Effects Of Low Temperature Stress On The MAPK Pathway And Lipid Metabolism In The Muscle Tissue Of Pufferfish

Fish is a typical thermophilic animal,the change of temperature will directly affect its physiological and biochemical state.Takifugu fasciatus is a typical migratory fish in the river and sea,which spawns from river in early spring,and falls into the sea in winter.The optimal growth temperature range of T.fasciatus was 23-32?,the feeding rate began to decline at 19?,and a large number of frostbite began at 13?.In the process of breeding,which will be affected by low temperature,especially the sudden cold wave will also cause significant economic losses.Therefore,it is very important to study the molecular mechanism of low temperature tolerance of T.fasciatus.Firstly,the pathological changes of muscle tissue of T.fasciatus were studied at different temperatures(25,19,and 13?)and different times(0,6,24,and96 h).Secondly,to explore the changes of antioxidant enzyme activities in muscle tissue under cold stress.Then,to analyze the response of MAPK signaling pathway and lipid metabolism related genes in muscle tissue under low temperature.Finally,to explore the changes of lipid metabolism in muscle tissue under low temperature.The results of this study provide theoretical data for clarifying the molecular mechanism of T.fasciatus under low temperature,and provide valuable reference for cold resistance breeding of teleost fish.The results are as follows:1.Effects of low temperature on muscle damage and oxidative stress level of T.fasciatusHematoxylin eosin staining(HE staining)was used to observe and analyze the muscle tissue of T.fasciatus under low temperature.The results showed that cold stress could change the morphology of muscle tissue,and with the extension of time,muscle fibers were broken and dissolved.With the temperature droping,the damage more serious.Superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),catalase(CAT),malondialdehyde(MDA)and triglyceride(TG)kits were used to determine the oxidative stress of T.fasciatus muscle under low temperature.The results showed that the enzyme activities of SOD and GSH-PX in 13? and 19?treatment groups reached the maximum at 6 h(p<0.05),SOD activity increased by2.1 and 1.7 times,GSH-PX activity increased by 2.3 and 1.5 times,respectively.The content of MDA in 13? and 19? groups also reached the maximum at 6 h(p<0.05),which was 2.3 times and 1.8 times higher than the control group,respectively.The cat activity of 13? group and 19? group reached the maximum at 96 h(p<0.05),which was 2.1 and 1.6 times higher than control group,respectively.These results indicate that lipid peroxidation occurs in muscle,and the activity of antioxidant enzymes is significantly up-regulated,indicating that the body quickly actives the antioxidant system to maintain body balance.The continuous increase of TG content indicates that the fish may mobilize fat synthesis to generate energy in response to low temperature.2.Time series response of MAPK signaling pathway and fat metabolism-related genes under low temperature stress in T.fasciatus.qRT PCR and Western blot were used to detect the expression of ERK?JNK and p38 MAPK in the MAPK family of T.fasciatus under low temperature.The results showed that the mRNA expressions of ERK?JNK and p38 MAPK were significantly up-regulated at 13 ? and 19 ? compared with 25 ?(control group).The mRNA expressions of ERK?JNK and p38 MAPK were continuously up-regulated at 19? and reached the maximum at 96h(p<0.05).Compared with control group,the mRNA expressions of ERK?JNK and p38 MAPK were up-regulated by 1.9,2.1 and 3.2 times,respectively.In the 13? group,the mRNA expression of ERK?JNK and p38 MAPK increased at first and then decreased,and reached the maximum at 6 h(p<0.05),which was 2.2,2.7 and 3.3 times higher than that in the control group.Western blot results showed that ERK?JNK and p38 MAPK were phosphorylated in different degrees under low temperature.The phosphorylation levels of ERK ?JNK and p38 MAPK in the 13? group reached the maximum at 24 h(p<0.05).Compared with the control group,the phosphorylation levels of ERK?JNK and p38 MAPK were increased by 3.5,2.1 and 3.8 times.qRT-PCR was used to detect the effect of low temperature on the fat metabolism-related genes in the muscle tissue of the T.fasciatus.The expression levels of fat synthesis genes and lipolysis genes were the highest at 96 h in 19? group(p<0.05),and the highest at 6 h in 13? group(p<0.05).The expression levels of transcription factors PPAR? and PPAR? in the 19?group were significantly up-regulated at 24 h compared with the control group(p<0.05).The expression levels of transcription factors PPAR? and PPAR? in the 13?group reached their peak at 6h.Compared with the control group,there is a significant difference(p<0.05).These results indicate that low temperature can not only induce the phosphorylation of MAPK,but also induce the changes of lipid metabolism related genes,and change the metabolic level of the body.3.Lipomics analysis of T.fasciatus muscle under low temperatureThe composition of lipid metabolites in T.fasciatus under low temperature was determined by lipomics.Principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were used to analyze the data.A total of 2495 lipids were identified,including 4 lipid classes(sterols,glycerols,glycerophospholipids and sphingolipids)and 43 subclasses(Phosphatidylcholines,phosphatidylethanolamines,oxidized Phosphatidylserines and lysophosphatidic inositols).The proportion of saturated fatty acids in muscle decreased with the decrease of temperature,and the proportion of monounsaturated fatty acids and polyunsaturated fatty acids increased with the decrease of temperature.Under low temperature stress,phospholipid metabolites(phosphatidylcholines,phosphatidylethanolamines,phosphatidylserines and phosphatidylinositols)related to cell membrane repair are significantly up-regulated,and oxidized phospholipid metabolites(oxidation phosphatidylcholines,oxidized phosphatidylserines,oxidized phosphatidylinositols and oxidized phosphatidylethanolamines)and lysophospholipid metabolites(lysophosphatidylcholines,lysophosphatidylserines,lysophosphatidylinositols)and lysophosphatidylethanolamine)significantly down-regulated.In the positive mode,the metabolic markers that were significantly up regulated were ACar 18:1,PE 16:0-18:2,and the significantly down regulated metabolites were PC 15:0-15:0,PMe OH12:0-24:4.The metabolites significantly down regulated in negative mode were Acyl Glc ADG16:2-16:4-16:4,Cer-AP t19:0/24:1,GM3 d27:0,HBMP 14:1-14:1-18:1,PC 16:0-16:0,PC 16:0-22:1,SHex Cer d44:2.The results showed that the phospholipid metabolites in lipid metabolism may play a key role in the response of T.fasciatus to low temperature.