Role Of Soluble Epoxide Hydrolase In Hyperhomocysteinemia-induced Hepatic Steatosis

Nonalcoholic fatty liver disease(NAFLD)is a chronic disease charactered with intrahepatic fat accumulation caused by non-alcoholic factors.It contains a series of histological changes,including hepatic steatosis,Nonalcoholic hepatitis,fibrosis,cirrhosis and even liver cancer.In recent years,the incidence of NAFLD has been relevant to several pathogenic factors of diabetes,hyperlipidemia and obesity.Homocysteine,Hcy is a sulfur-containing amino acid with lots of biological function,formed by hepatic metabolism of methionine in vivo.The elevation of plasma Hcy(?15 ?M)is considered as Hyperhomocysteinemia(HHcy).The occurrence of metabolic diseases,cardiovascular diseases and immune disease is closely related to HHcy.Clinical research shows that the level of plasma Hcy is posotive correlated with the incidence of NAFLD.Soluble Epoxide Hydrolase(sEH)is an important enzyme in cytochrome P450 metabolism pathway of Arachidonic acid(AA).sEH hydrolyzes epoxyeicosatrienoic acids(EET)to inactive Dihydroxyeicosatrienoic acids(DHETs)and inhibites their cardiovascular protective effects and anti-inflammatory effects to affect the development of diseases.Several studies have shown that Hcy could trigger the disorders of lipid metabolism in liver.Our previous studies found that sEH participate high fat-diet induced hepatic lipid deposition through increasing the inflammation of adipose tissue;HHcy induces hepatic steatosis by activating aryl hydrocarbon receptor-CD36 pathway.However,whether HHcy influence the hepatic lipid metabolism via affecting the expression of sEH has not been reported.The aim of this investigation was to illuminate the role and mechanism of sEH in HHcy-induced hepatic steatosis in order to provide a novel potential therapeutic target of fatty liver diseases.At first,we fed the C57BL/6 mice with 2% methionine diet(High-methionine diet,HMD)for 8 weeks,the level of plasma Hcy was significantly increased,it suggested that we successfully established HHcy mouse model.The results of Oil red O staining of the liver section and lipid extraction showed that the HHcy mice arised significant steatosis.Then,we detected the exprssion and activity of sEH and found that HHcy upregulated the expression and activity of sEH and reduced the hepatic EETs content.In vitro experimental results showed that treatment of Hcy arised hepatic steatosis in primary hepatocytes and upregulated the expression and activity of sEH in hepatocytes simultaneously.The above results indicated that sEH participates HHcy-induced hepatic steatosis.In order to confirm the key effects of sEH in HHcy-induced hepatic steatosis,we subsequnently added sEH Inhibitor,1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea,TPPU to HMD mice drinking water from the 5th week for 4 weeks.Inhibition of sEH significantly alleviated hepatic steatosis of HHcy mice and increased the expression of Cpt1 a,Acox1 and Mcad simultaneously,which are the target genes of Peroxisome proliferator-activated receptor alpha(PPAR?)and facilitate lipid oxidation in liver.Further,we added TPPU to primary hepatocytes which were treated with Hcy to inhibit sEH activity.We found inhibition of sEH significantly alleviated hepatic lipid depositon,and increased the gene expression of Cpt1 a,Acox1 and Mcad.And siPPAR? reversed TPPU arised activation of PPAR?.Results of luciferase reporter assay showed that TPPU significantly increased the activity of Peroxisome proliferator-activated receptor response element(PPRE)and it suggested that inhibiton of sEH activated PPAR? transcriptionally.Thus,inhibition of sEH activity promoted the hepatic lipid oxidation through activating PPAR? and then alleviated HHcy-induced steatosis.As an important substrate of sEH,EETs have crucial effects on protection of cardiovascular and anti-inflammatory.Four isomers of EETs are classified according to their structural differences: 5,6-;8,9-;11,12-;14,15-EET.Different EETs have different biological function in diverse diseases models.In this study,we found HHcy reduced the level of 11,12-EET in liver tissue and hepatocytes specifically.Inhibition of sEH activity markedly increased the level of 11,12-EET.Relevant research shows that EETs are endogenous PPAR? activators.To further confirm whether EETs could directly participate the activation of PPAR?,the primary hepatocytes were transfected with PPRE luciferase reporter plasmid and then were treated with four isomers of EETs.The result showed that 11,12-EET increased the activty of PPRE significantly.Thus,11,12-EET could be served as endogenous PPAR? activators to prevent from HHcy-induced hepatic steatosis.The research of this study shows that sEH plays an important role in the process of HHcy-induced hepatic steatosis.Inhibition of sEH activity promotes lipid oxidation through increasing the hepatic 11,12-EET content and alleviates HHcy-induced hepatic steatosis.TPPU effectively alleviates hepatic steatosis via decreasing the activity of sEH.It suggested that sEH may be a potently therapeutic target for fatty liver in clinic treatment.