. MATERIALS： The experiment was carried out at Emergency Department, Tongji Hospital, Tongji Medical College, Huazhong Univesity of Science and Technology from September 2001 to Februray 2002. Totally 45 of 3 months old Wistar rats of clean grade were selected, and randomly divided into 3 groups： sham operation group, model control group and peerarin treatment group with 15 rats in each group. METHODS： Acute global brain ischemia-reperfusion models were established in rats of puerarin treatment group and model control group. In rats of sham operation group, stigmata of both flanks of the first cervical vertebrae were isolated, but bilateral vertebral arteries were not electric coagulated, and blolateral common carotid arteries were only isolated without clamping close. Rats in pucrarin treatment group were given puerarin injection 100 mg/kg,1 hour before ischemia, and model control group were given normal saline in equivalence while rats in sham operation group were not given medicine. Death of rats in each group was performed separately in the 3^rd, 6^th, 12^th 24^th and 48^th hours after cerebral ischemia-reperfusion with 3 rats per group in each time. Hippocampus tissues of rats were isolated, and tissue slices were preparated. And the changes of.the protein expression levels and the number of apoptosis cells of rats in each group at different time point after cerebral ischemia-reperfusion were detected in inununo-histochemical method and end labelling in situ method. MAIN OUTCOME MEASURES： （1） The number of positive cells in protein expression of Fas and P53 in hippocampus CA1 region of rats in each group at different time point after cerebral ischemia-reperfosion was studied.（2） Comparison of the number of apoptosis cells in hippocampus CA1 region of rats between groups at different time point after cerebral ischemia-reperfusion were studied, too. RESULTS： All the 45 rats enrolled in researchwere entered the stage of result analysis： （1） The number of positive cells in protein expression of Fas in hippocampus CA1 region of rats in each group at different time point after cerebral ischemia-reperfusion： Obvious gene expression of Fas was not found in sham operation group. In contrast with model control group, obvious decrease was found at all time points after cerebral ischemia-reperfusion in puerarin treatment group, and in the 6^th, 12^th, 24^th and 48^th hour the differences were significant [（15.0±4.3）, （13.5±4.9）; （40.7±3.4）, （27.2±3.1）; （37.0±4.8）, （22.0±2.1）; （24.7±4.1）, （18.9±5.3）/mm; P 〈 0.05, P 〈 0.01l. （2） The number of positive cells in protein expression of P53 in hippocampus CA1 region of rats in each group at different time point after cerebral ischemia-reperfusion： Obvious gene expression of P53 was not found in sham operation group. In contrast with model control group, obvious decrease was found ＇in the 24^th and 48th hour after cerebral ischemia-reperfusion in poerarin treatment group [（25.3±4.4）, （12.8±2.7）; （24.3±3.6）, （10.9±3.0）/mm; P 〈 0.01]. （3） Comparison of the number of apoptosis ceils in hippocampus CA1 region of rats between groups at different time point after cerebral ischemia-reperfusion：In contrast with model control group, obvious decrease was found in the 12^th, 24^th and 48^th hour after cerebral ischemia-reperfusion in poerarin treatment group [（34.0±3.7）, （21.0±3.7）; （41.0±4.2）, （33.0±4.8）; （71.0±5.5）, （41.0±3.4）/mm; P〈0.01]. CONCLUSION： In rats which were given puerarin treatment, the expression of Fas decrease obviously in 6 to 48 hours after cerebral ischemiareperfusion, and the expression of P53 decreased obviously in the 24^th to 48^th hour after cerebral ischemia-reperfusion, and a descent tendency could be found in the number of apoptosis cells. These can further prove the cerebral proteetive effect of puerafin, and indicate that the inhibition of puerarin to cell apotosis after cerebral resuscitation is related to its effect on the decrease in protein expression of apoptosis-promoting gene, Fas and P53.