Supplementary Material for: Oxidative stress accompanies HIF1-dependent impairment of glucose metabolism in the hippocampus of adult rats survived prenatal severe hypoxia

Introduction: Many socially significant diseases are associated with prenatal developmental disorders. Previously, we showed the pathological role of hypoxia-inducible factor HIF1 in post-hypoxic reoxygenation. This study aimed to investigate the effect of prenatal severe hypoxia (PSH) on HIF1α protein expression as well as on HIF1-dependent activity of the pentose phosphate pathway (PPP) and anaerobic glycolysis in the hippocampus (HPC) of offspring that reached adulthood. Methods: PSH was induced during the critical period of fetal hippocampal formation on gestation days 14-16 in a hypobaric chamber (180 Torr, 5% oxygen, 3 hours). Subsequent studies were conducted on both the HPC of adult control and PSH rats under normal conditions, as well as in response to severe hypobaric hypoxia (SH) or psycho-emotional stress (“learned helplessness” model, LH). We evaluated HIF1α protein levels using both immunohistochemistry and western blotting techniques. The amount of glucose-6-phosphate dehydrogenase (G6PD) was also determined by western blotting. Colorimetric enzymatic assays were employed to analyze enzymatic activity of lactate dehydrogenase (LDH), the concentration of lactate, NADPH, reduced glutathione (GSHred), and malonic dialdehyde (MDA). Results: We showed that PSH caused a stable increase in the content of HIF1α protein in the HPC, which was accompanied by an increase in the efficiency of anaerobic glycolysis. This was confirmed by increased LDH activity and lactate concentration. At the same time, the amounts of G6PD, NADPH, and reduced glutathione decreased in the HPC of PSH rats, whereas the concentration of MDA, an oxidative stress marker, exceeded the control values. In a series of experiments using the LH or SH stress, it was shown that in the HPC of control rats, there was an increase in the amount of HIF1α in response to stress, which was also accompanied by more efficient anaerobic glycolysis and decreased of PPP-dependent NADPH production, similar to the intact PSH rats. In PSH rats, emotional stress resulted in higher HIF1α levels without affecting glycolysis or PPP. Conclusion: Therefore, the increased content and activity of the transcription factor HIF1α in the HPC of adult rats exposed to prenatal hypoxia leads to an imbalance between glycolysis and PPP, which is accompanied by oxidative stress.

引言：众多与社会意义重大的疾病均与孕前发育障碍相关。先前，我们揭示了低氧诱导因子HIF1在缺氧后复氧过程中的病理作用。本研究旨在探讨孕前严重低氧（PSH）对成年后海马体（HPC）中HIF1α蛋白表达以及HIF1依赖性磷酸戊糖途径（PPP）和厌氧糖酵解活性的影响。方法：在胎鼠海马体形成的关键期，即妊娠第14-16天，在低气压舱（180托，5%氧气，3小时）中诱导PSH。随后，在正常条件下对成年对照组和PSH大鼠的海马体进行了研究，并对其施加严重低气压缺氧（SH）或心理情绪压力（“习得性无助”模型，LH）。我们利用免疫组化和蛋白印迹技术评估了HIF1α蛋白水平，并通过蛋白印迹技术测定了葡萄糖-6-磷酸脱氢酶（G6PD）的含量。采用比色酶联免疫吸附测定法分析乳酸脱氢酶（LDH）的酶活性、乳酸浓度、NADPH、还原型谷胱甘肽（GSHred）和丙二醛（MDA）的浓度。结果：我们发现PSH导致HPC中HIF1α蛋白含量稳定升高，伴随厌氧糖酵解效率的增加，这通过LDH活性和乳酸浓度的升高得到证实。同时，PSH大鼠海马体中G6PD、NADPH和还原型谷胱甘肽的含量降低，而氧化应激标志物MDA的浓度超过对照组。在LH或SH压力的一系列实验中，发现对照组大鼠的海马体在应激反应中HIF1α含量增加，伴随厌氧糖酵解效率的提高以及PPP依赖性NADPH生产的减少，与完整的PSH大鼠相似。在PSH大鼠中，情绪压力导致HIF1α水平升高，但未影响糖酵解或PPP。结论：因此，成年大鼠孕前低氧暴露导致海马体中转录因子HIF1α的含量和活性增加，从而引起糖酵解与PPP之间的失衡，伴随氧化应激的发生。