摘要
目的探讨白血病抑制因子(LIF)抑制缺氧缺血性脑病(HIE)新生大鼠中神经元凋亡及神经炎症的分子机制。方法将7日龄SPF级Sprague-Dawley(SD)新生大鼠分为4组(n=15):假手术组、HIE组、HIE+LV-NC组和HIE+LV-LIF组。通过结扎新生大鼠的右侧颈总动脉建立HIE模型。假手术组大鼠进行相同的手术操作但不结扎右侧颈总动脉。HIE+LV-LIF组和HIE+LV-NC组在HIE建模前7 d,分别对新生大鼠脑室注射过表达LIF的慢病毒(LV-LIF)或对照慢病毒(LV-NC)。LV-LIF或LV-NC给药及HIE建模后,采用2%2,3,5-三苯基四氮唑氯(TTC)染色检测梗死体积,根据Longa评分进行神经行为评估,通过尼氏染色和TUNEL染色观察脑组织形态和神经元凋亡。通过RT-PCR或Western blot检测缺血半影区内LIF、NLRP3、ASC、Caspase-1、NF-κB p65和IκBα的表达。通过酶联免疫吸附试验(ELISA)检测脑组织匀浆中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)的水平。通过免疫荧光检测脑组织中活化的小胶质细胞标志物Iba-1的表达。结果与假手术组相比,HIE组LIF mRNA和蛋白表达水平均显著增加(P<0.05)。与HIE组和HIE+LV-NC组相比,HIE+LV-LIF组LIF mRNA和蛋白表达水平均升高(P<0.05),神经功能缺损评分、脑梗死体积、细胞凋亡指数均显著降低(P<0.05)。与HIE组和HIE+LV-NC组相比,HIE+LV-LIF组脑组织匀浆中TNF-α、IL-1β和IL-6的水平均降低(P<0.05)。与HIE组和HIE+LV-NC组相比,HIE+LV-LIF组脑组织中Iba-1的相对荧光强度显著降低(P<0.05)。与HIE组和HIE+LV-NC组相比,HIE+LV-LIF组大鼠脑缺血半影区内NLRP3、ASC、Caspase-1和NF-κB p65的蛋白表达水平均降低,IκBα蛋白表达水平升高(P<0.05)。结论LIF可通过抑制NLRP3炎症体和NF-κB信号通路的激活来有效抑制缺氧缺血性脑病后的小胶质细胞活化和神经炎症。
Objective To explore the molecular mechanism of leukemia inhibitory factor(LIF)inhibiting neuronal apoptosis and neuroinflammation in neonatal rats with hypoxic-ischemic encephalopathy(HIE).Methods The 7-day-old SPF Sprague-Dawley(SD)newborn rats were divided into 4 groups(n=15 in each group):sham operation group,HIE group,HIE+LV-NC group and HIE+LV-LIF group.HIE model was established by ligating the right common carotid artery of newborn rats.Rats in sham operation group underwent the same operation but the right common carotid artery was not ligated.Newborn rats in HIE+LV-LIF group and HIE+LV-NC group were injected with LIF overexpressing lentivirus(LV-LIF)and control lentivirus(LV-NC)into the cerebral ventricle 7 d before HIE modeling,respectively.After LV-LIF or LV-NC administration and HIE modeling,the infarct volume was detected by 2%2,3,5-triphenyltetrazolium chloride(TTC)staining.Neurobehavioral evaluation was performed according to the Longa score.Nissl staining and TUNEL staining were used to observe brain tissue morphology and neuronal apoptosis.The expression levels of LIF,NLRP3,ASC,Caspase-1,NF-κB p65 and IκBαin the ischemic penumbra were detected by RT-PCR and Western blot.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in brain tissue homogenate were detected by enzyme-linked immunosorbent assay(ELISA).The expression of activated microglia marker Iba-1 in brain tissue was detected by immunofluorescence.Results Compared with sham group,LIF mRNA and protein expression levels were increased significantly in HIE group(P<0.05).Compared with HIE group and HIE+LV-NC group,the expression levels of LIF mRNA and protein were increased significantly in HIE+LV-LIF group(P<0.05),while the neurological deficit score,the cerebral infarction volume,and the apoptosis index were significantly reduced(P<0.05).Compared with HIE group and HIE+LV-NC group,the levels of TNF-α,IL-1βand IL-6 in the brain tissue homogenate were decreased in HIE+LV-LIF group(P<0.05).Compared with HIE group and HIE+LV-NC group,the relative fluorescence intensity of Iba-1 in the brain tissue was significantly reduced in HIE+LV-LIF group(P<0.05).Compared with HIE group and HIE+LV-NC group,the protein expression levels of NLRP3,ASC,Caspase-1 and NF-κB p65 in the cerebral ischemic penumbra were decreased in HIE+LV-LIF group(P<0.05),while the expression of IκBαwas significantly increased(P<0.05).Conclusion LIF can effectively inhibit the activation of microglia and the neuroinflammation after HIE by inhibiting the activation of NLRP3 inflammasome and NF-κB signaling pathway.
作者
汤丽萍
闫瑾
王浩
范芳
TANG Liping;YAN Jin;WANG Hao;FAN Fang(Department of Pediatrics,Xijing Hospital,Air Force Military Medical University,Xi’an 710032,China;College of Medical Technology,Xi’an Medical University)
出处
《山西医科大学学报》
CAS
2021年第6期706-714,共9页
Journal of Shanxi Medical University
基金
陕西省自然科学基础研究计划项目(2019JM-514)。
关键词
白血病抑制因子
缺氧缺血性脑病
神经元凋亡
神经炎症
小胶质细胞活化
leukemia inhibitory factor
hypoxic-ischemic encephalopathy
neuronal apoptosis
neuroinflammation
microglia activation
