从炎症机制探讨Notch通路抑制剂对小鼠脑缺血模型的神经保护作用

The neuroprotective effect of notch pathway inhibitors on cerebral ischemia in mice from the mechanism of inflammation

目的探讨Notch通路抑制剂对小鼠脑缺血模型的神经保护作用。方法78只成年BALB/c小鼠按简单随机抽样方法分为Sham组、二甲基亚砜(DMSO)组、γ-分泌酶抑制剂(DAPT)组,每组26只;线栓法制作小鼠脑缺血模型,其中Sham组小鼠接受相同手术,但未插入缝线;DAPT组小鼠在大脑中动脉闭塞前3h腹腔注射DAPT溶液(5 mL·kg^(-1)),Sham组、二甲基亚砜(DMSO)组小鼠注射等剂量DMSO溶液,将Longa评分1~3分的小鼠作为实验小鼠;应用尼氏染色及TUNEL/NeuN免疫荧光双标染色鉴定右额叶皮质神经元,免疫荧光检测缺血半脑右半脑皮质Notch1、胶质纤维酸性蛋白(GFAP)阳性细胞,Westermblot检测缺血半脑右半脑皮质Hes1蛋白、Hes5蛋白表达,透射电镜观察各组右额叶皮质神经元超微结构变化,ELISA检测右前额叶皮质白细胞介素6(IL-6)、肿瘤坏死因子(TNF-α)水平。结果与Sham组比较,DMSO组、DAPT组皮质神经元存活数显著下降,皮质神经元凋亡数显著上升(P<0.05);与DMSO组比较,DAPT组皮质神经元存活数显著上升,皮质神经元凋亡数显著下降(P<0.05);与Sham组比较,DMSO组缺血半脑右半脑皮质Notch1、GFAP阳性细胞数显著上升(P<0.05),DAPT组缺血半脑右半脑皮质Notch1、GFAP阳性细胞数显著下降(P<0.05),DAPT组缺血半脑右半脑皮质Notch1、GFAP阳性细胞数显著低于DMSO组(P<0.05);与Sham组比较,DMSO组、DAPT组右半脑皮质Hes1蛋白、Hes5蛋白表达显著高于Sham组(P<0.05),DAPT组右半脑皮质Hes1蛋白、Hes5蛋白表达显著低于DMSO组(P<0.05);Sham组小鼠梗死周围皮质神经元超微结构正常,线粒体内外嵴结构正常;DMSO组小鼠神经元细胞核形状欠规则,核膜欠清晰,核周间隙扩大,染色质欠均匀呈聚集状,胞质内可见空泡,线粒体肿胀,线粒体内外嵴被破坏;DAPT组神经元超微及线粒体内外嵴结构接近正常;与Sham组比较,DMSO组、DAPT组右前额叶皮质IL-6、TNF-α因子水平显著上升(P<0.05),但DAPT组右前额叶皮质IL-6、TNF-α因子水平显著低于DMSO组(P<0.05)。结论Notch通路抑制剂的应用可阻断脑缺血后Notch信号通路传导,通过抑制Notch1及其下游Hes1蛋白、Hes5蛋白表达,下调IL-6、TNF-α水平来发挥神经元保护作用,值得临床重视。

Objective To investigate the neuroprotective effect of Notch pathway inhibitors on cerebral ischemia in mice based on the mechanism of inflammation.Methods By simple random sampling method78 adult BALB/c mice were divided into Sham group,Dimethylsurfoxide(DMSO)group,γ-glucodenase inhibitor(N-[N-(3,5-difluorophenacetyl)-L-alanyl]-s-phenylglcine t-butyl ester,DAPT)group,with 26 mice in each group;The model of cerebral ischemia in mice was established by thread embolism mice in the Sham group received the same operation but no suture was inserted;mice in DAPT group were intraperitoneally injected with DAPT solution(5mL·kg^(-1))3 hours before middle cerebral artery occlusion,and mice in Sham group and Dimethyl sulfoxide(DMSO)group were intraperitoneally injected with equal dose of DMSO solution.Mice with Longa score of 1 to 3 were selected as experimental mice;the neurons of the right frontal cortex were identified by Nigner staining and TUNEL/NeuN immunofluorescent staining.Positive cells of Notch1,glial fibrillary protein(GFAP)after ischemia in the right cerebral cortex were detected by immunofluorescence.The expression of Hes1 and Hes5 proteins in the right cerebral cortex was detected by Western-blot,and the ultrastructural changes of neurons in the right frontal cortex were observed by transmission electron microscopy.The levels of interleukin-6(IL-6)and tumor necrosis factor(TNF-α)in the right prefrontal cortex were measured by ELISA.Results Compared with Sham group,the survival number of cortical neurons in DMSO and DAPT groups significantly decreased,and the apoptosis number of cortical neurons significantly increased(P<0.05);Compared with DMSO group,the survival number of cortical neurons in DAPT group significantly increased,while the apoptosis number of cortical neurons significantly decreased(P<0.05);Compared with Sham group,the number of Notchl and GFAP positive cells in DMSO group significantly increased(P<0.05),and the number of Notchl and GFAP positive cells in DAPT group significantly decreased(P<0.05).The number of Notch1and GFAP positive cells in DAPT group was significantly lower than that in DMSO group(P<0.05);Compared with Sham group,the expressions of Hes1 protein and Hes5 protein in right cerebral cortex in DMSO group and DAPT group were significantly higher than those in Sham group(P<0.05),while the expressions of Hes1protein and Hes5protein in right cerebral cortex in DAPT group were significantly lower than those in DMSO group(P<0.05);in Sham group,the ultrastxucture of neurons in the cortex around infarction was normal,and normal mitochondrial and inner and outer crists;In DMSO group,the shape of the neurons nuclei was irregular,the nuclear membrane was unclear,the perinuclear space was enlarged,the chromatin was uneven and clustered,the vacuoles were visible in the cytoplasm,the mitochondria were swollen,and the mitochondrial internal and external cristae were destroyed;the ultrastructure and posterior ridge structure in mitochondria in DAPT group were close to normal;compared with Sham group,the levels of IL-6 and TNF-αin right prefrontal cortex in DMSO and DAPT groups significantly increased(P<0.05),but the levels of IL-6 and TNF-αin right prefrontal cortex in DAPT group were significantly lower than those in DMSO group(P<0.05).Conclusion The application of Notch pathway inhibitors can block the conduction of Notch signaling pathway after cerebral ischemia,and play a neuronal protective role by inhibiting the expression of Notch1and its downstream Hes1 and Hes5 proteins,and down-regulating the levels of IL-6 and TNF-α,which is worthy of clinical attention.