高血糖对大鼠局灶性脑缺血再灌注后神经细胞凋亡及细胞色素C、半胱氨酸天冬氨酸蛋白酶-3表达的影响

Effects of hyperglycemia on cell apoptosis and expression of cytochrome C and caspase-3 in focal cerebral ischemia reperfusion injury of rats

目的研究高血糖对大鼠局灶性脑缺血再灌注后神经细胞凋亡及细胞色素C(CytC)、半胱氨酸天冬氨酸蛋白酶-3(caspase-3)表达的影响,进一步探讨高血糖加重脑缺血损伤的作用机制。方法将30只雄性Sprague-Dawley大鼠随机分为3组:高血糖组(10只)、正常血糖组(10只)和假手术组(10只)。按体质量4 g/kg给予大鼠尾静脉注射25%葡萄糖,造成大鼠高血糖状态;继而制作大脑中动脉阻塞脑缺血再灌注模型。于缺血2 h再灌注24 h进行神经功能评分,采用脱氧核糖核苷酸末端转移酶介导的dUTP缺口末端标记法(TUNEL)检测大鼠脑组织细胞凋亡数,采用免疫组化染色及Western blot技术检测大鼠脑组织CytC和caspase-3表达。采用SPSS 17.0软件分析系统处理,计量资料以(x珋±s)表示,组间比较采用单因素方差分析,两两比较采用SNK-q法。以P<0.05为差异有统计学意义。结果 (1)高血糖组神经功能评分[(4.20±0.63)分]显著高于正常血糖组[(3.50±0.71)分,q=15.56,P<0.05]。(2)高血糖组凋亡细胞数[(19.10±0.99)个]明显多于正常血糖组[(17.50±1.08)个,q=14.84,P<0.05]。(3)高血糖组CytC蛋白的表达为1.369±0.055,显著高于正常血糖组(1.196±0.088,q=23.51,P<0.05)。(4)高血糖组caspase-3蛋白的表达(1.133±0.047)明显高于正常血糖组(1.085±0.033,q=27.58,P<0.05)。结论高血糖可增加缺血再灌注后脑神经细胞凋亡,CytC、caspase-3的激活及表达增强可能是高血糖加重脑缺血再灌注损伤的机制之一。

Objective To study the effects of hyperglycemia on cell apoptosis and expression of cytochrome C and caspase-3 in rats with focal cerebral ischemia-reperfusion injury and to discuss further the mechnism of cerebral ischemia injury caused by hyperglycemia. Methods Thirty adult male Sprague-Dawley rats were equally randomized into 3 groups ： the hyperglycemic group （ 10 cases ）, the normal glycemic group （10 cases） and the sham-operated group（ 10 cases）. Hyperglycemia models were made by injection of 25% glucose（4 g/kg）through the tail vein in rats and then focal cerebral ischemia injury was made by occluding the middle cerebral artery. The scores of neurological deficit were estimated after 2 h ischemia and 24 h reperfusion. The apoptotic neurons were detected with the terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling （TUNEL）. The number of apoptotic neurons were tested in the rats. Immunohistochemistry and Western blot technique were used to detect the expression level of cytochrome C and caspase-3. Data were expressed as x^- ± s. One-way analysis of variance was used for comparison among groups,and SNK test for comparison between two groups. Statistical significance was set at P 〈 0. 05. Results （ 1 ） The scores of neurological deficits of the hyperglycemic group rats （4. 20 ± 0. 63 ） were higher significantly than those of nonhyperglycemic group rats（3.50 ±0. 71 ,q = 15.56,P 〈0. 05）. （2） The number of apoptotic neurons of the hyperglycemic group rats（ 19. 10 ±0. 99）were increased significantly than those of nonhyperglycemic group rats（ 17.50 ＋ 1.08, q = 14. 84, P 〈 0. 05 ）. （ 3 ） The expression of cytochrome C in the hyperglycemic group rats （ 1. 369 ±0. 055 ） were higher significantly than those of nonhyperglycemic group rats （ 1. 196 ±0. 088, q = 23.51, P 〈 0. 05 ）. （4） The expression of caspases-3 in the hyperglycemic group rats （1. 133 ±0. 047 ）were higher significantly than those of nonhyperglycemic group rats （1. 085 ±0. 033, q = 27. 58,P 〈 O. 05）. Conclusion The higher expression of cytochrome C and caspase-3 may be one of the mechanisms in ischemia reperfusion injury aggravated by hyperglycemia.