| 万金铭,王映,陈良标.鳞头犬牙南极鱼LeptinB基因在抵御低温应激中的作用.渔业科学进展,2023,44(6):97-106 |
| 鳞头犬牙南极鱼LeptinB基因在抵御低温应激中的作用 |
| Role of the Dissostichus mawsoni LeptinB gene in cellular cold resistance |
| 投稿时间：2023-04-18 修订日期：2023-05-18 |
| DOI：10.19663/j.issn2095-9869.20230418001 |
| 中文关键词: 鳞头犬牙南极鱼 LeptinB 低温应激 细胞凋亡 脂质代谢 |
| 英文关键词: Dissostichus mawsoni LeptinB Low-temperature stress Apoptosis Lipid metabolism |
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| 中文摘要: |
| 为了探究瘦素基因(LeptinB, LepB)在斑马鱼肝脏细胞系(ZFL)低温应激中的作用，本研究根据鳞头犬牙南极鱼(Dissostichus mawsoni) LepB基因(DM-LepB)编码的氨基酸序列，克隆到构建的pTOL2-actin-EGFP质粒中(启动子为斑马鱼的β-actin)。选取EcoRⅠ和BamHⅠ为限制酶，设计构建了pTOL2-LepB+HIS-EGFP表达载体，并转染至ZFL细胞中。选择致死温度10 ℃进行实验，采用流式细胞术检测了低温胁迫下细胞的存活率，使用增强型CCK-8试剂盒检测了低温条件下细胞的生长增殖情况，使用荧光探针DHE检测了细胞活性氧(ROS)含量。结果显示，LepB基因的过表达能有效减少细胞ROS的产生，减轻细胞凋亡以应对低温胁迫。分析还显示，DM-LepB的过表达也有利于维持低温刺激下的胞内ATP水平与线粒体状态，有效减轻低温刺激对细胞的凋亡和坏死作用，对细胞冷应激起到保护作用。采用油红O染色和甘油三酯(TG)实验检测发现，DM-LepB基因能减缓低温刺激时细胞脂质的消耗，较多的脂质保留可减弱低温刺激对细胞的伤害，使得细胞能更好地抵抗低温损害。研究结果为揭示南极鱼类的低温适应分子机制提供了基础资料。 |
| 英文摘要: |
| Variation in fish temperature is determined by changes in the external aquatic environment, and temperature serves as a crucial regulatory factor for the behavior, physiology, and molecular processes of fish. A rapid decline in water temperature can elicit a strong stress response in fish, potentially inhibiting growth and even leading to mortality. Low temperature stress significantly impacts various life processes of organisms, including growth and development, energy metabolism, and reproductive development. In recent years, there has been increasing research on the effects of low-temperature stress on organisms, with a greater focus at the molecular level. Low temperatures induce a decrease in cellular activity and even growth arrest, resulting in apoptosis. Apoptosis is a form of programmed cell death ubiquitous in the biological world, and it plays a vital role in maintaining the normal physiological function of an organism. Once apoptosis occurs, it is irreversible and cannot be stopped. Temperature regulation is crucial for fish, and current knowledge of the role of leptin in fish primarily focuses on the regulation of feeding, lipid energy metabolism, and reproduction, with limited reports on its role in temperature regulation.
Leptin, a protein hormone produced by adipocytes, has long been recognized as a product of the obese gene, which regulates organismal metabolism, neuroendocrine function, and other physiological processes. In animals, it exerts diverse physiological functions related to cell growth, proliferation, apoptosis, and metabolism. The homology of the leptin gene in fish is relatively low compared to that in mammals, and multiple leptin proteins encoded by multiple leptin genes can be produced due to genomic duplication events. Regarding the role of leptin in temperature regulation, more research has been conducted on mammals, mainly related to the regulation of energy metabolism and the influence on temperature adaptation evolution. There has been limited research on the regulatory mechanism of leptin in fish during cold stress.
Dissostichus mawsoni belongs to the group of Antarctic vertebrates and has lived in the cold and isolated environment of the Southern Ocean at a temperature of –1.9 ℃ for ~30 million years. It has adapted to the extremely low temperatures of the surrounding Antarctic waters and is an excellent material and living fossil for studying temperature adaptation mechanisms in extreme environments. Compared with other fish species at the same depth, it has a large amount of lipid deposition in its subcutaneous and muscle tissues, mainly triglycerides (TGs). Predictions based on 3D structural models suggest that the partial absence of the lepB structure in D. mawsoni leads to only three α-helices, and lepA has four α-helices and a short and twisted E-helix as well as several irregular turns, forming a hollow barrel-like structure, which differs from the protein structure of all other known leptins. Moreover, previous studies have found a close correlation between lepA and temperature evolution, leading to speculation that Antarctic fish lepB may play an essential role in low-temperature adaptation.
In this study, a eukaryotic expression vector of the Antarctic toothfish lepb gene was constructed and transfected into ZFL cells to establish a model of overexpression of the D. mawsoni lepb (DMLB) gene in the ZFL cells. After conducting a cell culture temperature experiment, 10 ℃ was selected as the significant difference temperature. Following 2 weeks of cold stimulation, the DMLB experimental group cells remained viable, whereas the control group cells died. The results indicated that the overexpression of the DMLB gene provided strong resistance to low-temperature conditions. By detecting changes in cell proliferation, apoptosis, reactive oxygen species (ROS) level, and ATP content under low-temperature stress, it was discovered that DMLB maintained cell growth and proliferation, reduced ROS production, and inhibited cell apoptosis. DMLB effectively maintained ATP levels in cells under low-temperature stress, which helps maintain the mitochondrial status and reduce the effects of apoptosis and necrosis caused by low-temperature stress, thereby protecting cells from cold stress. Additionally, the results of Oil red O staining and TG detection suggested that the DMLB gene may slow down the depletion of cell lipids under low-temperature stress, perhaps by lowering lipid metabolism to preserve lipids to cope with low-temperature damage.
Consequently, the DMLB gene may be functional in the cold resistance of D. mawsoni by protecting cells from damage at low temperatures, but its specific molecular regulatory mechanism needs to be further explored. In this study, 10 ℃ was used as the key temperature, providing a basis for understanding the regulation of energy homeostasis by the lepb gene in D. mawsoni under low-temperature stress. This study explored the role of the lepB gene in the adaptation of D. mawsoni to extremely low-temperature environments and provided fundamental data for further study of the evolution of similar species in the Antarctic region. Furthermore, this study enriched the basic data related to low-temperature tolerance and provided reference materials for further investigation of the low-temperature tolerance mechanism of leptin in Notothenioids, as well as laid a foundation for further study of the mechanism of leptin in temperature regulation. |
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