The Effects Of Hypoxia Stress On Energy Metabolism, Blood Indexes And Gene Expression Of Takifugu Obscurus

Takifugu fasciatus is one of China's "Three Aquatic Foods in Yangtze River".In recent years,the development of domestic breeding industry of the globe fish has been very rapid.However,oxygen fluctuations and nourishing substances in the aquaculture water caused oxygen deprivation,which makes great economic losses in high-density farming.At present,many studies had reported the effects of ammonia toxicity,salinity on Takifugus,but little is known about the hypoxia stress on them.In this paper,T.fasciatus were treated with hypoxia stress(0h,2h,4h,6h,8h;1.51±0.2 mg/L)and recovery(4h,12h;7.06± 0.3 mg/L)to explore the relevant changes of oxygen sensing protein,energy metabolism and blood index.These data provide scientific basis for the control of dissolve oxygen in aquatic environment of T.fasciatus,and also provide reference for the breeding of the new varieties with low oxygen tolerance.The specific findings areas follows:(1)Effects of hypoxia stress and recovery on oxygen sensor gene expression in T.fasciatusT.fasciatus measured floating point and suffocation point by static chamber method,and the results were 1.42 mg/L and 0.49 mg/L,respectively.Inaddition,based on the HIF-1 signaling pathway,the expression of genes on Hypoxia inducible factor-1?(HIF-1?),HIF-Prolyl hydroxylase-2(PHD2)and Von Hippel-Lindau tumor suppressor protein(VHL)in different tissues of T.fasciatus was detected by qRT-PCR.The results showed that these three genes were distributed in the brain,liver,spleen,heart,intestine,sputum,muscle,and kidney.Meanwhile,HIF-1? and VHL expressed the highest in the heart,and PHD2 expressed in the liver.What's more,experimental result revealed that the expression levels of HIF-1?,PHD2,and VHL genes were significantly up-regulated under hypoxic conditions(P<0.001)and recovered after normal oxygen environment.VHL was still higher than that in the control group(P<0.01).Thus,it can be seen that HIF-1 signaling pathway is activated in the hypoxic brain and liver,and the up-regulation of VHL and PHD2 may serve as a feedback mechanism to terminate the hypoxic response.The hypoxic stress suffered by the brain and liver of T.fasciatus after hypoxia stress was minimized to terminate the hypoxia response period.(2)Effects of hypoxia stress and recovery on energy metabolism in T.fasciatusThe expression of energy metabolism-related genes on Glucose transporters2(GLUT2),HK(Hexokinase),Phosphoglycerate kinasel(PGK1),Phosphoglycerate mutase2(PGAM2),Malate dehydrogenasel(MDH1)and Citrate synthase(CS)in different tissues of T.fasciatus was tested by qRT-PCR.In the analysis of the results,PGK1,MDH1 and CS genes were all expressed in the brain,while GLUT2 and LDH genes have the highest expression levels in the liver.Moreover,HK gene has the highest expression in the heart and PGAM2 gene has the highest expression in muscle.The qRT-PCR,enzyme activity assay,and Western Blot technique were used to analyze the expression of energy metabolism-related genes in the brain and liver of hypoxia and restorer T.fasciatus.and the temporal expression of enzyme activity and protein.Under acute hypoxia and recovery conditions,GLUT2 gene,HK gene and its enzymatic activity,the mRNA and protein expression of PGK1 and PGAM2 genes all showed a trend of increasing firstly and then decreasing.The LDH gene of brain and its enzymatic activity only showed a significant difference(P<0.05)between 8 hours after hypoxia and 4 hours after normoxia recovery,the tendency to increase first then decrease in the liver.After hypoxia treatment,the CS gene of the brain and liver did not change significantly during hypoxia,but after normal oxygen recovery,the expression of CS gene was significantly upregulated and maintained at a high level Furthermore,the brain MDH1 gene,enzyme activity and its protein were obvious up-regulated at the early stage of hypoxia(P<0.05),while the liver was significantly up-regulated at 8 hours after hypoxia(P<0.05).After returning to a normal oxygen environment,the expression level remained high.The above results indicate that the oxidative metabolism ability of the brain of T.fasciatus is activated in the early stage of hypoxia,and energy can be obtained by oxidizing glucose.The liver is more likely to effectively use anaerobic glycolysis to provide energy for its own consumption.(3)Effects of hypoxia stress and recovery on blood indexes in T.faciatusThe expression and distribution of blood-related genes on Vascular endothelial growth factor-A(VEGF-A)and Erythropoietin(EPO)in different tissues of T.fasciatus were measured by qRT-PCR.The experiment result showed that the expression level of VEGF-A gene was highest in the liver and the EPO gene expression was highest in the heart.In the brain and liver of T.fasciatus,hypoxia stress and restoration of blood-related genes and proteins were studied by qRT-PCR and Western Blot techniques.Under the conditions of acute hypoxia and normoxia recovery,EPO gene and VEGF-A gene and their proteins have a tendency to increase firstly then decrease.Hypoxia is the cause of red blood cells and capillary blood vessels in the brain and liver,which leads to improved blood oxygen carrying capacity.At the same time,the results of blood index parameters showed that RBC,HCT,HGB,MCH,MCHC,and Glucose were lower in hypoxic conditions than in the control group when acute hypoxic exposure is prolonged,while MCV levels were low,indicating the increase of red blood cell proliferation and glucose content in the blood.In conclusion,with the up-regulation of HIF-1?,T.fasciatus can regulate the transport of oxygen and glucose to different tissue sites,achieving a switch from aerobic respiration to anaerobic respiration to maintain energy supply under hypoxic stress.