低氧胁迫对虹鳟心脏生化指标和低氧相关基因表达的影响

Effects of hypoxia stress on biochemical indices and expression of hypoxia-related genes in the heart of rainbow trout (Oncorhynchus mykiss)

关键词:  虹鳟  低氧胁迫  生化指标  代谢  基因表达

Key words:  Rainbow trout (Oncorhynchus mykiss)  Hypoxic stress  Biochemical indices  Metabolism  Gene expression

摘要:

虹鳟(Oncorhynchus mykiss)是一种低氧敏感性鱼类，低氧胁迫下其生长、行为、代谢和免疫等均会受到影响。为了解低氧胁迫对虹鳟心脏生化指标和低氧相关基因表达的影响，本研究用酶活性测定和实时荧光定量PCR (RT-qPCR)方法，分析中度低氧[(4.5±0.1) mg/L]和重度低氧[(3.0± 0.1) mg/L]胁迫4、8、12、24 h、中度低氧1个月(TMM)、重度低氧1个月(TMS)及复氧[(8.5±0.1) mg/L] 12 h和24 h后虹鳟心脏中生化指标变化以及低氧相关基因的表达情况。结果显示，在中度低氧胁迫下，虹鳟心脏中丙酮酸激酶(PK)、总胆固醇(TC)、乳酸(LD)和谷丙转氨酶(GPT)水平在8 h时升高，24 h时降低，复氧后仍显著高于对照水平(P<0.05)。在重度低氧胁迫下，琥珀酸脱氢酶(SDH)活性随低氧胁迫时间延长逐渐升高，在8 h时达到峰值(P<0.05)，24 h和复氧后均与对照无显著性差异(P>0.05)。三磷酸腺苷酶(ATPase)、脂肪酶(LPS)、TC、谷草转氨酶(GOT)和GPT水平在12 h时降低，复氧后均恢复至正常水平(P>0.05)。与对照组相比，TMM组和TMS组中PK、TC、乳酸脱氢酶(LDH)和GPT水平均显著升高(P<0.05)。在中度低氧胁迫下，sdh、fih1和hif-1α基因表达量在8 h显著升高(P<0.05)，复氧后均恢复至正常水平。在重度低氧胁迫下，ldh、pk、sdh、hif-1α、egln-1和vhl基因表达量在24 h时显著升高(P<0.05)。与对照组相比，TMM和TMS组中ldh、pk、sdh、fih1、egln-1和vhl基因表达量在中度低氧胁迫下降低但无显著性差异(P>0.05)，重度低氧胁迫下pk、sdh和vhl基因表达量显著升高(P<0.05)。本研究结果表明，低氧胁迫使虹鳟心脏发生代谢紊乱，影响体内正常的代谢水平，并对虹鳟心脏造成一定损伤。本研究可为进一步阐明虹鳟低氧胁迫的调控机制提供基础资料。

Abstract:

Rainbow trout (Oncorhynchus mykiss) is a hypoxia-sensitive fish, and its growth, behavior, metabolism, and immunity are affected in hypoxic environments. To elucidate the effects of hypoxic stress on the heart of rainbow trout, biochemical indices and hypoxia-related gene expression were measured during moderate (4.5±0.1 mg/L) and severe hypoxia (3.0±0.1 mg/L) stress for 4 h, 8 h, 12 h, and 24 h; moderate hypoxia for 1 month (TMM); severe hypoxia for 1 month (TMM); and reoxygenation (8.5±0.1 mg/L) for 12 h and 24 h using enzyme activity assays and quantitative real time polymerase chain reaction (RT-qPCR). The results showed that the pyruvate kinase (PK), total cholesterol (TC), lactic acid (LD), and glutamine aminotransferase (GPT) reactivates increased at 8 h, decreased at 24 h, and were significantly higher than the control levels after reoxygenation (P＜0.05) under moderate hypoxic stress. Under severe hypoxic stress, the succinate dehydrogenase (SDH) activities gradually increased and peaked at 8 h (P＜0.05). No significant difference was observed between the control at 24 h and after reoxygenation (P＞0.05). The adenosine triphosphatase (ATPase), lipase (LPS), TC, glutamic transaminase (GOT), and GPT reactivates decreased at 12 h and recovered to normal levels after reoxygenation (P＞0.05). The PK, TC, lactate dehydrogenase (LDH), and GPT reactivates were significantly higher in the TMM and TMS groups than those in the control group (P＜0.05). The expressions of succinate dehydrogenase gene (sdh), factor inhibiting hypoxia-inducible factor-1 (fih1), and hypoxia-inducible factor-1α (hif-1α) were significantly increased at 8 h compared to those of the control (P＜0.05) and returned to normal reactivates after reoxygenation. Under severe hypoxic stress, lactate dehydrogenase gene (ldh), pyruvate kinase gene (pk), sdh, hif-1α, proline hydroxylase domain protein 2 (egln-1), and von Hippel-Lindau (vhl) expressions were significantly increased at 24 h (P＜0.05) compared to those under moderate hypoxic stress. Compared with those of the control group, ldh, pk, sdh, hif-1α, egln-1, and vhl expressions were significantly decreased in the TMM and TMS groups under moderate hypoxic stress with no significant differences (P＞0.05). pk, sdh, and vhl were significantly increased under severe hypoxic stress (P＜0.05). This study indicated that varying levels of dissolved oxygen led to changes in biochemical indices and in the expression of hypoxic-related genes in the heart of rainbow trout. Hypoxic stress affected the cardiac metabolism of rainbow trout, which affected the normal metabolism level and caused damage. Under hypoxic stress, the rainbow trout were able to provide feedback regulation of hypoxic stress through the high expression pattern of hypoxic-related, which prevents the heart from being in a constant state of hypoxic stress, allowing the organism to quickly return to a stable state and perform its normal physiological function. This study provides basic data to further elucidate the regulatory mechanism of cardiac metabolism in rainbow trout under hypoxic stress, it has guiding significance for the intensive and healthy breeding of this fish and the selection and breeding of new hypoxia-tolerant species in the future.