摘要
目的探究坏死性凋亡相关蛋白大脑中动脉栓塞（MCAO）在脑组织缺血再灌注小鼠脑损伤中的作用及其机制。方法通过大脑中动脉栓塞（MCAO）构建C57BL／6小鼠缺血再灌注损伤模型，分别使用凋亡抑制剂z-VAD．fmk（zVAD，1．1g／kg）、受体相互作用蛋白3（RIP3）抑制剂GSK’872（0,7g／kg）以及二者联合进行干预，采用mNSS评估神经功能缺损状况，TTC染色检测脑组织梗死体积，免疫印迹（WesternBlot）和免疫荧光法分析受体相互作用蛋白1（RIPl）、RIP3和混合系列蛋白激酶样结构域（MLKL）表达变化和定位情况。结果MCAO小鼠发生明显的神经功能缺损和脑组织梗死，与MCAO组比较，zVAD、GSK’872及二者联合均能缓解神经功能缺损，降低脑组织梗死体积（P〈0．05或P〈0．01）。MCAO引起RIP1、RIP3和MLKL蛋白水平升高，而GSK’872以及联合干预能够明显下调上述蛋白表达[RIP1（GSK’872：0．64±0．02，MCAO：1．28±0．02，P〈0．01）；RIP3（GSK’872：1．08±0．02，MCAO：1．45±0．02，P〈0．01）；MLKL（GSK’872：0．54±0．01，MCAO：1．00±0．01，P〈0．01）]，但是zVAD除导致MLKL轻度降低外（P〈0．05），并未引起RIP1和RIP3表达变化（P〉0．05）。结论坏死性凋亡相关蛋白RIP1、RIP3与MLKL参与介导坏死胜凋亡的发生，促进脑组织缺血再灌注损伤的病理进展。
Objective To explore the effect and mechanism of necroptosis related proteins in middle cerebral artery occlusion （ MCAO ） induced brain ischemia/reperfusion injury in mice. Methods C57BL/6 mice were used to establish the brain ischemia/reperfusion injury model induced by MCAO. MCAO mice were treated with z-VAD.fmk （zVAD, 1.1 g/kg）,GSK＇ 872 （0.7 g/kg） and combined intervention of zVAD and GSK＇ 872, and neurological defect was evaluated by mNSS while brain infarct volume was measured by TTC staining. Western blot and immunofluorescence assay were used to detect protein expression and location of RIP1, RIP3 and MLKL, respectively. Results Neurological defect and brain infarction were caused by MCAO. Compared with MCAO group, zVAD, GSK＇872 and the combined intervention alleviated neurological defect and reduced brain infarct volume significantly （P〈0.05 or P〈0.01 ）. The protein levels of RIP3 and RIP1 MLKL were increased in mice of MCAO group,while GSK＇872 and the combined intervention obviously downregulated the aforementioned protein expression [ RIP1 （GSK＇ 872：0.64± 0.02 vs MCAO ： 1.28±0.02 ,P〈0.01 ） ; RIP3 （ GSK＇ 872 ： 1.08±0.02 vs MCAO ： 1.45±0.02 ,P〈0.01 ） ; MLKL （GSK＇ 872：0.54±0.01 vs MCAO： 1.00±0.01 ,P〈0.01 ）]. However, zVAD only slightly reduced protein expression of MLKL （P〈0.05） but didn＇t change the protein expression of RIP1 and RIP3 （P〉0.05）. Conclusion RIP1, RIP3 and MLKL are involved in the execution of necroptosis and contribute to the pathological progress of brain ischemia/reperfusion injury.
出处
《中华行为医学与脑科学杂志》
CAS
CSCD
北大核心
2017年第10期917-921,共5页
Chinese Journal of Behavioral Medicine and Brain Science
基金
国家自然科学基金项目（31200809）
天津市科技计划项目（15ZXLCSY00040）
武警后勤学院中心实验室开放基金项目（2015ZXKF09）
