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目的:观察和比较手性3-n丁基苯酞(NBP)对大鼠局灶性脑缺血再灌注诱导的凋亡的影响。方法:插线法制备大脑中动脉阻断(MCAO)模型,原位末端标记(TUNEL)和琼脂糖凝胶电泳检测DNA断裂,蛋白质印迹和免疫组化方法检测细胞色素c及caspase-3蛋白的表达。结果:大鼠局灶性脑缺血2小时,再灌注开始后24小时可见明显的TUNEL阳性染色和DNA ladder.s-(-)-NBP 5,10 mg/kg显著减少TUNEL阳性细胞数和DNA ladder,s-(-)-NBP 10 mg/kg几乎完全抑制DNA片段化,而r-(+)-NBP 10 mg/kg仅有轻度抑制作用。(±)-NBP(20 mg/kg)对DNA片段化的抑制作用介于s-(-)-NBP(10 mg/kg)和r-(+)-NBP(10 mg/kg)之间。脑缺血过程中可见到线粒体细胞色素c的释放及caspase-3的激活,s-(-)-NBP能明显抑制这一效应,r-(+)-NBP和(±)-NBP对细胞色素c和caspase-3作用的强弱与它们抑制DNA片段化的作用强度相似。结论:NBP,尤其是s-(-)-NBP能够抑制脑缺血诱导的DNA片段化,细胞色素c释放和caspase-3激活。
Objective: To observe and compare the effects of chiral 3-n butylphthalide (NBP) on apoptosis induced by focal cerebral ischemia-reperfusion in rats. Methods: Middle cerebral artery occlusion (MCAO) model was established by patch cord method. DNA fragmentation was detected by TUNEL and agarose gel electrophoresis. Western blotting and immunohistochemistry were used to detect the expressions of cytochrome c and caspase-3 expression. Results: TUNEL staining and DNA ladder s - (-) - NBP 5 and 10 mg / kg markedly decreased the number of TUNEL positive cells and DNA at 2 hours after focal cerebral ischemia in rats and 24 hours after reperfusion. ladder, s - (-) - NBP 10 mg / kg almost completely inhibited DNA fragmentation, whereas r - (+) - NBP 10 mg / kg showed only modest inhibition. The inhibitory effect of (±) -NBP (20 mg / kg) on DNA fragmentation was between s - (-) - NBP (10 mg / kg) and r - (+) - NBP (10 mg / kg). The release of mitochondrial cytochrome c and the activation of caspase-3 were observed during cerebral ischemia. The effect of r - (+) - NBP and (±) -NBP on cytochromes The strength of c and caspase-3 is similar to their effect of inhibiting DNA fragmentation. Conclusion: NBP, especially s - (-) - NBP can inhibit cerebral ischemia-induced DNA fragmentation, cytochrome c release and caspase-3 activation.