低氧胁迫和复氧对长吻鮠鳃组织低氧应答基因和生理生化指标的影响

Effects of Hypoxic Stress and Reoxygenation on Hypoxic Response Genes and Physiological and Biochemical Indexes in the Gill Tissue of Leiocassis longirostris

为了研究低氧胁迫(0.8±0.1)mg/L和复氧(7.3±0.5)mg/L对长吻鮠(Leiocassis longirostris)鳃组织氧传感蛋白、呼吸代谢、氧化应激、组织结构和细胞凋亡的影响。本研究对长吻鮠进行了低氧胁迫(0、2、4、6 h)和复氧(2、4、6 h)试验,采用qRT-PCR、酶活性测定、H&E染色和TUNEL切片检测等方法,比较和分析了该鱼鳃组织低氧应答基因、生理生化指标的变化。结果表明:在低氧胁迫下,长吻鮠鳃组织氧传感蛋白相关基因(HIF-1α、HIF-2α、PHDs和Vhl)表达量显著升高,复氧后较对照组仍有显著差异(P<0.05);糖酵解相关酶(PFK、HK和PK)、无氧呼吸酶(LDH)、抗氧化酶(GSH-Px、CAT和SOD)和氧化应激指标(MDA和LPO)活性和含量显著升高,TCA循环相关酶(SDH和MDH)活性显著降低,复氧后多数生理指标均逐渐恢复至对照水平。H&E染色显示低氧胁迫下长吻鮠鳃组织上皮出现了抬升,部分血细胞大量聚集使鳃小片顶端呈棒状,发生线粒体丰富细胞肿胀和增生等变化,在恢复溶氧后低氧引起的这些组织形态变化并未得到改善;TUNEL切片结果显示长吻鮠鳃组织凋亡细胞数量随低氧时间延长不断增加,凋亡相关基因(Bax、Caspase 3、p53和Apaf-1)表达量显著升高,Bcl-2表达量显著降低,恢复溶氧后细胞凋亡现象仍然存在。低氧胁迫和复氧对长吻鮠鳃组织氧传感蛋白、呼吸代谢、氧化应激、组织结构、细胞凋亡等具有明显的影响,该结果为探究鱼类低氧胁迫下分子调控机制奠定一定的理论基础,为今后开展长吻鮠耐低氧新品种选育提供基础资料。

In order to study the effects of hypoxic stress[(0.8±0.1)mg/L]and reoxygenation[(7.3±0.5 mg/L]on oxygen sensing,respiratory metabolism,oxidative stress,tissue structure,and apoptosis in the gill of L.longirostris,by using qRT-PCR,enzyme activity measurement,H&E staining and TUNEL slice detection,the experiments of hypoxic stress(0,2,4,6 h)and reoxygenation(2,4,6 h)on L.longirostris were conducted and the changes of hypoxic response genes,and physiological and biochemical indexes in the gill of L.longirostris were analyzed.The results showed that:the expressions of genes related to oxygen sensors(HIF1α,HIF-2α,PHDs and Vhl)were upregulated in the gill of L.longirostris during hypoxia,after reoxygenation,there was still a significant difference compared with the control(P<0.05).Expression activities and contents of glycolysis related enzymes(PFK,HK and PK),anaerobic respiration enzymes(LDH),antioxidant enzymes(GSH-Px,CAT and SOD)and oxidative stress indicators(MDA and LPO)significantly increased.The activities of TCA cycle-related enzymes(SDH and MDH)decreased significantly during hypoxia,but most physiological indexes gradually recovered to the level of the control upon reoxygenation.H&E staining showed that under hypoxic stress,the epithelium of the gill was uplifted,and some blood cells gathered in large numbers to make the gill small pieces have rod-like tips,and tissue changes such as swelling and proliferation of mitochondriarich cells occurred,but these changes in tissue morphology caused by hypoxia were not improved after the recovery of dissolved oxygen.TUNEL sections showed that the number of apoptotic cells in the gill of L.longirostris increased with the prolongation of hypoxia time,the mRNA levels of apoptosis-related genes(Bax,Caspase 3,p53 and Apaf-1)increased significantly and Bcl-2 decreased significantly during hypoxia,and the apoptosis still existed compared with control upon reoxygenation.Hypoxic stress and reoxygenation can significantly affect the gill oxygen sensors,respiratory metabolism,oxidative stress,tissue structure,and apoptosis of L.longirostris.The results lay a theoretical foundation for exploring the molecular regulation mechanism of fish under hypoxic stress,and provide basic data for future development of new hypoxia tolerant varieties of L.longirostris.