Effects Of Hypoxia On Physiological And Biochemistry Indexes And Cardiomyocyte Apoptosis In Silver Carp (Hypophthalmichthys Molitrix)

Silver carp(Hypophthalmichthys molitrix)is an economic important freshwater farming fish in China,occupying the second position in annual yield.During farming and transportation,silver carp is vulnerable to hypoxia stress,with gasping and asphyxia appearing,which might lead to a mass of death and cause economic loss.Therefore,the research of the physiological stress and mechanism of injure under hypoxia,would provide the basic data for the improvement of silver carp farming and transportation technology,therefore there were important application value.In present study,silver carp were kept in the closed containers for hypoxia treatment by spontaneous oxygen consumption(H groups)and returned to normal oxygen after moderate hypoxia(R groups).Take samples at 0,2,4,6,10,18 h under hypoxia and at 0,1,2,4,8h after reoxygenation respectively.The serum biochemical indexes of silver carp were measured by automatic biochemical analyzer and the oxidative stress-related indexes of heart,brain,liver and kidney were measured by kit,to analyze the effects of hypoxia stress on the physiological function of internal organs of silver carp.cardiomyocytes was detected by TUNEL and the expressions of bax and bcl-2 mRNA were analyzed by qPCR,to further study the hypoxia damage to the heart in silver carp.Because calcium is closely related to apoptosis and necrosis,it can participate in the apoptotic pathway,interact with apoptosis regulators,and accelerate apoptosis.The activities of Na+/K+-ATPase,Ca2+-ATPase were measured on spectrophotometer and the expressions of calm and LETM1 mRNA in the heart were detected by qPCR,to investigate the effects of hypoxia on the regulation of calcium homeostasis.The main results were as follows:(1)Effects of hypoxia and reoxygenation on serum biochemistry indexes of silver carp.Light and mild hypoxia treatment would cause the activity of CK increased in silver carp.The activities of CK-MB,LDH and HDBH showed a decreasing trend under hypoxia,and all the indexes could be recovered after reoxygenation.The activities of ALT,AST and ALP significantly decreased under hypoxia,gradually increased after normal oxygen,and H8 was significantly higher than the control group(P<0.05).The contents of TP?TG and TC had an obvious tendency of descend under hypoxia,then gradually return after reoxygenation.The concentration of UREA and UA were significantly increased after hypoxia(P<0.05).UREA was significantly increased and UA was significantly decreased when oxygen return to normal(P<0.05).There was no significant difference between H8 and H0.The contents of TBIL,CREA,Cl-and Ca2 + had no distinct changes by the dissolved oxygen impact.(2)Effects of hypoxia and reoxygenation on oxidative stress in heart,brain,liver and kidney of silver carp.(1)Under hypoxia stress,in heart,the activity of SOD and MDA gradually increased,the activity of CAT gradually decreased,the activity of SOD and the contents of GSH and MDA were the highest at H10(P<0.05).In brain,under hypoxia,the activity of SOD and the concentration of GSH rose and then fell,the concentration of MDA decreased significantly.Activity of CAT was the highest at H18.In liver,exposed to hypoxic condition,the activities of SOD,CAT and the content of MDA higher at H2 than H0(P<0.05),then they were decreasing,finally increased,except the content of MDA was gradually decreased.In kidney,under hypoxic stress,the activities of SOD and CAT were increasing significantly.(2)After reoxygenation treatment,in heart,the tendency of the activity of CAT was rising,contrary of SOD.Compared with NC(normoxia control group),the activity of SOD and the concentration of GSH had no significant difference at R8,the activity of CAT was significantly lower and the content of MDA was significantly higher at R8(P<0.05).In brain,tendency of the activities of SOD,CAT and the concentration of GSH was increasing firstly and then decreasing.All indexes reached maximum at R4.Compared with NC,the activity of CAT and the content of MDA were lower and the concentration of GSH was higher(P<0.05).In liver,antioxidants manifested fluctuation changes and the content of MDA gradually increased.There were no significant differences between R8 and NC on all indexes.In kidney,the activities of SOD and CAT were significantly decreasing.There were no significant differences between R8 and NC on mainly indexes.(3)Cardiomyocyte apoptosis was caused by excessive hypoxia.Results of TUNEL showed that hypoxia induced cardiomyocyte apoptosis and the cell number of apoptosis increased with dissolved oxygen decline.Apoptosis index was 7.69±2.96 in control group(C),47.94±1.87 in gasping group(G)and 85.39±2.61 in asphyxia group(A).There were significant differences among groups(P<0.01).Results of qPCR showed that expression of bax in A group were significantly increased than group C(P<0.05).Bcl-2 gene was significantly down-regulated in groupA and group G(P<0.05).The results of TUNEL showed that compared with the normal oxygen group,the apoptosis of myocardial cells in silver carp significantly increased under hypoxic stress.(4)Regulation of calcium ion homeostasis in cardiomyocyte was affected by hypoxia.Transcription levels of calmwere significantly lower in group G than in group A(P<0.05).LETM1 expression levels were significantly up-regulated in group A(P<0.05).Compared with group C,activities of Na+/K+-ATPase and Ca2+-ATPase significantly decreased in heart of group G,but increased in group A.Both the two enzymic activities and mRNA expression levels of above two genes in Group A were higher thangroup A.In summary,the present study showed that mild or moderate hypoxic stress would cause physiological dysfunction and oxidative stress to heart,brain,liver and kidney in silver carp.Physiological function of liver and kidney could be recovered,while oxygen levels were recovered to normal.However the heart and brain would be still subjected to oxidative stress which suggested that the organs were severely injured by hypoxia.With hypoxia,there was unbalance of calcium homeostasis in organism,and calm and LETM1 genes were up-regulated to defense calcium overload in cytoplasm or mitochondrial matrix of myocardial cell.In the meantime,bax was up-regulated and bcl-2 was down-regulated,giving rise to promote myocardial cell apoptosis.Organic injure of visceral tissue and a strong oxidative stress response,even death,probably occurred,as a result of severe hypoxic condition.Asphyxia death may be associated with a large number of myocardial cell apoptosis and impaired cardiac function.