缺血后处理对脑缺血再灌注损伤后Caspase-3、Caspase-9激活和细胞色素C释放的影响

Effects of Ischemic Postprocessing on Activation of Caspase-3 and Caspase-9 and Release of Cytochrome C in Reperfusion Injury after Cerebral Ischemia

目的探讨缺血后处理对大鼠脑缺血再灌注损伤后Caspase-3、Caspase-9激活和细胞色素C(Cyt C)释放的影响。方法选择成年健康雄性SD大鼠60只,随机分为假手术组、缺血再灌注组、缺血后处理组,每组20只。线栓法建立大脑中动脉闭塞再灌注模型。缺血再灌注24 h后留取大脑组织,采用原位末端标记法检测脑内神经细胞凋亡情况,采用酶活性测定、Western blot和免疫组化等方法检测Caspase-3、Caspase-9及Cyt C含量。结果缺血再灌注组、缺血后处理组较假手术组细胞凋亡增多,差异均有统计学意义(P<0.05);缺血后处理组较缺血再灌注组凋亡细胞减少,差异有统计学意义(P<0.05)。缺血再灌注组、缺血后处理组Caspase-3、Caspase-9活性较假手术组高,缺血后处理组活性显著低于缺血再灌注组,差异均有统计学意义(P<0.05)。缺血再灌注组、缺血后处理组Caspase-3、Caspase-9前体蛋白proCaspase-3、proCaspase-9含量较假手术组减少(P<0.05),而缺血后处理组含量高于缺血再灌注组,差异有统计学意义(P<0.05)。在胞浆中假手术组无明显Cyt C释放,缺血再灌注组、缺血后处理组胞浆内Cyt C释放较假手术组增多,差异有统计学意义(P<0.05);缺血后处理组Cyt C释放较缺血再灌注组明显减少,差异有统计学意义(P<0.05)。线粒体中缺血再灌注组、缺血后处理组Cyt C较假手术组外漏明显,而缺血后处理组Cyt C漏出较缺血再灌注组明显减少,差异均有统计学意义(P<0.05)。结论缺血后处理可以减轻脑缺血再灌注损伤后神经细胞凋亡,抑制Caspase-3、Caspase-9的激活和Cyt C的释放。

Objective To investigate the effect of ischemic postprocessing on activation of Caspase-3 and Caspase-9 and release of cytochrome C in reperfusion injury after cerebral ischemia in rats.Methods Sixty adult SD male rats were randomly divided into sham opration group（group A,n =20）,ischemia and reperfusion group（group B,n = 20） and ischemic postprocessing group（group C,n =20）.The model of focal ischemic reperfusion was induced by intraluminal middle cerebral artery occlusion（MCAO） with a nylon monofilament suture.After 24 h of ischemia and reperfusion,the brain tissues were obtained for detecting.Apoptosis of intracephalic nerve cells was detected by in situ end labeling,and contents of Caspase-3,Caspase-9 and cytochrome C were detected by enzyme activity assay,Western blot and immunohistochemisty method.Results The amount of apoptosis cell in group B and C increased when compared with that in group A,and the differences were statistically significant（P 0.05）;the amount of apoptosis cell in group C was significantly reduced when compared with that in group B（P 0.05）.The activity rate of Caspase-3 and Caspase-9 was higher in group B and C than that in group A,while the activity rate in group C was abated when compared to that in group B,and the differences were statistically significant（P 0.05）.Contents of pro-Caspase-3 and pro-Caspase-9 in group B and C were significantly reduced when compared to those in group A（P 0.05）,and the contents in group C were significantly increased when compared to those in group B（P 0.05）.In the cytoplasm,Cyt C was no obviously released in group A,while it was released obviously in group B and C,and the differences were statistically significant（P 0.05）;Cyt C was released more obviously in group C than that in group B（P 0.05）.In mitochondria,Cyt C leakage was more obvious in group C than that in group A,and it was significantly decreased in group C when compared to that in group B,and the differences were statistically significant（P 0.05）.Conclusion Ischemic postprocessing may reduce apoptosis of nerve cells in reperfusion injury after cerebral ischemia,and may restrain the release of cytochrome C and activation of Caspase-3 and Caspase-9 in cerebral I/R injury.