磷酸化细胞外信号调节蛋白激酶加重糖尿病大鼠脑缺血性损伤

Phosphorylation of extracellular signal-regulated protein kinase induced neuronal death under brain ischemia in diabetic rats

目的探讨高血糖加重脑缺血性损伤的分子机制。方法采用大鼠全脑缺血模型,通过免疫组化、末端脱氧核苷酸转移酶介导的dUTP原位切口末端标记方法和Western blot技术,对比研究Streptozotocin诱导的糖尿病大鼠脑缺血时神经元细胞外信号调节蛋白激酶1/2(ERK1/2)磷酸化和神经元凋亡的关系。结果糖尿病组脑缺血30min和再灌注1、3、6h后,扣带皮质和海马CA3区ERK1/2阳性细胞数和神经元凋亡明显高于正常血糖组(P<0.05)。ERK1/2抑制剂U0126可明显减少ERK1/2的磷酸化和凋亡神经元的数量。Western blot分析可见,在缺血脑组织中磷酸化ERK1/2明显增高,再灌注3和6h糖尿病组显著高于正常血糖缺血组(P<0.05)。结论糖尿病高血糖加重缺血性脑损伤与MAPK家族激活有关,特别是ERK1/2参与了脑细胞的损伤。

Objective To investigate the molecular mechanism of hyperglycemia on brain damage caused by ischemia. Methods Sixty-five male SD rats were divided into normoglycemia ＋ ischemia group （ n = 20）, diabetes ＋ ischemia group （ n =20）, diabetes ＋ ischemia ＋ U0126 group （ n =20） and normal control （ n =5 ）. Streptozotocin （60 mg/kg） was injected through vena caudalis to induce diabetes. The global cerebral ischemia was made by ligating bilateral common carotid arteries for 30 min and reperfusing for 1,3, or 6 h. ERK inhibitor U0126 （4 g/kg） was injected through vena caudalis 30 min before ischemia, while normal saline was as substitute in other groups. The TUNEL, immunohistochemistry rons in the cingulate cortex and phosphorylation of ERK1/2 expression and neuronal apoptosis were studied by and Western blot. Results Increased phospho-ERK1/2 immunoreactive neuhippocampal CA3 were detected in euglycemia ＋ ischemia group. The number of phospho-ERK1/2 positive and apoptotic neurons was much more in diabetes ＋ ischemia group than normoglycemia ＋ ischemia group （ P 〈 0. 05 ）. Pretreatment with U0126 in diabetes ＋ ischemia group significantly decreased ERK1/2 immunoreactive ceils and apoptotic neurons. Western blot analysis confirmed that phospho- ERK1/2 increased significantly after ischemia and reperfusion as compared to normal controls. The amount of phospho-ERK1/2 was obviously increased after 3-hour or 6-hour reperfusion in diabetes ＋ ischemia group in comparison with normoglycemia ＋ ischemia group （P 〈 0. 05 ）. Treatment with U0126 significantly reduced phospho-ERK1/2 in the diabetic rats. Conclusion ERK1/2 may play a role in mediating the apoptosis of neuronal ceils under hyperglycemic condition.