| In recent years,high-density and intensive aquaculture models have become a common phenomenon,which is also the main cause of fatty liver.As we all know,fatty liver is often accompanied by a large number of negative effects and thus seriously hinder the development of aquaculture.Therefore,exploring the mechanism of fatty liver in fish and finding a suitable approach to prevent or treat it are very benefit of the development of fish farming.In order to carry out this experiment,we need to establish a culture method for the primary hepatocytes of the blount snout bream in the first step.Then we can build a primary hepatocyte steatosis model to provide materials for the study of fatty liver.The experiment consists of four parts:(1)The culture method for primary hepatocytes from blunt snout breamThis experiment aimed to establish a stable method for isolation and culture of primary hepatocytes from the blunt snout bream.The hepatocytes were isolated by tissue separation and trypsin digestion and then purified by Percoll layered liquid combined with density gradient centrifugation.After re-suspending hepatocytes in L-15 medium containing 15% FBS,cell viability and concentration were determined by trypan blue staining.Then,cells were cultured in an incubator.Hepatocytes were cultured for 24,48,and 72 hours respectively.The status of cell were assessed using the activities of AST,ALT and LDH in the supernatant.The results showed that the activity of hepatocytes reached over 90% after isolation and purification.In addition,the hepatocytes were tightly connected in an island shape and the morphology tends to be stable after 48 hours of culture.The activities of AST,ALT and LDH in the supernatant were the highest at 24 h,and gradually decreased with the prolongation of culture time(P<0.05).Howere,there was no significant difference between 48 h and 72 h.Therefore,we applied trypsin digestion to isolate hepatocytes at room temperature,then cultured for 48 h in a constant temperature incubator.Based the above,cells with physiologically and morphologically stable were obtained.(2)Establishment of the steatosis of primary hepatocyte from blunt snout breamIn this study,the oleic acid was used to induce the steatosis of primary hepatocytes.Firstly,the primary hepatocytes were cultured with different concentrations of oleic acid(0,0.2,0.4,0.8 mM)for 48 h.Then,we measured triglyceride(TG)and total cholesterol(TC)levels of hepatocytes,and activities of alanine aminotransferase(AST),aspartate aminotransferase(ALT)and lactate dehydrogenase(LDH)in supernatants.The results showed that 0.4 mM oleic acid for 48 h significantly elevated(P<0.05)the levels of TG and TC of hepatocytes and the activities of AST,ALT and LDH in supernatants.Moreover,there are obvious lipid droplets in the cells,and the survival rate of hepatocytes is higher.In summary,hepatocyte steatosis model was obtained by the treatment with 0.4 mM oleic acid for 48 h.(3)The effects of AMP-activated protein kinase on lipid metabolism of hepatocytes from blunt snout breamBased on the primary hepatocyte steatosis model,this experiment aimed to explore the role of AMPK in lipid metabolism of hepatocytes.There were three treatments: control group(normal medium),oleic acid group(0.4 mM oleic acid incubation),metformin group(200 μM metformin and 0.4 mM oleic acid incubation).After 48 h treatment,the cells and supernatants were collected for analysis.The results showed significant enhancement of cell triglyceride and total cholesterol concentrations in the oleic acid group,and metformin group significantly reduced the values(P<0.05).Also,the oleic acid group exhibited significantly higher AST activity than both control and metformin groups(P<0.05).The lowest AMPK and phospho-AMPK protein expression was found in the oleic acid group while metformin addition significantly up-regulated their expression levels(P<0.05).Expression of genes associated with lipid metabolism such as PPAR-α,CPT I,AOX and TFAM was suppressed in the oleic acid group,and metformin supplementation up-regulated their expression levels(P<0.05).The opposite trend was true for the expression of ACC2 gene.Also,the results showed down-regulation of FAS and SREBP-1C genes in oleic acid group,and metformin addition resulted in further reduction of their expression level(P<0.05).The lowest activities of mitochondrial complexes(Ⅰ-Ⅲ)were found in the oleic acid group and metformin prevented oleic acid-induced reduction of mitochondrial complexes activity.Notably increased concentrations of ROS and MDA were found in the oleic acid group,and metformin treatment reduced their concentrations(P<0.05).Moreover,metformin group exhibited higher GPX activity than the oleic acid group(P<0.05).The findings in this study showed clearly that metformin activated AMPK in blunt snout bream hepatocytes,which contributed to enhanced lipid metabolism and attenuated lipid deposition in the cells incubated with oleic acid.(4).The effects of hydroxytyrosol on lipid metabolism in liver of blunt snout breamTo explore the role of hydroxytyrosol in lipid metabolism in the liver of blunt snout bream,this part was carried on both in vitro and in vivo experiments.In vitro: There were three treatments: control group(normal medium),oleic acid group(0.4 mM oleic acid incubation),HT group(10 mM hydroxytyrosol and 0.4 mM oleic acid incubation).After 48 h of treatment,the cells and supernatants were collected for analysis.The results showed significant reduction of cell triglyceride and total cholesterol concentrations in the HT group over oleic acid group(P<0.05).and decrease the number of lipid droplets in cells.Also,HT can reduce the activities of AST,ALT and LDH in the supernatant(P<0.05),thereby alleviate cell damage.Moreover,HT significantly up-regulated the genes expression of PGC-1β,PPAR-α,CPTI and AOX for lipidolysis,while inhibited lipid synthesis related genes ACC2,FAS and SREBP-1c expression(P<0.05).In addition,HT can also regulate mitochondria-associated genes as PGC-1α,NRF1,TFAM and UCP4 and promote their expression(P<0.05).HT prevented oleic acid-induced reduction of mitochondrial ability like mitochondrial complexes activity,Membrane potential level,SDH activity(P<0.05)and increase the abundance of mitochondria.HT also improves the redox state of cells by reducing the levels of ROS and MDA;meanwhile,increasing the activity of glutathione peroxidase and superoxide dismutase in hepatocytes.In vivo: Contained three treatments: 5% fat group(L5),5% fat with 200 mg/Kg HT group(L5/HT),15% fat group(L15),15% fat with 200 mg/Kg HT group(L15/HT);After the experiment was finished,samples were collected for analysis.The results showed that the addition of HT at low-fat level reduced the weight gain rate of the blunt snout bream;the oposite results occured in L15/HT which significantly raised the weight gain rate(P<0.05).However,the survival rate,specific growth rate and protein deposition rate of the blunt snout bream were significantly improved by adding HT at high-fat level(P<0.05).Besides,both low-fat and high-fat levels of HT all can up-regulate the expression of lipolysis-related genes(PPAR-α,CPTI,AOX)and mitochondria-related genes(PGC-1α,NRF1,TFAM,UCP2,UCP4).Meanwhile,HT also could promote mitochondrial production and decrease the deposition of lipid in liver(P<0.05).The addition of HT at low-lipid level had no significant effect on the expression of lipid synthesis-related genes(ACC2,FAS,SREBP-1c)(P>0.05),but significantly inhibited the expression of PPAR-γ;while HT was added at high-lipid level,the expressions of genes(ACC2,FAS,SREBP-1c,PPAR-γ)were all significantly down-regulated(P<0.05).Moreover,HT also can improve the activity of complex(Ⅰ-Ⅴ).But,there is no statistically significant of complex Ⅳ in L15/HT group(P>0.05).In summary,the results of in vitro and in vivo experiments indicating: HT can up-regulate the genes expression of lipidolysis and improve mitochondrial function in liver.So,HT significantly alleviated the dysfunction of lipid metabolism caused by high-fat and promote the growth of blunt snout bream. |
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