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。 |
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