Abstract：
The increasing frequency of high-temperature extremes threatens largemouth bass Micropterus salmoides, a significant fish for freshwater ecosystems and aquaculture. Our previous studies at the transcript level suggested that heat stress induces hepatic apoptosis in largemouth bass. In the current study, we sought to validate these findings and further investigate the role of the c-Jun N-terminal kinase (JNK)/P53 signaling in hepatic apoptosis under heat stress. First, heat treatments were conducted in vivo and in vitro under different temperatures: 28 °C, 32 °C, and 37 °C. In primary hepatocytes subjected to heat treatment, cell viability was evaluated via the Cell Counting Kit-8, while mitochondrial membrane potential and nuclear morphology were assessed through JC-1 and Hoechst 33258 staining, respectively. We observed reductions in both cell viability and mitochondrial membrane potential (ΔΨm), along with alterations in nuclear morphology, in primary hepatocytes exposed to heat stress at temperatures of 32 °C and 37 °C. Quantitative real-time PCR revealed significant alterations in the expression profiles of intrinsic apoptosis-related genes within liver tissues under heat stress. Immunohistochemistry analysis revealed that JNK1 signaling increased as the temperature increased, JNK2 expression increased only at 37 °C, and JNK3 expression did not change with temperature. We speculate that JNK1 and JNK2 have pro- and anti-apoptotic effects, respectively. Western blot analysis conducted on cultured hepatocytes further validated these findings. JNK inhibition reduced hepatocyte apoptosis, improved nuclear morphology, and maintained ΔΨm even after 37 °C treatment. These results not only confirm that heat stress led to intrinsic apoptosis of hepatocytes but also indicated that JNK1 could mediate P53 expression and activate caspase-dependent intrinsic apoptosis in largemouth bass hepatocytes under such conditions. This study illuminates the physiological responses of largemouth bass to acute heat stress, offering valuable insights into the potential impacts of climate change on freshwater fishes and the sustainability of aquaculture.
摘要：
高温极端事件的频率增加威胁着大嘴鲈鱼Micropterussalmoides，淡水生态系统和水产养殖的重要鱼类。我们先前在转录水平上的研究表明，热应激可诱导大口鲈鱼的肝细胞凋亡。在目前的研究中,我们试图验证这些发现,并进一步研究c-JunN末端激酶(JNK)/P53信号在热应激下肝细胞凋亡中的作用.首先,在不同温度下进行体内和体外热处理：28°C，32°C,37°C在经过热处理的原代肝细胞中，通过细胞计数试剂盒-8评估细胞活力，同时通过JC-1和Hoechst33258染色评估线粒体膜电位和核形态，分别。我们观察到细胞活力和线粒体膜电位（ΔkWm）的降低，随着核形态的改变，在32°C和37°C的温度下暴露于热应激的原代肝细胞中。定量实时PCR显示，在热应激下，肝组织内固有凋亡相关基因的表达谱发生了显着变化。免疫组织化学分析显示，JNK1信号随着温度的升高而增加，JNK2表达仅在37°C时增加，JNK3表达不随温度变化。我们推测JNK1和JNK2具有促凋亡和抗凋亡作用，分别。对培养的肝细胞进行的Western印迹分析进一步验证了这些发现。JNK抑制减少肝细胞凋亡，改善核形态，并且即使在37°C处理后仍保持ΔkW。这些结果不仅证实了热应激导致肝细胞的内在凋亡，而且还表明JNK1可以介导P53表达并激活在这种条件下大口鲈鱼肝细胞中依赖caspase的内在凋亡。本研究阐明了大嘴鲈鱼对急性热应激的生理反应，提供有关气候变化对淡水鱼和水产养殖可持续性的潜在影响的宝贵见解。
