低氧胁迫和恢复对长吻鮠脑组织低氧应答基因、生理生化指标和食欲的影响
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S 965

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江苏省种业振兴“揭榜挂帅”项目(JBGS [2021] 034);国家自然科学基金青年基金(32102754,32102760);江苏省研究生科研与实践创新计划 (SJCX22_0605)


Effects of hypoxia stress and reoxygenation on appetite, hypoxic response genes and physiological and biochemical indexes in the brain tissues of Leiocassis longirostris
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    摘要:

    为了解长吻鮠脑组织应对低氧胁迫的调控机制,实验运用酶活性测定、H.E染色、qRT-PCR和TUNEL检测等方法,分析比较了低氧胁迫[(0.8±0.1) mg/L] 0、2、4、6 h(标示为H0、H2、H4和H6)和恢复[(7.3±0.5) mg/L]2、4、6 h (标示为R2、R4和R6)下长吻鮠脑组织低氧应答基因、生理生化指标和食欲基因的变化。结果显示,在低氧胁迫和恢复下,长吻鮠脑组织氧传感蛋白相关基因(HIFsPHDsVhl)表达量整体呈现出先上升后下降趋势;呼吸代谢酶(HK、PK和LDH)活性在H0时显著升高,SDH和MDH活性在H6时显著降低,恢复溶解氧后,代谢模式由无氧呼吸逐渐转变为有氧呼吸;抗氧化酶(GSH-Px、CAT和SOD)和应激指标(MDA和LPO)在低氧2 h后逐渐升高,恢复溶解氧后氧化应激现象仍然存在。观察脑组织形态发现,在低氧胁迫下长吻鮠脑组织出现了神经细胞肿胀、空泡等受损现象,恢复溶解氧6 h后脑组织受损并未得到有效改善。随着低氧时间延长,脑组织细胞凋亡程度不断增加,凋亡相关基因(BaxCaspase-3和p53)表达量显著升高,而Bcl-2基因表达量降低,恢复溶解氧后较对照组仍有显著差异。另外,在低氧胁迫0 h和2 h时,长吻鮠摄食率分别下降54%和98%,检测到低氧胁迫能显著抑制促食基因(NPY)和诱导抑食基因(PYY、CCKNUCB2)表达。研究表明,低氧胁迫和恢复对长吻鮠脑组织氧传感蛋白、呼吸代谢、氧化应激、结构形态、细胞凋亡以及食欲等指标均具有显著影响。本研究结果为阐明低氧胁迫和恢复下长吻鮠脑组织分子调控机制提供一定的理论依据,对今后开展该鱼集约化健康养殖和耐低氧新品种选育工作具有指导意义。

    Abstract:

    Affected by natural factors such as temperature, season and weather, or human factors such as eutrophication and high stocking density, oxygen concentration in the water environment where fish live is unpredictable, and they are often faced with a low-oxygen environment. Studies have found that fish have evolved a complex physiological and biochemical system to adapt to the stress response caused by hypoxia in the water environment. However, severe and acute hypoxia can cause a large number of fish to suffocate and die in a short period of time. Because of its delicious taste, no intermuscular spines and high nutritional value, the L. longirostris has become one of the important freshwater aquaculture species in China. Currently, studies have been carried out at home and abroad on the growth characteristics, product processing and nutritional evaluation of this fish. There are few reports on its important ecological impact factor "hypoxia". In order to understand the regulatory mechanism of the brain tissue in response to hypoxic stress. In this study, enzyme activity determination, H.E staining, qRT-PCR, TUNEL detection and other methods were used. Changes in brain tissue of L. longirostris hypoxia response genes, physiological and biochemical indicators and appetite genes were analyzed and compared under hypoxic stress [(0.8±0.1) mg/L] 0, 2, 4 and 6 hour (labeled as H0, H2, H4 and H6) and recovery [(7.3±0.5) mg/L] 2,4 and 6 hour (labeled as R2, R4 and R6). The results showed that: Under hypoxic stress and recovery, the expression levels of oxygen-sensing protein-related genes (HIFs, PHDs and Vhl) in the brain tissue of L. longirostris showed a trend of first rising and then falling as a whole. Respiratory metabolic enzymes (HK, PK and LDH) activities were significantly increased at H0, SDH and MDH activities were significantly reduced at H6. After reoxygenation, the metabolic mode gradually changes from anaerobic respiration to aerobic respiration. Antioxidative enzymes (GSH-Px, CAT and SOD) and stress indicators (MDA and LPO) gradually increased after 2 h of hypoxia, and oxidative stress persists after the recovery of dissolved oxygen. Through the observation of brain tissue morphology, it was found that under hypoxia stress, neuronal cell swelling and vacuoles were damaged in the brain tissue of L. longirostris, which were not effectively improved after 6 hours of reoxygenation with dissolved oxygen. However, with the prolongation of hypoxia time, the degree of apoptosis of brain tissue cells increased, and the expression of apoptosis-related genes (Bax, Caspase-3 and p53) increased significantly, while the expression of Bcl-2 gene decreased, there are still significant differences in expression compared with the control group after the recovery of dissolved oxygen. In addition, it was found that the feeding rate of L. longirostris decreased by 54% and 98% at 0 h and 2 h of hypoxia stress, respectively. Hypoxic stress was detected to significantly inhibit the expression of appetite-promoting genes (NPY) and inducible food-suppressive genes (PYY, CCK and NUCB2). This experiment showes that hypoxic stress and reoxygenation have significant effects on oxygen sensor proteins, respiratory metabolism, oxidative stress, structural morphology, apoptosis and appetite in the brain tissues of L. longirostris. The results of this study provide a theoretical basis for elucidating the molecular regulation mechanism of L. longirostris brain tissues under hypoxia stress and reoxygenation, 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.

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李谣,杨智茹,程景颢,李杰,宁先会,王涛,张凯,暨杰,张国松,尹绍武.低氧胁迫和恢复对长吻鮠脑组织低氧应答基因、生理生化指标和食欲的影响[J].水产学报,2023,47(1):019611

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  • 收稿日期:2022-01-14
  • 最后修改日期:2022-08-25
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  • 在线发布日期: 2023-01-16
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