Pigment Epithelium-derived Factor Improves TNF?-induced Hepatic Steatosis In Grass Carp(Ctenopharyngodon Idella)

Pigment epithelium-derived factor(PEDF)is a well known angiogenesis inhibitor and neuroprotective factor,as well as anti-inflammatory factor.Studies of mammals have shown that PEDF is directly involved in the regulation of lipid metabolism.However,so far people do not clear its role in the bony fish may exist.Tumor necrosis factor-?(TNF?)is an important cytokine in the body,regulating cell differentiation,proliferation,apoptosis,and participate in the regulation of liver steatosis process.In this study,the PEDF gene was cloned on grass carp(Ctenopharyngodon idella)for the first time.The methods of prokaryotic expression,immunoprecipitation,cell culture,in vivo injection,etc.were used in the trial.The basic characteristics of PEDF and its role and mechanism in relieving TNF?-induced hepatocyte steatosis in grass carp were explored in vivo and in vitro.The results were as follows:1.Sequence analysis showed that PEDF gene,which possesses 8 exons and 7 introns,encodes a protein with 409 amino acids.The N-terminus of the protein has a signal peptide,and the C-terminal is a serine protease inhibitor domain.The intracellular PEDF is localized to the cytoplasm and binds to the adipose triglyceride lipase(ATGL).2.PEDF was a critical determinant of the transcriptional response to nutrient availability in grass carp.PEDF has a wide range of tissue expression and is highest in adipose tissue.Its expression increases with the differentiation and maturation of adipocytes.PEDF expression was affected by starvation in vivo and vitro.The expression of PEDF was significantly increased after oleic acid and insulin treatment of hepatocytes in vitro.3.PEDF promotes triglyceride degradation.The PEDF recombinant protein with biological activity was obtained by prokaryotic expression.The WB assay showed that HIS tag-labeled exogenous PEDF can be uptake by cells.The results of triglyceride assay demonstated that PEDF promoted lipolysis.4.PEDF inhibits TNF?-induced hepatic steatosis,and PPAR? is involved in the regulation of this process.TNF? significantly promoted lipid accumulation in vivo and vitro,accompanied with a decrease in mRNA levels of PEDF and peroxisome proliferator-activated receptor alpha(PPAR?).Recombinant PEDF and PPAR? agonist diminished the TNF?-induced hepatic steatosis.Meanwhile,PPAR? agonist caused an increase in PEDF expression.This study shows that PEDF reduces lipid accumulation and inhibits TNF?-induced hepatic steatosis to protect the homeostasis of triglycerides.