长托宁对急性全脑缺血再灌注损伤大鼠细胞色素和Caspase-3表达的影响

Effects of Penehyclidine Hydrochloride on the Expression of Cytochrome C and Caspase-3 in Rat's Brain After Acute Focal Cerebral Ischemia/Reperfusion

目的:从细胞凋亡及其信号转导通路角度,探讨长托宁对全脑缺血再灌注损伤大鼠的脑保护作用及部分机制。方法:雄性健康SD大鼠96只,随机分成3组:假手术组(S组,n=32)、缺血再灌注组(IR组,n=32)、长托宁干预组(P组,n=32),3组按再灌注时间再分为4个亚组,即缺血10 min后分别再灌注3,12,24,48 h,每个亚组动物均为8只。采用四血管闭塞法制造大鼠全脑缺血模型,应用免疫组化SP法动态观察不同时间点海马CA1区细胞色素C(CytC)和Caspase-3表达的变化;电镜和光镜观察再灌注24 h亚组海马CA1区神经细胞病理形态和线粒体超微结构的改变。结果:①大鼠脑缺血再灌注后海马CA1区CytC在再灌注3 h即有明显表达,于再灌注12h达高峰,以后表达逐渐降低;Caspase-3在再灌注3 h开始表达,12 h明显升高,24 h达高峰,48 h仍有较高表达。②与S组比较,IR组和P组各时间点海马CA1区的CytC、Caspase-3蛋白表达均明显升高(P<0.01);再灌注24 h亚组海马CA1区神经细胞病理形态和线粒体形态结构受损明显;③与IR组比较,P组各时间点海马CA1区的CytC、Caspase-3蛋白表达均明显降低(P<0.05或P<0.01);再灌注24 h亚组海马CA1区神经细胞病理形态和线粒体形态结构均有不同程度的改善。结论:长托宁对Caspase依赖的线粒体凋亡通路有干预作用,通过保护线粒体的形态功能、稳定线粒体膜、抑制CytC和Caspase-3的释放和激活,从而减少细胞凋亡的发生,发挥脑保护作用,这可能是其改善缺血再灌注损伤的部分作用机制。

Objective： To evaluate the effect of penehyelidine hydrochloride（PHC）resisting acute focal cerebral ischem/reperfusion （IR） injury in rats, and to explore the mechanism through apoptosis and signal transduction pathway. Methods： Healthy male SD rats were randomly divided into sham operation group （S group,n=32） ,ischemia/reperfusion group （IR group, n=32） and PHC treated group （P group,n=32）. Each group was divided equally into four subgroups according to the reperfusion time of 3, 12, 24 or 48 hours after 10 minutes of ischemia. The model of focal cerebral ischemia reperfusion injury was established by using Pulsinelli＇s method. Immunohistochemical SP method was performed to examine the expression of cytochrome C （CytC） and Caspase 3 at different time points after reperfusion. The pathological changes of nerve cells and mitochondria ultrastructure at hippocampus CA1 region were observed at 24 h after reperfusion by light microscope and electron microscope. Results. （1）The activation of CytC began to increase at 3 h after reperfusion, reached the peak at 12 h, and then gradually decreased. Caspase-3 began to increase at 3 h after reperfusion and reached the peak at 24 hours, and remained at highlevels till 48 h. （2）Compared with that in S group, the expression of CytC and Caspase-3 in IR and P group was increased significantly （P〈0.01）. The pathological changes of nerve cells and mitochondria ultrastructure at hippocampus CA1 region were exacerbated at 24 h after reperfusion. （3）Compared with that in IR group, the expression of CytC and Caspase-3 in P group were decreased significantly （P〈0.05 or P〈0.01）. The pathological changes of nerve cells and mitochondria ultrastructure at hippocampus CA1 region were improved at 24 hours after reperfusion. Conclusion： PHC shows protective effects on focal cerebral I/R injury partly through intervening in mitochondria mediated and Caspase-dependent apoptopic pathway. PHC reduces apoptosis, plays a role of protecting nerve by maintaining mitochondrial basical form, stabilizes mitochondrialmembrane and inhibits the release of cyt C and activation of Caspase-3.