School of Biology and Basic Medical Sciences, Suzhou Medical College of Soochow University, 215000, China.
School of Biology and Basic Medical Sciences, Suzhou Medical College of Soochow University, 215000, China.
Sci Total Environ. 2024 Oct 15;947:174664. doi: 10.1016/j.scitotenv.2024.174664. Epub 2024 Jul 10.
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.
高温极值的频率不断增加，对大口黑鲈 Micropterus salmoides 这一淡水生态系统和水产养殖中重要的鱼类构成威胁。我们之前的转录组水平研究表明，热应激会诱导大口黑鲈的肝凋亡。在本研究中，我们试图验证这些发现，并进一步研究在热应激下 c-Jun N-末端激酶 (JNK)/P53 信号在肝凋亡中的作用。首先，我们在不同温度下进行了体内和体外的热处理：28°C、32°C 和 37°C。在接受热处理的原代肝细胞中，通过 Cell Counting Kit-8 评估细胞活力，同时通过 JC-1 和 Hoechst 33258 染色分别评估线粒体膜电位和核形态。我们观察到，在 32°C 和 37°C 的热应激下，原代肝细胞的细胞活力和线粒体膜电位（ΔΨm）均降低，同时核形态发生改变。实时定量 PCR 显示，热应激下肝组织中内在凋亡相关基因的表达谱发生显著改变。免疫组织化学分析显示，JNK1 信号随着温度的升高而增加，JNK2 的表达仅在 37°C 时增加，而 JNK3 的表达不受温度变化的影响。我们推测 JNK1 和 JNK2 分别具有促进和抑制凋亡的作用。对培养的肝细胞进行的 Western blot 分析进一步验证了这些发现。JNK 抑制减少了肝细胞凋亡，改善了核形态，并在 37°C 处理后维持了 ΔΨm。这些结果不仅证实了热应激导致了肝细胞的内在凋亡，还表明 JNK1 可以介导 P53 表达，并在这种情况下激活大口黑鲈肝细胞中 caspase 依赖性内在凋亡。本研究阐明了大口黑鲈对急性热应激的生理反应，为气候变化对淡水鱼类的潜在影响以及水产养殖的可持续性提供了有价值的见解。
