Regulation Mechanism Of Liver Fat Metabolism By FXR In Dairy Cows With Fatty Liver

Fatty liver is a metabolic disease that is highly prevalent in perinatal cows.The main pathological basis for its occurrence is the negative energy balance in perinatal cows,which leads to fat mobilization and the release of non esterified fatty acids（NEFA）in large quantities,which are then esterified in the liver to form triglycerides（TG）.Very low density lipoprotein（VLDL）is a means of transporting TG to extrahepatic tissues.Cholesterol is the main component of VLDL synthesis,and the synthesis of bile acids（BA）is one of the main modes of cholesterol metabolism.Studies have shown that the synthesis of BA in perinatal cows increases.Moreover,in recent years,BA is an important intracellular signaling molecule,which plays a role in liver and extrahepatic tissues and is involved in the regulation of lipid metabolism and energy balance in the body.However,as a BA synthesis receptor,the effect of FXR on the regulation mechanism of BA anabolic metabolism in the liver caused by fatty liver of dairy cows remains unclear.First,in order to clarify the metabolic characteristics of fatty acids and bile acids in the liver of dairy cows with fatty liver,this study collected liver tissues of dairy cows with fatty liver,isolated primary calf hepatocytes and added 1.2 m M NEFA to establish a liver fat accumulation model.Then,changes of lipid and bile acid metabolism in liver tissues of cows with fatty liver and hepatocyte of calves with 1.2 m M NEFA were detected.The results showed as follows:1)The TG content and fluorescence intensity of lipid drops in liver and hepatocytes supplemented with 1.2m M NEFA were increased,the fatty acid synthesis factors Sterol regulatory element binding protein 1c（SREBP1C）,Fatty acid synthase（FAS）and Acetyl-Co A carboxylase 1（ACC1）were increased,and the expression of fatty acid oxidation factor CPT1A was decreased（P<0.05 or P<0.01）.2)Cholesterol 7α-hydroxylase（CYP7A1）,cholesterol12α-hydroxylase（CYP8B1）and oxysterol 7α-hydroxylase（CYP7B1）were increased in liver of dairy cows with fatty liver and liver cells supplemented with 1.2 m M NEFA.Regulating the expression of BA synthesis factor FXR and BA transporter bile salt output pump（ABCB11）and multi-drug resistance associated protein 2（ABCC2）decreased（P<0.05 or P<0.01）.The metabolomics of BA in blood and feces of cows with fatty liver supplemented with 1.2m M NEFA hepatocyte supernatant showed that the contents of primary BA,secondary BA and total bile acid increased.The results showed that lipid and bile acids accumulated in the liver of cows with fatty liver.Secondly,in order to clarify the regulatory mechanism of FXR on lipid metabolism in hepatocytes with high NEFA,the effects of FXR on hepatocytes added with 1.2 m M NEFA were examined in this experiment.Calf hepatocytes were treated with FXR activator and inhibitor,and treated with 1.2m M NEFA.Fatty acid,cholesterol and bile acid metabolism and liver injury were detected.The results show that:1)Compared with the 1.2 m M NEFA alone group,the TG content and lipid drop fluorescence of calf hepatocytes supplemented with1.2m M NEFA and the FXR activator GW4064 were decreased,and the expressions of lipid synthesis factors SREBP1C,FAS and ACC1 were decreased（P<0.05 or P<0.01）.2)The expressions of cholesterol synthesis factors 3-hydroxy-3-methyl-glutaryl coenzyme A reductase（SREBP2C）and Sterol regulatory element binding protein 2（HMGCR）were increased（P<0.05 or P<0.01）.3)The expressions of BA synthesis factors such as CYP7A1,CYP8B1,CYP27A1 and CYP7B1 were decreased,while the expressions of BA synthesis factor FXR and BA transporter factors ABCB11 and ABCC2 were increased（P<0.05 or P<0.01）.4)The contents of H₂O₂and ROS in lipid oxidation were decreased（P<0.05 or P<0.01）.5)Hepatocytes TG content and lipid drop fluorescence were increased after adding 1.2m M NEFA and FXR inhibitor（Z）-Guggulsterone compared with 1.2m M NEFA treatment group alone.The expressions of lipid synthesis factors SREBP1C,FAS and ACC1 were increased（P<0.05 or P<0.01）.6)The expressions of cholesterol synthesis factors SREBP2C and HMGCR were decreased（P<0.05 or P<0.01）.7)The expressions of bile acid synthesis factors CYP7A1,CYP8B1 and CYP7B1 were increased,and bile acid transporters ABCB11 and ABCC2 were decreased（P<0.05 or P<0.01）.8)The contents of H₂O₂and ROS in lipid oxidation level were increased（P<0.05 or P<0.01）.The addition of FXR activator GW4064 alleviated hepatocyte injury,promoted cholesterol synthesis,inhibited lipid and bile acid synthesis,and reduced the accumulation of hepatocyte lipid and bile acid.FXR inhibitor（Z）-Guggulsterone aggravated hepatocyte damage,inhibited cholesterol synthesis and increased lipid and bile acid synthesis to increase lipid and bile acid accumulation in hepatocytes.Finally,in order to determine whether cholesterol metabolism regulates liver lipid metabolism by influencing FXR,this study added HMGCR overexpressed adenovirus and 1.2m M NEFA into calf hepatocytes,and then detected lipid,cholesterol and BA metabolism and liver damage in calf hepatocytes.The results showed as follows:1)Compared with the 1.2m M NEFA treatment group alone,the TG content and lipid drop fluorescence of calf hepatocytes were decreased after the addition of 1.2 m M NEFA and the addition of HMGCR overexpressed adenovirus,and the expressions of lipid synthesis factors SREBP1C,FAS and ACC1 were decreased（P<0.05 or P<0.01）.2)Increased cholesterol synthesis factor SREBP2C and HMGCR（P<0.05 or P<0.01）.3)BA synthesis factors CYP7A1,CYP8B1,CYP27A1 and CYP7B1 regulate the expression of BA synthesis factor FXR and BA transporter factors ABCB11 and ABCC2（P<0.05 or P<0.01）.4)The contents of H₂O₂and ROS in lipid oxidation were decreased（P<0.05 or P<0.01）.The addition of HMGCR overexpressed adenovirus alleviated hepatocyte injury,promoted the synthesis of cholesterol in hepatocytes,inhibited the synthesis of lipids and bile acids,and thus reduced the accumulation of lipids and bile acids.In conclusion,this study indicates that the low expression of BA receptor FXR in liver cells of dairy cows with fatty liver plays an important role in promoting liver lipid synthesis and BA synthesis.Activation of FXR can effectively reduce the expression of fatty acid synthesis factors,lipid accumulation,ROS and H₂O₂content in calf hepatocytes under high NEFA,thus alleviating liver injury.This study provides an important reference for the in-depth interpretation of the pathogenesis of fatty liver in dairy cows and the research and development of targeted prevention and treatment technology of fatty liver in dairy cows.