瘦素在小鼠脑缺血/再灌注损伤神经元凋亡中的作用

The role of Leptin on neuron apoptosis in mice with cerebral ischemia/reperfusion injury

目的 研究瘦素（Leptin）在脑缺血性损伤神经元凋亡中的作用及机制.方法 按照完全随机数字表法将75只小鼠均分为3组,通过大脑中动脉闭塞（MCAO） 2 h后再灌注方式制作小鼠局灶性脑缺血/再灌注（I/R）损伤模型.Leptin干预组于缺血即刻腹腔注射Leptin 1 μg/g;模型组腹腔注射等量磷酸盐缓冲液.于再灌注24 h采用原位末端缺刻标记法（TUNEL）检测凋亡神经元,用逆转录-聚合酶链反应（RT-PCR）和免疫组化法检测凋亡相关基因天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）和bcl-2的mRNA及蛋白表达.结果 模型组脑缺血中心区神经元以坏死为主.与假手术组比较,模型组半影区神经元凋亡率显著增高,caspase-3、bcl-2的mRNA和蛋白表达水平均显著升高[凋亡率：（68.65±0.79）%比（4.40±0.00）%,caspase-3 mRNA：2.563±0.250比0.153±0.020,bcl-2 mRNA：0.337±0.100比0.125±0.030,caspase-3 蛋白（吸光度值,A值）：0.57±0.05 比 0.37士0.03,bcl-2蛋白（A值）：0.51±0.04比0.35±0.01,均P〈0.01].与模型组比较,Leptin干预组半影区神经元凋亡率显著降低[（42.30±8.45）%比（68.65±0.79）%,P〈0.01],caspase-3 mRNA和蛋白表达显著降低[caspase-3 mRNA：2.267±0.040比2.563±0.250,caspase-3蛋白（A值）：0.45±0.04比0.57±0.05,P〉0.05和P〈0.013,bcl-2 mRNA和蛋白表达显著升高[bcl-2 mRNA：0.662±0.040比0.337±0.100,bcl-2蛋白（A值）：0.76±0.09比0.51±0.04,均P〈0.013.结论 Leptin能够通过下调促凋亡基因caspase-3表达、上调抑凋亡基因bcl-2表达,从而抑制神经元凋亡,在脑缺血性损伤中发挥神经保护作用.

Objective To study the effect of Leptin on neuron apoptosis in mice with cerebral ischemia injury and its mechanism.Methods Seventy-five mice were randomly divided into three groups.Focal cerebral ischemia/reperfusion injury model in mice was reproduced by middle cerebral artery occlusion for 2 hours followed by reperfusion.In Leptin intervention group mice were given Leptin 1μg/g during cerebral ischemia by intraperitoneal injection.Mice in the model group were given equal amount of phosphate buffer saline. After reperfusion for 24 hours,the neuron apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） staining.The mRNA and protein expression of apoptosis relative gene caspase-3 and bcl-2 were determined by reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry.Results Most of neuron necrosis was obseved in cerebral ischemia center in model group. Compared with sham-operation group,neuron apoptosis rate,mRNA and protein expression of caspase-3 and bcl-2 in model group increased significantly [apoptosis rate：（68.65±0.79）%vs.（4.40±0.00）%,caspase-3 mRNA：2.563±0.250 vs.0.153±0.020,bcl-2 mRNA：0.337±0.100 vs.0.125±0.030,caspase-3 protein （absorbance value,A value）：0.57±0.05 vs.0.37±0.03,bcl-2 protein （A value）：0.51±0.04 vs.0.35±0.01,all P〈0.01].The apoptosis rate of penumbra neurons was reduced in Leptin intervention group significantly compared with model group [（42.30±8.45）% vs.（68.65±0.79）%,P〈0.01].Compared with model group,the mRNA and protein expression of caspase-3 in Leptin intervention group were reduced significantly [caspase-3 mRNA：2.267±0.040 vs.2.563±0.250,caspase-3 protein（A value）：0.45±0.04 vs.0.57±0.05,P〉0.05 and P〈0.01],and the mRNA and protein expression of bcl-2 in Leptin intervention group upregulated significantly [bcl-2 mRNA：0.662±0.040 vs.0.337±0.100,bcl-2 protein （A value）：0.76±0.09 vs.0.51±0.04,both P〈0.01].Conclusion Leptin could reduce apoptosis of neurons through down-regulation of the expression of caspase-3 and up-regulation of the expression of bcl-2.The results suggest that Leptin plays a neuroprotective role in cerebral ischemia injury.