黄皮酰胺对高血压局灶性脑缺血-再灌注大鼠Bcl-2蛋白表达和细胞凋亡的影响

Effect of clausenamide on the expression of Bcl - 2 protein and apoptosis after focal cerebral ischemia/reperfusion in renovascular hypertensive rats

目的观察黄皮酰胺对肾性高血压大鼠(RHR)脑缺血-再灌注后Bcl-2蛋白表达及细胞凋亡的影响,探讨其脑保护作用机制。方法75只RHR被随机分成假手术组、单纯缺血-再灌注组和黄皮酰胺组。采用线栓法制作局灶性脑缺血-再灌注模型,假手术组不造成缺血。用免疫组化法和原位末端脱氧核苷酸转移酶标记法(TUNEL法)分别观察缺血2h后再灌注6、12、24、48和72h脑细胞Bcl-2蛋白表达和凋亡细胞数。结果①再灌注6h即可见Bcl-2蛋白表达较多,24h达高峰,此后逐渐下降。与单纯缺血-再灌注组比较,黄皮酰胺组Bcl-2蛋白表达于再灌注后各时间点均明显增高,差异均有显著性(P均<0.01)。②再灌注6h后有较多凋亡细胞,于72h达高峰。与单纯缺血-再灌注组比较,再灌注6-24h黄皮酰胺组凋亡细胞均显著减少(P均<0.01),但再灌注48h后细胞凋亡的抑制作用不明显(P均>0.05)。假手术组及缺血-再灌注组的非缺血侧无Bcl-2阳性细胞,仅见0-2个凋亡细胞。结论局灶性脑缺血-再灌注后Bcl-2表达显著增高,细胞凋亡参与了脑缺血-再灌注损伤的过程。黄皮酰胺能显著上调Bcl-2表达,抑制细胞凋亡,并可能与Bcl-2协同抑制细胞凋亡,这可能是黄皮酰胺脑保护作用的机制。

Objective To observe the effect and mechanism of clausenamide on the expression of Bcl - 2 and apoptosis after focal cerebral ischemia/reperfusion in renovascular hypertensive rats. Methods Seventy-five renovascular hypertensive rats were randomly divided into three groups (25 in each group): clausenamide intervention group,single ischemia/reperfusion model group and sham -operated group. Focal cerebral ischemia was reproduced by ligature for 2 hours and loosening of the ligature in the rats. No arterial ligature was applied in sham - operated group. Computerized pathological image analyzer was used to determine the number of cells positive for Bcl - 2 by immunohistochemical staining, and also the counts of apoptotic cells after TdT - mediated dUTP nick end labeling (TUNED staining respectively in coronal sections of brain after reperfusion (6, 12, 24, 48 and 72 hours). Results (1)The expression of Bcl - 2 protein was detected 6 hours after reperfusion, peaking at 24 hours, then declined gradually. The Bcl - 2 protein positive cell counts at every time point in clausenamide intervention group were significant higher than simple ischemia/reperfusion model group (all P<0. 01). (2)The number of apoptotic cells was increased with reperfusion, reaching its peak at 72 hours. The apoptosis counts in clausenamide intervention group were significant lower than single ischemia/reperfusion model group (all P<0. 01). At all time points, except at 48 hours after reperfusion, as there was no significant difference (all P>0. 05). No Bcl -2 positive cells and only 0-2 apoptotic cells could be discernible in brain sections from sham - operated animals or in the contrateral side of ischemia in animals of the other groups. Conclusion Expression of Bcl - 2 protein is enhanced and apoptosis appears after focal cerebral ischemia/reperfusion in rat brain. Clausenamide can enhance the expression of Bcl -2 protein and inhibit apoptosis remarkably. Clausenamide may coordinate with Bcl - 2 in inhibiting apoptosis. This may be the mechanism of protection of brain cells from ischemic damage of clausenamide treatment.