Sun Yat-sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
Brain Behav. 2024 Oct;14(10):e70108. doi: 10.1002/brb3.70108.
The presence of hypoxic-ischemic brain damage (HIBD) in neonates triggers a strong neuroinflammatory reaction. Pyroptosis, a programmed cell death mechanism associated with inflammation, plays a crucial role in HIBD. Pyruvate kinase M2 (PKM2) plays a significant role in connecting metabolic processes and inflammatory responses, but whether it affects hippocampus pyroptosis in HIBD is unclear. The aim of this study is to elucidate the role of PKM2 in HIBD and to propose a novel therapeutic approach for neonatal ischemic-hypoxic encephalopathy.
In this study, we employed neonatal 7-day-old Sprague Dawley rats to establish a model of HIBD using the Rice-Vannucci surgical technique and a hypoxia device. To inhibit the elevation of PKM2, we utilized the PKM2 inhibitor shikonin. The rats were categorized into four groups: Sham, Shikonin, HIBD, and Shikonin + HIBD. Behavioral tests, hematoxylin eosin staining, immunofluorescence staining, ELISA (IL-1β, IL-18), and LDH were conducted in each group to evaluate neurological function, hippocampal damage, the occurrence of neuronal pyroptosis, and the neuroinflammation. Western blot was used to assess the expression levels of PKM2, NLRP3, Caspase-1, Cleaved Caspase-1, GSDMD, GSDMDN, and IL-1β.
The expression of PKM2 elevated in hippocampal tissues of the HIBD model and the localization of PKM2 in the hippocampus was activated in neurons instead of microglia during the HIBD. Meanwhile, the inhibition of PKM2 improved the behavioral test scores and the body weight of rats, the neuronal damage in the CA1 region of hippocampal tissue was also attenuated. In addition, inhibiting PKM2 alleviated neuronal pyroptosis by decreasing the expression of PKM2, NLRP3, Caspase-1, Cleaved Caspase-1, GSDMD, GSDMDN. Furthermore, serum levels of LDH and inflammatory factors IL-1β and IL-18 decrease with PKM2 inhibition.
Based on these findings, we can conclude that PKM2 plays a crucial role in regulating hippocampal neuronal pyroptosis of HIBD rats via NLRP3/Caspase-1/GSDMD pathway. Therefore, inhibiting PKM2 could be a promising therapeutic strategy for the treatment of neonatal ischemic-hypoxic encephalopathy.
新生儿缺氧缺血性脑损伤（HIBD）的存在会引发强烈的神经炎症反应。细胞焦亡是一种与炎症相关的程序性细胞死亡机制，在 HIBD 中起着至关重要的作用。丙酮酸激酶 M2（PKM2）在连接代谢过程和炎症反应方面发挥着重要作用，但它是否会影响 HIBD 中海马体的细胞焦亡尚不清楚。本研究旨在阐明 PKM2 在 HIBD 中的作用，并为新生儿缺氧缺血性脑病提出一种新的治疗方法。
本研究采用新生 7 天龄 Sprague Dawley 大鼠，采用 Rice-Vannucci 手术技术和缺氧装置建立 HIBD 模型。为了抑制 PKM2 的升高，我们使用了 PKM2 抑制剂紫草素。将大鼠分为四组：假手术组、紫草素组、HIBD 组和紫草素+HIBD 组。每组均进行行为测试、苏木精-伊红染色、免疫荧光染色、ELISA（IL-1β、IL-18）和 LDH 检测，以评估神经功能、海马损伤、神经元细胞焦亡的发生和神经炎症。Western blot 用于评估 PKM2、NLRP3、Caspase-1、Cleaved Caspase-1、GSDMD、GSDMDN 和 IL-1β 的表达水平。
HIBD 模型中海马组织中 PKM2 的表达升高，PKM2 在 HIBD 期间在海马体中的定位在神经元中被激活，而不是小胶质细胞中。同时，抑制 PKM2 可改善大鼠的行为测试评分和体重，减轻海马组织 CA1 区的神经元损伤。此外，抑制 PKM2 通过降低 PKM2、NLRP3、Caspase-1、Cleaved Caspase-1、GSDMD、GSDMDN 的表达，减轻神经元细胞焦亡。此外，抑制 PKM2 可降低血清 LDH 水平和炎症因子 IL-1β 和 IL-18 的水平。
基于这些发现，我们可以得出结论，PKM2 通过 NLRP3/Caspase-1/GSDMD 通路在 HIBD 大鼠的海马神经元细胞焦亡中发挥关键作用。因此，抑制 PKM2 可能是治疗新生儿缺氧缺血性脑病的一种有前途的治疗策略。
