促红细胞生成素对全脑缺血再灌注后神经细胞凋亡和诱导型一氧化氮合酶蛋白的影响

Effects of erythropoietin on nerve cell apoptosis and induced nitricoxide synthase expression after global cerebral ischemia and reperfusion

目的:观察促红细胞生成素(EPO)对短暂性全脑缺血再灌注后海马CA1区iNOS蛋白表达的影响,探讨其对缺血脑组织保护的可能机制。方法:应用四动脉血流阻断法制作大鼠短暂性全脑缺血再灌注模型;于再灌注开始时经腹腔注射EPO(3000U/kg);48h灌注取脑。苏木精—伊红染色和TUNEL法染色观察海马CA1区神经细胞坏死和凋亡。应用免疫组织化学方法检测iNOS蛋白的表达。结果:短暂性全脑缺血15min再灌注后48h,苏木精—伊红染色海马CA1区存活神经元细胞数正常组(211.28±7.95)个/视野,假手术组为(209.28±11.34)个/视野,EPO治疗组海马CA1区存活神经元细胞数为(170.28±8.12)个/视野,缺血组为(146.84±8.35)个/视野。EPO治疗组与缺血组比较差异有显著性意义(P<0.01)。TUNEL法凋亡细胞测定,正常组无阳性细胞,假手术组阳性细胞(152.48±18.52)个/视野,EPO治疗组有(1797.51±151.35)个/视野,缺血组有(2250.41±180.06)个/视野,两者比较差异有显著性意义(P<0.01)。iNOS蛋白的表达测定,正常组无阳性细胞,假手术组海马CA1区阳性细胞区灰度值为5.36±1.54,EPO治疗组为31.80±6.42,缺血组为49.46±8.96。EPO治疗组与缺血组比较,两者差异有显著性意义(P<0.01)。结论:EPO可减少大鼠短暂性全脑缺血再灌注后神经元的死亡和凋亡。

AIM:To observe the effects of erythropoietin(EPO) on induced nitricoxide synthase(iNOS) expression in hippocampal CA1 region after transient global cerebral ischemia and reperfusion in rats,and to explore the neuroprotective mechanism of EPO. METHODS:The model of transient global cerebral ischemia and reperfusion was produced by four vessel occlusion in Sprague Daewley rats.The animals were injected intraperitoneally with EPO(3 000 U/kg).The brains were removed after 48 hours so as to make the paraffin sections. Some sections were used to observe the neuron necrosis and apoptosis in the hippocampual CA1 region by the method of HE staining and TUNEL.The expression of iNOS was detected by immunochemistry. RESULTS:Forty eight hours after transient global cerebral ischemia and reperfusion, the number of survival neurons in the hippocampal CA1 region after HE staining was(211.28±7.95) per visual field in the normal group,(209.28±11.34) per visual field in the sham operated group,(170.28±8.12) per visual field in the EPO group and(146.84±8.35) per visual field in the ischemic group. There was distinct difference between the EPO group and ischemic group(P< 0.01).No TUNEL positive cell was seen in the normal group.The number of TUNEL positive cells was(152.48±18.52),(1 797.51±151.35) and(2 250.41±180.06) per visual field in the sham operated group,EPO group and ischemia group,respectively.Compared with the ischemic group, the number of TUNEL positive cells in the EPO group was greatly decreased(P< 0.01).There were no positive cells in the normal group.The gray values of iNOS expression in the hippocampal CA1 region were 5.36±1.54,31.80±6.42 and 49.46±8.96 in the latter three groups,respectively.Compared with the ischemia group, the gray value of iNOS expression in the EPO group had the significant difference(P< 0.01). CONCLUSION:EPO could reduce neuron necrosis and apoptosis and inhibit iNOS expression after transient global cerebral ischemia and reperfusion in rats.