Study On Effects Of SORT1 On Liver VLDL Synthesis And Lipid Metabolism Of Dairy Cows With Ketosis

The pathological characteristics of perinatal dairy cow ketosis caused by negative energy balance are non-esterified fatty acid(NEFAs)and large amount of triglyceride accumulation in liver,which seriously reduce the performance of dairy cows and cause great economic loss.Very low density lipoprotein(VLDL)particles,as the main route of TAG transport to extrahepatic tissue,play a key role in liver lipid transport and accumulation during the occurrence of ketosis in dairy cows.Sorted protein 1(SORT1),as an intracellular protein transporter,has recently been found to be involved in a variety of lipid-related diseases,but its effects on VLDL synthesis,assembly and secretion and indirect effects on ketosis in dairy cows via VLDL remain unclear.Therefore,this study was aimed at clarifying the characteristics of VLDL secretion in cows with ketosis and the effects of SORT1 on the synthesis and secretion of VLDL in hepatocyte stimulated by high concentration of fatty acids.In this study,liver tissues of healthy cows and ketosis cows were collected by liver puncture in vivo,and lipid accumulation and steatosis in liver tissue were detected by pathological tissue section method.The expression of lipid biosynthesis related gene SREBP-1C and its downstream target genes in tissues were detected.The content of VLDL-APOB and lipid composition of VLDL particles in blood of dairy cows were determined by ELISA and LC-MS metabolomics.In order to study the effect of SORT1 on VLDL synthesis and secretion under ketosis state,this study isolated the primary hepatocyte of calves in vitro and stimulated the primary hepatocyte of calves with high concentration of fatty acid to establish a liver fat deposition model.Meanwhile,SORT1 adenovirus overexpression vector,SORT1 inhibitor and SORT1 sh RNA silencing plasmid were added to change the expression of SORT1 in hepatocytes.At the same time,the secretion of VLDL-Apo B in cell culture supernatant and the expression of ApoB100 and MTP,the important components of VLDL synthesis in hepatocyte were detected.There are different opinions on the effect of SORT1 expression inhibition on hepatic lipid metabolism in non-ruminant animals.Therefore,we used SORT1 inhibitor and sh RNA silencing plasmid to investigate the effects of their expression inhibition on lipid synthesis and metabolism in primary hepatocytes of calves.The results of in vivo study showed that the liver tissues of cows with ketone disease showed obvious lipid accumulation,steatosis and even fibrosis,and the expression levels of lipid synthesis related genes SREBP-1c,FASN,ACC1,DGAT1 and DGAT2 were significantly higher than those of healthy controls.However,the expression levels of CPT1 A,a key gene for fatty acid oxidation,and APOB100,an important component protein of VLDL synthesis,and MTP were significantly lower than those in the control group.At the same time,VLDL-Apo B content in blood of keto disease cows was significantly lower than that of healthy control group,and PE content in lipid composition of VLDL pellets and PE related subtypes were significantly reduced,and the pellets were significantly larger.The results showed that liver lipid accumulation and liver damage were observed in the liver tissues of cows with ketone disease,and VLDL secretion and metabolism were abnormal in the liver of cows with ketone disease.In vitro stimulation and induction treatment results showed that high concentration of Fatty acid stimulation could promote the expression of hepatocyte lipid synthesis genes SREBP-1c,FASN,ACC1,DGAT1 and DGAT2,and reduce the expression of the key gene of Fatty acid oxidation,CPT1 A,leading to excessive deposition of TAG and lipid droplets in hepatocytes.Under the action of high concentration of Fatty acid,the secretion of VLDL-Apo B is impaired and the expression of its assembled synthetic protein is extremely significantly reduced.Meanwhile,the related indexes of oxidative stress induced by Fatty acid decreased,while MDA and ROS increased significantly.The changes of the above indexes were stimulated by the addition of Fatty acid after the overexpression of SORT1 gene in hepatocyte.It was found that the overexpression of SORT1 gene further promoted the increased expression of lipid synthesis genes and lipid accumulation induced by Fatty acid.At the same time,it aggravated the VLDL synthesis disorder.On the contrary,when SORT1 was silenced and inhibited for 48 or 12 hours,the Fatty acid was added to stimulate for 12 hours.The results showed that the inhibition or silencing of SORT1 significantly alleviated the accumulation of hepatocyte TAG and lipid droplets induced by Fatty acid.Meanwhile,the increased expressions of Fatty acid-induced lipid synthesis related genes SREBP-1c and FASN were decreased.It reduced the oxidative stress of MDA,SOD and ROS induced by Fatty acid.Importantly,the inhibition or silence of SORT1 gene significantly increased the secretion of VLDL-Apo B and the expression levels of the key lipid proteins ApoB100 and MTP under the action of fatty acid.In conclusion,SORT1 regulates high concentration of Fatty acid to stimulate the assembly and secretion of VLDL particles in hepatocyte,and then regulates metabolic processes such as lipid synthesis and accumulation in primary hepatocyte of calves.