急性拥挤胁迫对草鱼肌肉泛素-蛋白酶体系统活性的影响
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摘要
泛素-蛋白酶体系统(theubiquitin-proteasomesystem,UPS)是细胞内调节蛋白质代谢的重要系统。在水温(17.0±0.5)℃下,将草鱼(Ctenopharyngodon idellus)暴露于100 kg/m2的养殖密度条件进行急性拥挤胁迫。在胁迫0 h、1 h、6 h、12 h、24 h和48 h以及取消胁迫后(养殖密度10 kg/m2)的6 h和168 h采集血清和背部肌肉样品,检测血清皮质醇水平、肌肉质构特性以及nrf2、hsp70和UPS相关基因表达,并定量测定了肌肉组织泛素化蛋白水平。结果表明,拥挤胁迫导致血清皮质醇含量显著上升,肌肉硬度和凝聚性显著性下降,以上各指标在取消胁迫后168 h恢复到对照水平,弹性、胶黏性、咀嚼性和回复性在恢复168h时反而高于对照组水平; hsp70和nrf2的表达量在胁迫48 h时显著高于对照组,后期恢复到对照水平;肌肉ub、psma2、psmc1、mafbx和chip的mRNA表达量在胁迫过程中显著性上升,胁迫后恢复168 h,还是表现出较高的表达量;处理组泛素化蛋白水平在胁迫6 h和12 h时显著升高,在恢复168 h时显著降低。研究表明,急性拥挤胁迫使草鱼产生明显的应激反应,对肌肉质构特性产生了显著性影响,同时提高了肌肉UPS的活性。经过168h的恢复,鱼体逐渐恢复到正常生理水平,但机体UPS活性仍呈现出被激活状态。急性拥挤胁迫对草鱼生理功能和肌肉质构特性的有害影响是可逆的,建议恢复时间大于168 h以帮助鱼体重新建立UPS的稳态。
The ubiquitin-proteasome system(UPS) is an important system for regulating protein degradation and function. In order to evaluate the effects of acute crowding stress on flesh quality and UPS in fish, we randomly distributed Ctenopharyngodon idellus(initial body weight: 56.88 g ± 10.54 g) into 6 aquarium tanks(length:200 cm, wide: 100 cm, depth of water: 55 cm) at two stocking density(three replicates for each group). Water temperature(17.0±0.5)℃, pH(7.5±0.3) and dissolved oxygen(7.0 mg/L) were maintained at the same level in each group. Fish serum and muscle samples were collected at 0 h, 1 h, 6 h, 12 h, 24 h, and 48 h after crowding stress as well as at 6 h and 168 h after the crowding stress was removed(treatment group stocking density changed from 100 kg/m~2 to 10 kg/m~2). The serum cortisol, muscle texture, the mRNA expressions of nrf2 and hsp70, and genes related to UPS as well as the content of ubiquitinated protein in fish muscle were investigated. The results showed that the serum cortisol level significantly increased after acute crowding stress, suggesting the crowding environment led to fish stress. The serum concentration of cortisol recovered to the control level after 168 h of recovery. Crowding stress also exerted an influence on muscular texture characteristics of cultured grass carp.Muscular hardness and cohesiveness decreased significantly in grass carp suffering from cute crowding stress, and the two flesh quality indexes recovered to the level of the control group after 168 h of post-stress recovery. However, muscular adhesiveness, chewiness and resilience were significantly higher than those in the control group at the end of the experiment. Compared with the control group, the mRNA expressions of hsp70 and nrf2 in muscle of fish stocked at the treatment group were significantly increased after 48 h of crowding stress, and then returned to the control level after 168 h of post-stress recovery. It is implied that the acute crowding environment might cause cellular stress in fish. In the UPS pathway, the mRNA levels of ubiquitin(ub), psma2, psmc1, mafbx, and chip were significantly increased after crowding stress. The transcriptional levels of these genes did not returned to the control level after 168 h of post-stress recovery. Moreover, a significant higher content of ubiquitinated proteins occurred in the dorsal muscle of fish in the treatment group at 6 h and 12 h after crowding stress. Subsequently, there were no significant difference in ubiquitinated proteins of fish stocked at two densities, while ubiquitinated proteins in the treatment group significantly declined compared to the control group at 168 h of recovery.It was concluded that the acute crowding environment induced the stress response and altered the muscular texture as well as improved muscle UPS activity. The deleterious effects of acute crowding stress on fish physiological function and muscular texture were reversible. Post-stress recovery time should be extended more than 168 h so that the stressed fish reestablish the homeostasis on UPS.
