Thiamine deficiency activates hypoxia inducible factor-1α to facilitate pro-apoptotic responses in mouse primary astrocytes

Thiamine is an essential enzyme cofactor required for proper metabolic function and maintenance of metabolism and energy production in the brain. In developed countries, thiamine deficiency (TD) is most often manifested following chronic alcohol consumption leading to impaired mitochondrial function, oxidative stress, inflammation and excitotoxicity. These biochemical lesions result in apoptotic cell death in both neurons and astrocytes. Comparable histological injuries in patients with hypoxia/ischemia and TD have been described in the thalamus and mammillary bodies, suggesting a congruency between the cellular responses to these stresses. Consistent with hypoxia/ischemia, TD stabilizes and activates Hypoxia Inducible Factor-1α (HIF-1α) under physiological oxygen levels. However, the role of TD-induced HIF-1α in neurological injury is currently unknown. Using Western blot analysis and RT-PCR, we have demonstrated that TD induces HIF-1α expression and activity in primary mouse astrocytes. We observed a time-dependent increase in mRNA and protein expression of the pro-apoptotic and pro-inflammatory HIF-1α target genes MCP1, BNIP3, Nix and Noxa during TD. We also observed apoptotic cell death in TD as demonstrated by PI/Annexin V staining, TUNEL assay, and Cell Death ELISA. Pharmacological inhibition of HIF-1α activity using YC1 and thiamine repletion both reduced expression of pro-apoptotic HIF-1α target genes and apoptotic cell death in TD. These results demonstrate that induction of HIF-1α mediated transcriptional up-regulation of pro-apoptotic/inflammatory signaling contributes to astrocyte cell death during thiamine deficiency.硫胺素（Thiamine）是一类必需的酶辅因子，对维持大脑正常代谢功能、代谢稳态与能量产生不可或缺。在发达国家，硫胺素缺乏症（TD）最常因慢性酒精摄入引发，可导致线粒体功能受损、氧化应激、炎症反应与兴奋性毒性。上述生化损伤可致使神经元与星形胶质细胞发生凋亡性细胞死亡。缺氧缺血与硫胺素缺乏症患者的丘脑及乳头体中，均观察到相似的组织病理学损伤，提示二者对应激的细胞应答具有一致性。与缺氧缺血模型一致，硫胺素缺乏症可在生理氧浓度下稳定并激活缺氧诱导因子-1α（HIF-1α）。但目前尚不明确硫胺素缺乏诱导的HIF-1α在神经损伤中的作用。本研究通过蛋白质印迹（Western blot）与逆转录聚合酶链反应（RT-PCR）实验，证实硫胺素缺乏可在原代小鼠星形胶质细胞中诱导HIF-1α的表达与活性上调。研究期间观察到，促凋亡与促炎性HIF-1α靶基因MCP1、BNIP3、Nix及Noxa的mRNA与蛋白表达呈时间依赖性升高。本研究同时通过碘化丙啶/膜联蛋白V染色、TUNEL检测及细胞死亡ELISA实验，证实硫胺素缺乏可引发细胞凋亡性死亡。采用YC1对HIF-1α活性进行药理学抑制，以及补充硫胺素，均可降低硫胺素缺乏模型中促凋亡HIF-1α靶基因的表达，并减少细胞凋亡性死亡。上述结果表明，硫胺素缺乏期间，HIF-1α介导的促凋亡/炎性信号转录上调，是导致星形胶质细胞死亡的重要机制。