脂联素在糖尿病大鼠脑缺血再灌注损伤中的表达及其影响

Expression of adiponecfin in diabetic rats with focal cerebral ischemia-reperfusion injury and its influence in the rats

目的探讨脂联素（APN）在糖尿病大鼠脑缺血再灌注损伤后血清及脑组织中的表达变化及其作用。方法104只SD大鼠按随机数字表法分成正常血糖组（8只）、糖尿病组（8只）、单纯脑缺血再灌注损伤组（I／R组，44只）、糖尿病合并脑缺血再灌注损伤组（DM＋I/R组，44只）。采用给予糖尿病饮食方法制备糖尿病模型，采用线栓法制备大脑中动脉阻塞（MCAO）缺血再灌注损伤模型。I／R组和DM＋UR组大鼠采用Zea Longa法进行神经行为学评分，并各取4只于脑缺血再灌注损伤前及不同再灌注时间点（3h、6h、24h、48h、72h、7d）连续取血测定血清APN浓度变化；剩余40只大鼠再按不同再灌注时间点分为再灌注3h、6h、24h、72h、7d5个亚组（每个亚组8只），HE染色观察大鼠脑组织形态学变化，免疫组化染色及Western blotting法检测大鼠缺血脑组织APN蛋白表达。结果（1）DM＋I／R组大鼠神经功能受损严重，其神经功能学评分[（2．79±0．41）分]明显较I／R组[（1．27±0．45）分]高，差异有统计学意义（P〈0．05）。（2）与I／R组相比，DM＋I/R组大鼠血清APN水平在再灌注3h、6h、24h、48h、72h、7d时均明显减少，差异均有统计学意义（P〈0．05）。（3）DM＋I／R组大鼠HE染色示脑组织损伤程度较I／R组明显严重，包括细胞变性、坏死明显。（4）免疫组化染色显示：I／R组和DM＋UR组大鼠缺血脑组织APN表达在再灌注3h时明显增加达高峰，6h时逐渐降低，24h时再次增高并在7d时维持在较高的水平；DM＋UR组大鼠在相同再灌注时间点APN表达均较I／R组明显减少，差异有统计学意义（P〈0．05）。Western blotting法检测到的APN蛋白含量变化与免疫组化染色结果相同。结论APN在糖尿病大鼠脑缺血再灌注损伤后表达降低，其可能是糖尿病加重脑缺血再灌注损伤的内源性机制之一。

Objective To investigate the expression of adiponectin （APN） and the clinical significance in type 2 diabetic rats with cerebral ischemic reperfusion damage. Methods One hundred and four SD rats were randomly divided into two groups, including normal diet group （n=52） and diabetic diet group （n=52）; middle cerebral artery occlusion was performed in some of the rats to induce ischemia reperfusion injury models. Normal diet group was then randomly divided into euglycemia group （n=8） and ischemia reperfusion group （I/R, n=44）; diabetic diet group was randomly divided into diabetes groups （n=8） and diabetes complicated with ischemia reperfusion group （DM＋I/R, n=44）. The blood was collected to detect the APN changes from the tail vein of rats from diabetes group （n=4） and DM＋I/R group （n=4） before MCAO surgery and at 3, 6, 24, 48 and 72 h, and 7 d after reperfusion. The rest 40 rats were randomly divided into subgroups subjected to 90 rain of focal ischemia followed by 3, 6, 24 and 72 h, and 7 d reperfusion （n=8）; the neuroethology assessment was determined by Zea Longa method; morphology of brain tissue was observed by HE staining. APN expression in infarction cores was detected by immunohistochemistry and Western blotting. Results The assessment scores of DM＋I/R group （2.79±0.41） were significantly higher than those of UR group （1.27±0.45, P〈0.05）. As compared with that in the UR group, the serum APN level in the DM＋I/R group was significantly decreased 3, 6, 24, 48 and 72 h, and 7 d after reperfusion （P〈0.05）. Cerebral tissue damage （cellular degeneration and necrosis） in DM＋I/R group was more serious as compared with I/R group at the same reperfusion time. In the ischemic hemisphere, APN expression increased at 3 h, decreased at 6 h, and increased again till 24 h after reperfusion, and then it remained at high level up to 7 days after reperfusion. The expression of APN in DM＋I/R group was significantly lower than that in the UR group （P〈0.05）. These findings were consistent with the results of Western blotting. Conclusion APN expression decreases after focal cerebral ischemia-reperfusion injury, indicating that APN plays an important role in aggravating ischemic reperfusion injury by diabetes.