半滑舌鳎响应急性高温胁迫的生理变化和相关基因表达

Physiological changes and related gene expression of Cynoglossus semilaevis in response to acute high temperature stress

中文关键词: 半滑舌鳎  急性高温胁迫  肝脏  细胞凋亡  抗氧化酶  热休克蛋白基因

英文关键词: Cynoglossus semilaevis  Acute heat stress  Liver  Apoptosis  Antioxidant enzymes  Heat shock protein genes

中文摘要:

      夏季高温会引起半滑舌鳎(Cynoglossus semilaevis)的应激，甚至造成死亡，是工厂化养殖的重要影响因素之一。为探究高温胁迫对半滑舌鳎肝脏氧化损伤及热应激相关基因的影响，本研究选取半滑舌鳎一个全同胞家系为实验对象，通过连续升温达到高温胁迫条件(35 ℃)后，分别在0、3、6、12和24 h采集肝脏组织，进行苏木精–伊红染色法(HE)和TUNEL染色并观察细胞损伤情况，测定抗氧化酶活性及丙二醛(MDA)含量并检测应激相关基因heat shock protein family A member 1A (hspa1a)、heat shock protein 90 beta family member 1 (hsp90b1)和dual-specificity phosphatase 1 (dusp1)的表达变化。结果显示，急性高温胁迫会造成半滑舌鳎肝脏组织发生明显病理变化并出现细胞凋亡；超氧化物歧化酶(SOD)活性在高温胁迫6 h时显著高于对照组(P<0.05)，谷胱甘肽过氧化物酶(GPx)活性在高温胁迫12 h时显著高于对照组(P<0.05)，过氧化氢酶(CAT)活性在高温胁迫0 h时显著高于对照组(P<0.05)，MDA含量在高温胁迫24 h时显著高于对照组(P<0.05)；热休克蛋白基因hspa1a和hsp90b1分别在高温胁迫0 h和3 h时显著上调表达，热应激相关基因dusp1在高温胁迫3 h时显著上调表达。综上所述，急性高温胁迫下，半滑舌鳎肝脏发生氧化应激，短期内机体可调动抗氧化系统加速清除活性氧，并激活热应激相关基因表达。该研究可为解析半滑舌鳎对高温胁迫的响应机制、预防夏季高温大规模死亡的发生以及开展耐高温良种的选育等提供参考。

英文摘要:

      The Chinese tongue sole (Cynoglossus semilaevis) belonging to Pleuronectiformes, Cynoglossidae, Cynoglossus, is distributed in the sea areas of Korea, Japan and China. C. semilaevis has limited natural resources and no long-distance migration, which is suitable for the development of aquaculture in coastal areas. After more than ten years of artificial domestication, C. semilaevis has become one of the main mariculture species. Water temperature is an important environmental factor affecting the growth and development of fish. High temperature in summer can cause stress and even death of C. semilaevis, which is one of the important factors affecting factory farming and the promotion in the southern coast. However, the physiological and molecular changes of C. semilaevis in response to acute high temperature stress was still unclear to date. When fish are stimulated by high temperature, it can cause oxidative stress, resulting in a variety of toxic effects. It has been found that there is a significant correlation between high temperature and the activity of antioxidant enzymes in many aquatic animals. Therefore, changes in the activities of antioxidant enzymes can be used as an important indicator when fish are in a state of oxidative stress under high temperature stress. As a tissue related to detoxification metabolism and immunity of fish, liver plays an important role in response to high temperature stress, thus the tissue structure and apoptosis are also important indicators of liver health status after high temperature stress. Heat shock proteins (HSPs) are a class of common biological stress proteins, which have biological functions such as anti-stress, anti-oxidation, and regulation of apoptosis. HSPs are also involved in the resistance of fish to heat stress. Recently, it has been found that zebrafish (Danio rerio) is extremely sensitive to temperature changes after knockout of dual-specificity phosphatase 1 (dusp1) gene, and dusp1 has the possibility of maintaining redox homeostasis, so it was speculated that dusp1 gene may plays an important role in fish resistance to heat stress. In this study, heat shock protein family A member 1A (hspa1a) in the heat shock protein HSP70 family, heat shock protein 90 beta family member 1 (hsp90b1) in the HSP90 family, and dusp1 gene were selected to study the temporal expression characteristics under high temperature stress. In order to explore the effects of high temperature stress on physiological and molecular changes in the liver of C. semilaevis, a full-sib family of C. semilaevis was selected as the experimental object to detect the oxidative damage and heat stress-related gene expressions. After continuous heating to high temperature stress conditions (35 ℃), liver tissues were collected at 0 h, 3 h, 6 h, 12 h and 24 h, respectively. Hematoxylin and eosin (HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to observe cell damage, antioxidant enzyme activity and malondialdehyde (MDA) content were measured, and the expression changes of stress-related genes hspa1a, hsp90b1 and dusp1 were detected. The results showed that acute high temperature stress could cause obvious pathological changes and apoptosis in the liver tissue of C. semilaevis. The activity of antioxidant enzyme superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and the content of MDA in the high temperature stress group was significantly higher than that of the control group at 6 h, 12 h, 0 h and 24 h, respectively (P<0.05). The expression of heat shock protein genes hspa1a and hsp90b1 were significantly up-regulated at 0 h and 3 h after high temperature stress, respectively. Heat stress-related gene dusp1 was significantly up-regulated at 3 h after high temperature stress. In conclusion, oxidative stress occurs in the liver of C. semilaevis under acute high temperature stress, in the short term, the body can mobilize the antioxidant system to accelerate the removal of reactive oxygen species and activate the expression of heat stress-related genes. This study provides a reference for analyzing the response mechanism of C. semilaevis to high temperature stress, preventing the occurrence of massive death in summer, and carrying out the breeding of high temperature resistant varieties.