大豆异黄酮对全脑缺血/再灌注大鼠海马神经细胞凋亡的影响

Effects of soybean isoflavones on apoptosis of rat hippocampal neurons after global cerebral ischemia/reperfusion

目的研究大豆异黄酮(SI)对全脑缺血/再灌注大鼠海马神经细胞凋亡的影响及作用机制。方法将60只成年健康SD大鼠随机分为假手术组(Sham组)、缺血/再灌注组(I/R组)、SI低剂量组(SI-L组,60 mg/kg)、SI中剂量组(SI-M组,120 mg/kg)、SI高剂量组(SI-H组,240 mg/kg),每组12只。采用三血管阻断法制备全脑缺血/再灌注损伤模型。缺血1 h再灌注1 h后处死大鼠,取海马组织,光学显微镜下观察海马神经元形态学变化,透射电子显微镜下观察超微结构变化,Annexin V-FITC/PI双染法检测海马神经细胞凋亡率,化学比色法检测SOD、i NOS活性及MDA、NO含量,半定量RT-PCR法测定海马组织NF-κB/p65 mRNA的表达,免疫组织化学法测定海马组织TNF-α、NF-κB/p65蛋白的表达。结果 I/R组海马神经元皱缩,胞核内染色质边集,核膜不完整,部分线粒体膜崩解、嵴消失,神经元部分凋亡。与I/R组比较,SI各组海马神经元超微结构的损伤明显改善,细胞凋亡率降低(P<0.01)。I/R组SOD、i NOS活性及MDA、NO含量明显高于Sham组(P<0.01);与I/R组比较,SI各组SOD、i NOS活性及MDA、NO含量降低(P<0.05)。I/R组TNF-α、NF-κB/p65蛋白及NF-κB/p65 mRNA的表达明显高于Sham组(P<0.01);与I/R组比较,SI各组TNF-α、NF-κB/p65蛋白及NF-κB/p65mRNA的表达明显降低(P<0.01)。结论 SI可能通过抑制氧化应激、减少TNF-α的表达进而抑制NF-κB/p65的活性,减少全脑缺血/再灌注后海马神经元的凋亡。

Objective To investigate the effects of soybean isoflavones（ SI） on the apoptosis of rat hippocampal neurons after global cerebral ischemia/reperfusion and relevant mechanisms.Methods A total of 60 adult healthy SDrats were randomly divided into the shamgroup, ischemia/reperfusion group（ I/R group）, SI lowdose group（ SI-L group, 60 mg/kg）, SI mediumdose group（ SI-M group, 120 mg/kg）, and SI high dose group（ SI-H group, 240 mg/kg）（ n = 12 in each）.The global ischemia/reperfusion model was established by 3-vessel occlusion.Rats were sacrificed after being reperfused for 1 h after 1 h of ischemia and the hippocampal tissue was collected.The morphological changes of hippocampal neurons were observed under the light microscope.Changes of the ultra-structure of hippocampal neurons were observed by the transmission electron microscope.Annexin V-FITC/PI double staining was adopted to detect the apoptosis rate of hippocampal neurons.The chemical colorimetry was employed to detect the activity of superoxide dismutase（ SO D） and inducible nitric oxide synthase（ i N O S） and contents of methane dicarboxylic aldehyde（ M DA） and nitric oxide（ N O）.The expression of N F-κB/p65 mRN A of hippocampal tissue was detected by the semi-quantitative RT-PCR.The protein expressions of TN F-α and N F-κB/p65 of hippocampal tissue were detected by the immunohistochemical method.Results Shrivel of hippocampal neurons, marginalization of nucleus chromatin, incompletion of nuclear membrane, disintegration of partial mitochondria membrane, disappearance of partial mitochondrial cristae, and apoptosis of partial neurons were observed in I/R group.Compared with the I/R group, the damage of ultra-structure of hippocampal neurons of SI-L, SI-M, and SI-H groups was relieved significantly and the rate of cell apoptosis decreased（ P〈0.01）.The activity of SO Dand iN O S and contents of M DA and N O of the I/R group were obviously higher than those of the Shamgroup（ P〈0.01）.Compared with the I/R group, the activity of SO Dand iN O S and contents of M DA and N O of SI-L, SI-M,and SI-H groups decreased（ P〈0.05）.The mRN A expression of N F-κB/p65 and protein expressions of TN F-α and NF-κB /p65 of the I /R group were significantly higher than those of the Shamgroup（ P〈0.01）.Compared with the I/R group, the mRN A expression of N F-κB/p65 and protein expressions of TN F-α and N F-κB /p65 of SI-L, SI-M, and SI-H groups decreased significantly（ P〈0.01）.Conclusion SI can decrease the apoptosis of rat hippocampal neurons after global cerebral ischemia/reperfusion, which may be relevant to inhibiting the oxidative stress, decreasing the expression of TN F-α, and inhibiting the activity of N F-k B/p65.