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PKM2对新生HIE小鼠神经元凋亡的影响
作者:罗玲英1  许静2  唐瑾3 
单位:1. 宝鸡市妇幼保健院 新生儿科, 陕西 宝鸡 721000;
2. 西安交通大学第一附属医院 特诊室, 陕西 西安 710061;
3. 宝鸡市人民医院 新生儿科, 陕西 宝鸡 721000
关键词:M2型丙酮酸激酶同工酶 缺氧缺血性脑病 皮质神经元 细胞凋亡 蛋白激酶B信号 小鼠 
分类号:R722.19;R-33
出版年·卷·期(页码):2020·39·第三期(274-280)
摘要:

目的:探讨M2型丙酮酸激酶同工酶(PKM2)对新生缺氧缺血性脑病(HIE)小鼠皮质神经元凋亡的影响。方法:根据改良Rice-Vannucci法建立新生小鼠HIE模型,另设假手术组。建模24 h后麻醉小鼠并分离大脑组织,2,3,5-氯化三苯基四氮唑(TTC)染色法检测脑梗死体积,Western blotting、免疫组化和免疫荧光检测皮质组织PKM2、裂解的半胱氨酸蛋白酶-3(cleaved caspase-3)、磷酸化蛋白激酶B(p-AKT)和蛋白激酶B(AKT)的表达,TUNEL染色检测神经元细胞凋亡。通过糖氧剥夺(OGD)实验和PKM2-siRNA转染来评价PKM2在体外对神经元凋亡的影响。3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)方法测定存活细胞数。结果:HIE组小鼠的脑梗死体积比显著高于假手术组(43.26% vs 0.71%)。与假手术组相比,HIE组小鼠大脑皮质组织和神经元中PKM2蛋白的表达水平显著升高。HIE组小鼠大脑皮质神经元的TUNEL阳性率显著高于假手术组(37.68% vs 8.95%);HIE组小鼠大脑皮质中凋亡相关蛋白cleaved caspase-3的表达水平显著增加,而p-AKT/AKT之值明显降低。体外研究中,OGD处理可显著减少活皮质神经元数量,但下调PKM2的表达可上调活神经元数量。OGD处理显著上调PKM2和cleaved caspase-3蛋白表达,但抑制AKT的磷酸化。敲除PKM2的表达则可显著抑制cleaved caspase-3蛋白的上调,并促进AKT的磷酸化。结论:PKM2在HIE发病过程中表达上调。PKM2表达的上调可能导致AKT信号通路被抑制,进而诱导皮质神经元的凋亡。

Objective:To investigate the effect of pyruvate kinase isozymes M2 (PKM2) on apoptosis of cortical neurons in neonatal mice with hypoxic ischemic encephalopathy (HIE). Methods:A neonatal mouse HIE model was established according to the modified Rice-Vannucci method, and a sham group was set up. After 24 hours of modeling, the mice were anesthetized and brain tissue was isolated. The volume of cerebral infarction was detected by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The expressions of PKM2, cleaved caspase-3, phosphorylated protein kinase B (p-AKT) and protein kinase B (AKT) in cortex were detected by Western blotting, immunohistochemistry and immunofluorescence. TUNEL staining was used to detect neuronal apoptosis. The effect of PKM2 on neuronal apoptosis in vitro was evaluated by glucose oxygen deprivation (OGD) assay and PKM2-siRNA transfection. The number of viable cells was determined by the 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Results:The cerebral infarction volume was significantly higher in the HIE group than in the sham group (43.26% vs 0.71%). Compared with the sham group, the expression level of PKM2 protein in the cerebral cortex tissues and neurons of the HIE group was significantly increased. The TUNEL positive rate of cerebral cortical neurons in HIE mice was significantly higher than that in the sham group (37.68% vs 8.95%). The expression level of apoptosis-related protein cleaved caspase-3 in the cerebral cortex of HIE mice was significantly increased, while the p-AKT/AKT ratio was significantly reduced. In vitro studies, OGD treatment significantly reduced the number of living cortical neurons but knocking down PKM2 up-regulated the number of living cortical neurons. OGD treatment significantly up-regulated PKM2 and cleaved caspase-3 protein expression, while inhibited AKT phosphorylation. Knockdown of PKM2 significantly inhibited the upregulation of cleaved caspase-3 protein and promoted phosphorylation of AKT. Conclusion:PKM2 is up-regulated during the pathogenesis of HIE. Upregulation of PKM2 leads to inhibition of the AKT signaling pathway, which in turn induces apoptosis in cortical neurons.

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