| In this experiment,the Japanese seabass(Lateolabrax japonicas)was treated as the objects of research with the plant protein blend as the protein source for fish meal replacement,conducted to investigate the effects of plant protein diet on growth performance,nutrition metabolism,immunity,inflammatory reaction and the liver heath of Japanese seabass,a typical carnivorous fish.In addition,the study explored the role of the c AMP-JNK/NF-k B-caspase signaling pathway in the liver injury of the Japanese seabass.The results of the research provide technical support for the optimization of compound feed and the prevention and treatment of liver disease for the Japanese seabass.The results of the experiment are as follows:Two isonitrogenous and isoenergetic diets were formulated.A basal diet containing 54% low-temperature steam-dried fishmeal was used as the control(FM),whereas the fishmeal was totally replaced with a plant protein blend(PP,soybean protein concentrate: cottonseed protein concentrate = 1:1.66),the crystalline amino acids and monocalcium phosphate were added into the PP diet according to the ideal amino acids profile of Japanese seabassl to balance the main essential amino acid and phosphorus.Each diet was fed to 4 replicates with 30 fish(initial body weight = 10.42 ± 0.01 g)each replicate.Fish were fed to apparent satiation twice a day at 8:00 and 20:00 for 10 weeks.Food intake was measured daily,the fish was weighted and sampled at the growth trial termination.The results showed that Japanese seabass could ingest the PP diet.Compared with FM group,significantly higher feeding rate could be observed in PP group.Although the survival rate was not affected by PP diet,the fish in PP group showed the significantly lower final body weight,weight gain rate and specific growth rate(P<0.05).A significantly higher feed conversion ratio and significantly lower productive protein value could be observed in fish of PP group(P<0.05)but the fat efficiency ratio had no significant difference between two groups.The feed hardness was very significantly increased(P<0.01)and whole body ash content was significantly increased in fish of PP group(P<0.05).In addition,the hepatosomatic index was significantly increased(P<0.05),and the metabolic disorder and liver disease appeared in PP group.Compared with fish fed FM diet,fish fed PP diet had protein deficiency supported by significantly lower plasma free amino acid(FAA)level.Glucose metabolism disorder was expressed as the uncontrollable fasting glycolysis and pyruvate aerobic oxidation at postprandial 24 h,significantly lower plasma glucose(GLU),hepatic glycogen(GLY)and higher plasma glucagon(GC)levels were observed in PP group(P<0.05),correspondingly,the hepatic m RNA levels of glycolytic enzymes including glucokinase(GK)and pyruvate kinase(PK),and the key enzyme of aerobic oxidation,pyruvate dehydrogenase(PDH),were very significantly up-regulated in the fish of PP group(P<0.01),which potentially over-produced acetyl-Co A as the substrate for protein and lipid synthesis.Plasma triglyceride(TG)and hepatic non-esterified fatty acid(NEFA)contents were significantly decreased,but the hepatic total cholesterol(TC)content was very significantly increased in the PP group,in addition,histological examination of the liver morphology with H&E staining showed the PP diet resulted in the ballooning and nuclei migration of hepatocytes.Hepatic m RNA expression level of cholesterol synthesis gene(HMGCR)was significantly up-regulated,but bile acid synthesis gene(CYP7A1)was significantly down-regulated(P<0.05),indicating that cholesterol accumulation could be attributed to the inadequate bile acids synthesis in fish fed PP diet.Both lipogenesis(FASN,PPARγ)and lipolysis related genes(HSL,CPT1α and PPARα)genes expression were significantly up-regulated(P<0.05),but the lipolysis had a deeper degree in PP group.Compared with FM group,the extremely increased hepatic cyclic adenosine monophosphate(c AMP)content(P<0.01)and he significantly up-regulated m RNA level of c AMP responsive element binding protein(CREB)(P<0.05),together with the significantly higher plasma hyroid hormones T3 and T4 levels(P<0.05),indicated that the accelerated energy metabolism process appeared and decreased the growth performance of fish in PP group.Compared with FM group,the plasma immunoglobulin M(Ig M)content of fish in PP group was significantly decreased,glutamic-pyruvic transaminase(ALT)activity was significantly increased(P<0.05),and the hepatic total antioxidant capacity(T-AOC)and activity of glutathione peroxidase(GSH-Px)were significantly decreased(P<0.05).The hepatic TNFα,IL1β,IL10 and caspase8 m RNA levels were significantly up-regulated in PP group(P<0.05),together with more cleaved-caspase3 and p65NF-k B protein signal,revealing that the inflammation and apoptosis occured in the hepatocyte of fish in PP group.The activated p65NF-k B may promote the hepatocytes apoptosis via the extrinsic pathway(caspase8/caspase3).Simultaneously,a "self-saving" response could be observed that activated c AMP promoted the lipolysis/β-oxidation process and up-regulated gene expression of anti-inflammatory cytokine IL10 via promoting CREB expression,further inhibited the over-phosphorylation of JNK protein,which might impede the intrinsic apoptosis pathway(caspase9/caspase3).In conclusion,the nutrient and energy metabolic disorder induced fatty liver related to the cholesterol accumulation in Japanese seabass fed full PP diet,which was under the regulation by c AMP-JNK/NF-k B-caspase signaling pathway.The hemostasis phosphorylation of JNK protein protected the liver tissues from more serious damage. |
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