The Model Establishment of Lipid Deposition in Primary Chicken Embryo Liver Cells Induced by Oleic Acid
[Background]Fatty liver syndrome (FLS) is a common nutritional metabolic disease in laying hens during the laying period,which includes a multi-stage pathogenesis process,such as "simple steatosis,steatohepatitis,liver fibrosis,and cirrhosis",causing substantial economic losses in poultry industry.The construction of fatty liver model is the key to exploring the pathogenesis of FLS,screening nutritional regulation measures and subsequent disease prevention and evaluation.However,there are few studies on poultry hepatocyte steatosis models.[Objective]Chicken embryo liver (CEL) cells were used to establish a liver lipid deposition model by oleic acid induction,aiming to explore the induction time required for different stages of FLS.[Method]CEL at the age of 18 embryo was used and when CEL cells grew to about 80％,and the medium containing 400 μmol·L-1 oleic acid was replaced for treatment.After 24 h,48 h and 72 h of culture,the cells were stained with oil red O,and the lipid content was measured.At the same time,the mRNA and protein levels of genes related to lipid metabolism,inflammation and fibrosis were analyzed.[Result]Compared with the Control group,there were a large number of red lipid droplets in 24 h group,48 h group and 72 h group,and the red color gradually deepened with the increase of induction time (P<0.05),indicating that fat droplets in cells were increased.Meanwhile,the content of TG and TC in the liver were higher than those in the Control group (P<0.05).RT-PCR analysis revealed a significant upregulation of ACC,FAS,ELOVL6,and SREBP gene expression related to lipid synthesis following 24 h of oleic acid induction (P<0.05),indicating that oleic acid lipid deposited on cells after 24 h of processing.ELISA results showed that the content of IL-6,IL-1β and TNF-α were not significantly changed in 24 h (P>0.05).However,an upward trend was observed in IL-6 level with prolonged induction durations (P=0.052).Notably,following 72 h of oleic acid treatment,the levels of IL-6 and IL-1β were significantly increased in Control group (P<0.05),which indicated that acid treatment after 48 h cells began to inflammation reaction and increased with the extension of induction time.In addition,compared with the Control group,the expression level of IL-6 protein in the 48 h group was significantly increased,which was consistent with the ELISA results,while the protein expression levels of COL1A1 and FIBRONECTIN were increased (P<0.05).On the contrary,the protein expression levels of IL-6,COL1A1 and FIBRONECTIN were significantly higher than those in the Control group at 72 h (P<0.05).[Conclusion]The early lipid deposition model of hepatocytes could be established by 400 μmol·L-1 oleic acid induction for 24 h.In addition,inflammation and fibrosis would occurr after 48 h and 72 h induction,respectively.
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