The ubiquitin-proteasome system(UPS) is an important system for regulating protein degradation and function. In order to evaluate the effects of acute crowding stress on flesh quality and UPS in fish, we randomly distributed Ctenopharyngodon idellus(initial body weight: 56.88 g ± 10.54 g) into 6 aquarium tanks(length:200 cm, wide: 100 cm, depth of water: 55 cm) at two stocking density(three replicates for each group). Water temperature(17.0±0.5)℃, pH(7.5±0.3) and dissolved oxygen(7.0 mg/L) were maintained at the same level in each group. Fish serum and muscle samples were collected at 0 h, 1 h, 6 h, 12 h, 24 h, and 48 h after crowding stress as well as at 6 h and 168 h after the crowding stress was removed(treatment group stocking density changed from 100 kg/m~2 to 10 kg/m~2). The serum cortisol, muscle texture, the mRNA expressions of nrf2 and hsp70, and genes related to UPS as well as the content of ubiquitinated protein in fish muscle were investigated. The results showed that the serum cortisol level significantly increased after acute crowding stress, suggesting the crowding environment led to fish stress. The serum concentration of cortisol recovered to the control level after 168 h of recovery. Crowding stress also exerted an influence on muscular texture characteristics of cultured grass carp.Muscular hardness and cohesiveness decreased significantly in grass carp suffering from cute crowding stress, and the two flesh quality indexes recovered to the level of the control group after 168 h of post-stress recovery. However, muscular adhesiveness, chewiness and resilience were significantly higher than those in the control group at the end of the experiment. Compared with the control group, the mRNA expressions of hsp70 and nrf2 in muscle of fish stocked at the treatment group were significantly increased after 48 h of crowding stress, and then returned to the control level after 168 h of post-stress recovery. It is implied that the acute crowding environment might cause cellular stress in fish. In the UPS pathway, the mRNA levels of ubiquitin(ub), psma2, psmc1, mafbx, and chip were significantly increased after crowding stress. The transcriptional levels of these genes did not returned to the control level after 168 h of post-stress recovery. Moreover, a significant higher content of ubiquitinated proteins occurred in the dorsal muscle of fish in the treatment group at 6 h and 12 h after crowding stress. Subsequently, there were no significant difference in ubiquitinated proteins of fish stocked at two densities, while ubiquitinated proteins in the treatment group significantly declined compared to the control group at 168 h of recovery.It was concluded that the acute crowding environment induced the stress response and altered the muscular texture as well as improved muscle UPS activity. The deleterious effects of acute crowding stress on fish physiological function and muscular texture were reversible. Post-stress recovery time should be extended more than 168 h so that the stressed fish reestablish the homeostasis on UPS.
引文
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[14]Dobly A,Martin S A M,Blaney S C,et al.Protein growth rate in rainbow trout(Oncorhynchus mykiss)is negatively correlated to liver 20S proteasome activity[J].Comparative Biochemistry and Physiology A:Molecular&Integrative Physiology,2004,137(1):75-85.
[15]Lamarre S G,Blier P U,Driedzic W R,et al.White muscle20S proteasome activity is negatively correlated to growth rate at low temperature in the spotted wolffish Anarhichas minor[J].Journal of Fish Biology,2010,76(7):1565-1575.
[16]Wang W B,Li A H,Wang J G,et al.Effects of crowding stress on non-specific immune function of grass carp[J].Journal of Fisheries of China,2004,28(2):139-144.[王文博,李爱华,汪建国,等.拥挤胁迫对草鱼非特异性免疫功能的影响[J].水产学报,2004,28(2):139-144.]
[17]Li A H.Effects of crowding stress on plasma cortisol,blood glucose and ascorbic acid in liver of grass carp[J].Acta Hydrobiologica Sinica,1997,21(4):384-386.[李爱华.拥挤胁迫对草鱼血浆皮质醇、血糖及肝脏中抗坏血酸含量的影响[J].水生生物学报,1997,21(4):384-386.]
[18]Lushchak V I.Environmentally induced oxidative stress in aquatic animals[J].Aquatic Toxicology,2011,101(1):13-30.
[19]Benarroch E E.Heat shock proteins:multiple neuroprotective functions and implications for neurologic disease[J].Neurology,2011,76(7):660-667.
[20]Kirschke E,Goswami D,Southworth D,et al.Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles[J].Cell,2014,157(7):1685-1697.
[21]Yarahmadi P,Miandare H K,Fayaz S,et al.Increased stocking density causes changes in expression of selected stress-and immune-related genes,humoral innate immune parameters and stress responses of rainbow trout(Oncorhynchus mykiss)[J].Fish&Shellfish Immunology,2016,48:43-53.
[22]Dodson M,Redmann M,Rajasekaran N S,et al.Correction:KEAP1-NRF2 signalling and autophagy in protection against oxidative and reductive proteotoxicity[J].Biochemical Journal,2015,469(3):347-355.
