Ras/Raf/MEK/ERK信号传导通路在2型糖尿病大鼠局灶性脑缺血早期作用的研究

The study of the primary effects of Ras/Raf/MEK/ERK signaling pathway on focal cerebral ischemia in type 2 diabetes rats

目的探讨高血糖加重脑梗死机制与Ras/Raf/丝裂原激活蛋白激酶(MEK)/细胞外信号调节激酶(ERK)信号传导通路的关系。方法通过大鼠尾静脉注射链脲佐菌素(STZ)及大脑中动脉闭塞(MCAO)术造成大鼠糖尿病脑缺血模型。将大鼠分成假手术组、健康大鼠脑缺血组、2型糖尿病(T2DM)大鼠脑缺血组、PD98059组,每组12只。PD98059组于MCAO术前30min尾静脉注射PD98059(3mL/kg)。MCAO术后2h给各组大鼠做神经学评分,取脑组织标本。应用免疫组织化学、蛋白质印迹法(Western blotting)测定大鼠磷酸化细胞外信号调节激酶1/2(p-ERK1/2)蛋白的表达,应用TUNEL检测神经细胞凋亡情况。结果除假手术组外,其他3组均有不同程度的神经功能缺损、神经细胞凋亡,且差异有统计学意义(P<0.01)。p-ERK1/2蛋白在假手术组偶见表达,在健康大鼠脑缺血组、T2DM大鼠脑缺血组表达明显升高(P<0.01),且T2DM大鼠脑缺血组升高更加明显(P<0.01)。PD98059组p-ERK1/2蛋白表达较少。免疫组织化学和Western blotting结果基本一致。结论 Ras/Raf/MEK/ERK信号传导通路在局灶性脑缺血早期能促进神经细胞的凋亡,糖尿病加重脑梗死与Ras/Raf/MEK/ERK信号传导通路有关。

Objective To study the relationship between Ras/Raf/MEK/ERK signaling pathway and the mechanism of hyperglycemia aggravating cerebral infarction.Methods Constract cerebral ischemia models of diabetic rats by rat tails vein injection of streptozotocin（STZ） and middle cerebral artery occlusion（MCAO）surgery.Rats were divided into four groups,namely the sham group,normal rat cerebral ischemia group,type 2 diabetes cerebral ischemia group and PD98059 group（n=12）.The rats in PD98059 group underwent tail vein injection of PD98059（3 mL/kg） preoperative 30 minutes before MCAO.Two hours after MACO,rats of each group were measured with neurological score and then brain tissue samples were extracted.The expression of p-ERK1/2 protein was detected by immunohistochemistry and Western blotting;the cell apoptosis was detected by TUNEL.Results In addition to the sham operation group,the other three groups have different levels of neurological deficit and neuronal cells apoptosis（P0.01）.There was occasional expression of p-ERK1/2 protein in sham operation and significantly increased expression in normal rats cerebral ischemia group and T2DM cerebral ischemia group（P0.01）.The increase in the T2DM cerebral ischemia group was more distinct（P0.01）.There were less expression of p-ERK1/2 protein in PD98059 group,and there were no difference between the result from immunohistochemistry and Western blotting.Conclusion The Ras/Raf/MEK/ERK signaling pathway could promote neuronal apoptosis in the early focal cerebral ischemia;diabetes aggravating cerebral infarction is related to Ras/Raf/MEK/ERK signaling pathways.