远端缺血后处理对新生缺氧缺血性脑病小鼠组织型纤溶酶原激活物/脑源性神经营养因子通路及海马突触可塑性的影响

Effects of distal ischemic postconditioning on tissue plasminogen activator/brain-derived neurotrophic factor pathway and hippocampal synaptic plasticity in neonatal hypoxic-ischemic encephalopathy mice

目的 探究远端缺血后处理(RIPoC)对新生缺氧缺血性脑病(HIE)小鼠组织型纤溶酶原激活物(tPA)/脑源性神经营养因子(BDNF)通路的调控作用及对海马突触可塑性的影响。方法 将60只7日龄C57BL/6j小鼠用随机数字表法分为假手术组、模型组、RIPoC组,每组20只。模型组小鼠通过结扎右侧颈总动脉并给予低氧处理建立HIE模型,假手术组除不结扎颈总动脉外其余同模型组,RIPoC组在模型组基础上行夹闭双侧股动脉行缺血5 min/再灌注5 min,3个循环。造模后24 h,采用2,3,5-氯化三苯基四氮唑(TTC)染色法检测脑梗死体积;HE法观察海马组织中神经元病理变化;透射电镜观察海马组织突触体数量及结构变化;Western Blotting法检测海马组织突触相关蛋白突触后致密物-95(PSD95)、突触囊泡膜蛋白(SYP)、钙离子结合蛋白鉴定蛋白复合体S100A10(p11)、tPA、BDNF、BDNF前体蛋白(Pro-BDNF)、酪氨酸蛋白激酶B(TrKB)相对表达水平。结果 与假手术组相比,模型组小鼠海马区神经元肿胀、胞体固缩、模糊、丢失坏死严重,突触数量较少;脑梗死体积、Pro-BDNF蛋白表达升高(均P<0.05),PSD95、SYP、p11、tPA、BDNF、TrKB蛋白表达均降低(均P<0.05)。与模型组相比,RIPoC组小鼠海马区组织神经元肿胀等损伤缓解,突触数量增多;脑梗死体积、Pro-BDNF蛋白表达降低(均P<0.05),PSD95、SYP、p11、tPA、BDNF、TrKB蛋白表达均升高(均P<0.05)。结论 RIPoC可能通过激活p11/tPA通路,促进Pro-BDNF向BDNF转化并激活BDNF/TrKB通路,提高HIE小鼠海马组织中突触可塑性,改善HIE所致脑损伤。

Objective To investigate the regulatory effect of remote ischemic postconditioning(RIPoC) on tissue plasminogen activator(tPA)/brain-derived neurotrophic factor(BDNF) pathway in neonatal hypoxic-ischemic encephalopathy(HIE) mice and its effect on hippocampal synaptic plasticity.Methods Sixty seven-day-old C57 BL/6 j mice were divided into sham operation group,model group and RIPoC group according to the random number table,with 20 mice in each group.In the model group,the HIE model was established by ligation of the right common carotid artery and hypoxia treatment,the sham operation group was the same as the model group except that the common carotid artery was not ligated,in the RIPoC group,bilateral femoral arteries were clamped for 5 min ischemia/5 min reperfusion based on the model group,the distal limb ischemic postconditioning(RIPoC) was achieved by repeated circulation for 3 times.At 24 h after modeling,cerebral infarct volume was detected by 2,3,5-triphenyl tetrazolium chloride(TTC) staining;HE staining was used to observe the pathological changes of neurons in the hippocampus;the number and structure of synaptosomes in the hippocampus were observed by transmission electron microscope;Western blotting was used to detect the relative expression levels of synaptic associated protein postsynaptic density 95(PSD95) and synaptophysin(SYP),identification of calcium-binding protein complex S100 A10(p11),tPA,BDNF,Pro-BDNF and Tyrosine-protein kinase B(TrKB) in the hippocampus.Results Compared with those in the sham group,the hippocampal neurons in the model group were swollen,pyknosis,fuzzy,loss and necrosis,and the number of synapses was less than in the model group;the cerebral infarct volume and the expression of Pro-BDNF protein were significantly higher(all P<0.05),the protein expression of PSD95,SYP,p11,tPA,BDNF and TrKB was significantly lower(all P<0.05).Compared with those in the model group,the swelling of neurons in the hippocampus of the RIPo C group was alleviated,the number of synapses was increased;the cerebral infarct volume and Pro-BDNF protein expression were significantly lower( all P < 0.05),and the protein expression of PSD95,SYP,p11,t PA,BDNF,Tr KB were significantly higher( all P < 0.05).Conclusion RIPo C may promote the transformation of Pro-BDNF to BDNF and activate BDNF/Tr KB pathway by activating p11/t PA pathway,which can improve synaptic plasticity in the hippocampus of HIE mice and improve brain injury induced by HIE.