[23]Sherman M Y,Goldberg A L.Cellular defenses against unfolded proteins[J].Neuron,2001,29(1):15-32.
[24]Cassidy A A,Saulnier R J,Lamarre S G.Adjustments of protein metabolism in fasting Arctic charr,Salvelinus alpinus[J].PLoS ONE,2016,11(4):e0153364.
[25]Todgham A E,Crombie T A,Hofmann G E.The effect of temperature adaptation on the ubiquitin-proteasome pathway in notothenioid fishes[J].Journal of Experimental Biology,2017,220(3):369-378.
[26]Bodine S C,Latres E,Baumhueter S,et al.Identification of ubiquitin ligases required for skeletal muscle atrophy[J].Science,2001,294(5547):1704-1708.
[27]Fuentes E N,Ruiz P,Valdes J A,et al.Catabolic signaling pathways,atrogenes,and ubiquitinated proteins are regulated by the nutritional status in the muscle of the fine flounder[J].PLoS ONE,2012,7(9):e44256.
[28]Lagirand-Cantaloube J,Offner N,Csibi A,et al.The initiation factor e IF3-f is a major target for Atrogin1/MAFbx function in skeletal muscle atrophy[J].EMBO Journal,2008,27(8):1266-1276.
[29]Gao Q J,Li Z Z,Ma X D,et al.A review of skeletal muscle cytoskeletal proteins[J].Journal of Beijing Sport University,2005,28(10):1382-1385.[高前进,李壮志,马新东,等.骨骼肌细胞骨架蛋白研究综述[J].北京体育大学学报,2005,28(10):1382-1385.]
[30]Li Y Q,Kong B H,Xia X F,et al.Effect of hydroxyl radical oxidation on emulsifying and gelation properties of myofibrillar protein[J].Food Science,2012,33(9):31-35.[李艳青,孔保华,夏秀芳,等.羟自由基氧化对鲤鱼肌原纤维蛋白乳化性及凝胶性的影响[J].食品科学,2012,33(9):31-35.]
[31]Van Hekken D L,Tunick M H,Park Y W.Rheological and proteolytic properties of monterey jack goat’s milk cheese during aging[J].Journal of Agricultural and Food Chemistry,2004,52(17):5372-5377.
[32]Ramsay J M,Feist G W,Varga Z M,et al.Whole-body cortisol response of zebrafish to acute net handling stress[J].Aquaculture,2009,297(1-4):157-162.
[2]Refaey M M,Li D P,Tian X,et al.High stocking density alters growth performance,blood biochemistry,intestinal histology,and muscle quality of channel catfish Ictalurus punctatus[J].Aquaculture,2018,492:73-81.
[3]Li D P,Liu Z D,Xie C X.Effect of stocking density on growth and serum concentrations of thyroid hormones and cortisol in Amur sturgeon,Acipenser schrenckii[J].Fish Physiology and Biochemistry,2012,38(2):511-520.
[4]Lupatsch I,Santos G A,Schrama J W,et al.Effect of stocking density and feeding level on energy expenditure and stress responsiveness in European sea bass Dicentrarchus labrax[J].Aquaculture,2010,298(3-4):245-250.
[5]Flick K,Kaiser P.Protein degradation and the stress response[J].Seminars in Cell and Developmental Biology,2012,23(5):515-522.
[6]Johnston I A,Bower N I,Macqueen D J.Growth and the regulation of myotomal muscle mass in teleost fish[J].Journal of Experimental Biology,2011,214:1617-1628.
[7]Valenzuela C A,Zuloaga R,Mercado L,et al.Chronic stress inhibits growth and induces proteolytic mechanisms through two different non-overlapping pathways in the skeletal muscle of a teleost fish[J].American Journal of Physiology-Regulatory,Integrative and Comparative Physiology,2018,314:102-113.
[8]Amm I,Sommer T,Wolf D H.Protein quality control and elimination of protein waste:The role of the ubiquitin-proteasome system[J].Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,2014,1843(1):182-196.
[9]Rom O,Reznick A Z.The role of E3 ubiquitin-ligases Mu RF-1 and MAFbx in loss of skeletal muscle mass[J].Free Radical Biology and Medicine,2016,98:218-230.
[10]Arndt V,Rogon C,H?hfeld J.To be,or not to bemolecular chaperones in protein degradation[J].Cellular and Molecular Life Sciences,2007,64:2525.
[11]Pickart C,Eddins M.Ubiquitin:structures,functions,mechanisms[J].Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,2004,1695(1-3):55-72.
[12]Todgham A E,Crombie T A,Hofmann G E.The effect of temperature adaptation on the ubiquitin-proteasome pathway in notothenioid fishes[J].Journal of Experimental Biology,2016,220(3):369-378.
