Hif-1α/Hsf1/Hsp70 signaling pathway regulates redox homeostasis and apoptosis in large yellow croaker (<i>Larimichthys crocea</i>) under environmental hypoxia

Oxygen is an essential molecule for animal respiration, growth, and survival. Unlike in terrestrial environments, contamination and climate change have led to the frequent occurrence of hypoxia in aquatic environments, thus impacting aquatic animal survival. However, the adaptative mechanisms underlying fish responses to environmental hypoxia remain largely unknown. Here, we used large yellow croaker ( Larimichthys crocea) and large yellow croaker fry (LYCF) cells to investigate the roles of the Hif-1α/Hsf1/Hsp70 signaling pathway in the regulation of cellular redox homeostasis, and apoptosis. We confirmed that hypoxia induced the expression of Hif-1α, Hsf1, and Hsp70 in vivo and in vitro. Genetic Hsp70 knockdown/overexpression indicated that Hsp70 was required for maintaining redox homeostasis and resisting oxidative stress in LYCF cells under hypoxic stress. Hsp70 inhibited caspase-dependent intrinsic apoptosis by maintaining normal mitochondrial membrane potential, enhancing Bcl-2 mRNA and protein expression, inhibiting Bax and caspase3 mRNA expression, and suppressing caspase-3 and caspase-9 activation. Hsp70 suppressed caspase-independent intrinsic apoptosis by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and disturbed extrinsic apoptosis by inactivating caspase-8. Genetic knockdown/overexpression of Hif-1α and dual-luciferase reporter assay indicated that Hif-1α activated the Hsf1 DNA promoter and enhanced Hsf1 mRNA transcription. Hsf1 enhanced Hsp70 mRNA transcription in a similar manner. In summary, the Hif-1α/Hsf1/Hsp70 signaling pathway plays an important role in regulating redox homeostasis and anti-apoptosis in L. crocea under hypoxic stress.氧气是影响动物呼吸、生长和生存的重要环境因子。与陆地环境不同，水生环境受污染和气候变化等因素的影响，低氧现象频发，进而影响水生动物的生存。然而，迄今为止，鱼类应对环境低氧的适应机制尚未探明。该研究中，我们以大黄鱼（ Larimichthys crocea）和大黄鱼仔鱼（LYCF）细胞为研究对象探讨了Hif-1α/Hsf1/Hsp70信号通路在调节细胞氧化还原稳态和细胞凋亡中的作用。结果显示，体内及体外低氧模型中，低氧诱导了大黄鱼Hif-1α、Hsf1及Hsp70的表达； Hsp70敲降及过表达分析表明，Hsp70是低氧胁迫下LYCF细胞维持氧化还原稳态及抗氧化应激所必需的；Hsp70可通过维持线粒体膜电位的正常、增强 Bcl-2 mRNA及蛋白的表达、抑制 Bax和 Caspase-3 mRNA的表达以及抑制Caspase-3和Caspase-9的活化等方式来抑制低氧胁迫下大黄鱼LYCF细胞的Caspase依赖性的线粒体途径凋亡；Hsp70可通过抑制AIF入核的方式来抑制低氧胁迫下大黄鱼LYCF细胞的非Caspase依赖性的线粒体途径凋亡，并通过抑制Caspas-8活性的方式来抑制低氧胁迫下大黄鱼LYCF细胞的死亡受体途径凋亡； Hif-1α敲降及过表达及双荧光素酶报告基因分析表明，Hif-1α激活了 Hsf1的DNA启动子并增强了 Hsf1 mRNA的转录，Hsf1又以同样的方式增强了 Hsp70 mRNA的转录。综上所述，Hif-1α/Hsf1/Hsp70信号通路在调节低氧胁迫下大黄鱼氧化还原平衡及抗凋亡中发挥重要作用。.