[13]Sun Y Q,Liang X,Chen J,et al.Change in ubiquitin proteasome system of grass carp Ctenopharyngodon idellus reared in the different stocking densities[J].Frontiers in Physiology,2018,9:837.
[14]Dobly A,Martin S A M,Blaney S C,et al.Protein growth rate in rainbow trout(Oncorhynchus mykiss)is negatively correlated to liver 20S proteasome activity[J].Comparative Biochemistry and Physiology A:Molecular&Integrative Physiology,2004,137(1):75-85.
[15]Lamarre S G,Blier P U,Driedzic W R,et al.White muscle20S proteasome activity is negatively correlated to growth rate at low temperature in the spotted wolffish Anarhichas minor[J].Journal of Fish Biology,2010,76(7):1565-1575.
[16]Wang W B,Li A H,Wang J G,et al.Effects of crowding stress on non-specific immune function of grass carp[J].Journal of Fisheries of China,2004,28(2):139-144.[王文博,李爱华,汪建国,等.拥挤胁迫对草鱼非特异性免疫功能的影响[J].水产学报,2004,28(2):139-144.]
[17]Li A H.Effects of crowding stress on plasma cortisol,blood glucose and ascorbic acid in liver of grass carp[J].Acta Hydrobiologica Sinica,1997,21(4):384-386.[李爱华.拥挤胁迫对草鱼血浆皮质醇、血糖及肝脏中抗坏血酸含量的影响[J].水生生物学报,1997,21(4):384-386.]
[18]Lushchak V I.Environmentally induced oxidative stress in aquatic animals[J].Aquatic Toxicology,2011,101(1):13-30.
[19]Benarroch E E.Heat shock proteins:multiple neuroprotective functions and implications for neurologic disease[J].Neurology,2011,76(7):660-667.
[20]Kirschke E,Goswami D,Southworth D,et al.Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles[J].Cell,2014,157(7):1685-1697.
[21]Yarahmadi P,Miandare H K,Fayaz S,et al.Increased stocking density causes changes in expression of selected stress-and immune-related genes,humoral innate immune parameters and stress responses of rainbow trout(Oncorhynchus mykiss)[J].Fish&Shellfish Immunology,2016,48:43-53.
[22]Dodson M,Redmann M,Rajasekaran N S,et al.Correction:KEAP1-NRF2 signalling and autophagy in protection against oxidative and reductive proteotoxicity[J].Biochemical Journal,2015,469(3):347-355.
[23]Sherman M Y,Goldberg A L.Cellular defenses against unfolded proteins[J].Neuron,2001,29(1):15-32.
[24]Cassidy A A,Saulnier R J,Lamarre S G.Adjustments of protein metabolism in fasting Arctic charr,Salvelinus alpinus[J].PLoS ONE,2016,11(4):e0153364.
[25]Todgham A E,Crombie T A,Hofmann G E.The effect of temperature adaptation on the ubiquitin-proteasome pathway in notothenioid fishes[J].Journal of Experimental Biology,2017,220(3):369-378.
[26]Bodine S C,Latres E,Baumhueter S,et al.Identification of ubiquitin ligases required for skeletal muscle atrophy[J].Science,2001,294(5547):1704-1708.
[27]Fuentes E N,Ruiz P,Valdes J A,et al.Catabolic signaling pathways,atrogenes,and ubiquitinated proteins are regulated by the nutritional status in the muscle of the fine flounder[J].PLoS ONE,2012,7(9):e44256.
[28]Lagirand-Cantaloube J,Offner N,Csibi A,et al.The initiation factor e IF3-f is a major target for Atrogin1/MAFbx function in skeletal muscle atrophy[J].EMBO Journal,2008,27(8):1266-1276.
[29]Gao Q J,Li Z Z,Ma X D,et al.A review of skeletal muscle cytoskeletal proteins[J].Journal of Beijing Sport University,2005,28(10):1382-1385.[高前进,李壮志,马新东,等.骨骼肌细胞骨架蛋白研究综述[J].北京体育大学学报,2005,28(10):1382-1385.]
[30]Li Y Q,Kong B H,Xia X F,et al.Effect of hydroxyl radical oxidation on emulsifying and gelation properties of myofibrillar protein[J].Food Science,2012,33(9):31-35.[李艳青,孔保华,夏秀芳,等.羟自由基氧化对鲤鱼肌原纤维蛋白乳化性及凝胶性的影响[J].食品科学,2012,33(9):31-35.]
[31]Van Hekken D L,Tunick M H,Park Y W.Rheological and proteolytic properties of monterey jack goat’s milk cheese during aging[J].Journal of Agricultural and Food Chemistry,2004,52(17):5372-5377.
[32]Ramsay J M,Feist G W,Varga Z M,et al.Whole-body cortisol response of zebrafish to acute net handling stress[J].Aquaculture,2009,297(1-4):157-162.