Regulation Mechanism Of Cholesterol On High Fatty Acid-Induced Lipid Deposition In Calf Hepatocytes

Under the condition of negative energy balance,it is required to mobilize fat to meet perinatal dairy cows ’ energy needs,which produce too much non-esterified fatty acids(NEFA)to accumulate triglycerides in the liver,leading to fatty liver.Moreover,the accumulation of liver fat due to high concentration of NEFA under negative energy balance involving with the insufficient synthesis of cholesterol,affects the assembly of very low density lipoprotein(VLDL),the main way to transport triglycerides out of the liver.However,due to the unclear regulatory mechanism of cholesterol metabolism in the liver of dairy cows under negative energy balance,it is of great significance for the regulatory mechanism to the pathogenesis of fatty liver in perinatal dairy cows and improve its prevention and control measures.Therefore,the lipid accumulation of primary calf hepatocytes mediated with high NEFA as a model,the study analyzes characteristics of related indicators of cholesterol liver lipid metabolism by adding or inhibiting cholesterol synthesis to clarify the metabolic regulation mechanism of cholesterol in high NEFA mediated lipid accumulation in calf hepatocytes,and provide a theoretical basis for clinical prevention and treatment of fatty liver in dairy cows,with great significant to complete the pathogenesis of perinatal dairy cow fatty liver.We first studied the characteristics of cholesterol and fatty acid metabolism in calf hepatocytes mediated by high NEFA.Liver puncture carried out in normal cows and cows with fatty liver in perinatal period and the stimulation of primary calves hepatocytes with 1.2 mm NEFA for 12 h was to detect the indexes related to fatty acid synthesis and cholesterol anabolism.In order to elucidate the metabolic regulation mechanism of cholesterol in lipid deposition of calf hepatocytes mediated with high NEFA,added 10 μM MβCD-CHO(βCyclodextrin cholesterol),100 μM MβCD-CHO,10 μM cholesterol synthesis inhibitor SIM,6μM cholesterol transport inhibitor U18666 A to the lipid deposition model to stimulate for 12 h,was to detect fatty acid synthesis,cholesterol anabolism and oxidative stress.The results of in vivo test showed that the expression of hepatic fatty acid synthesis factors in fatty liver cows including SREBP-1c,FAS and ACC1 were significantly increased,while the m RNA expression levels of SREBP-2c,HMGCR and ACAT2 were significantly decreased(P <0.05 or P < 0.01).Test results in vitro indicated,compared with the control group,the expressions of SREBP-1c,FAS,ACC1 and the content of TG and lipid droplets in NEFA group were significantly increased,while the expressions of SREBP-2c,HMGCR,ACAT2,MTP,APOB100,ABCA1,ABCG5,ABCG8 and CYP27A1 and the TC content has been reduced(P <0.05 or P < 0.01),which suggested increased lipidosis,decreased synthesis,transport and excretion of cholesterol,as well as reduced the synthesis of VLDL.After adding 10,30,60,100μM MβCD-CHO for stimulating the cells,the m RNA expression levels of SREBP-1c,FAS,ACC1,and content of TG and lipid droplets dose-dependent increased with the dose of CHO,and 10 μM and100 μM MβCD-CHO were screened for follow-up test.The expression of SREBP-1c,FAS and ACC1 were significantly decreased in10 μM MβCD-CHO+NEFA group compared with NEFA group,as well as and the contents of TG and lipid droplets(P < 0.05 or P< 0.01);while the expression of MTP,APOB100 and ACAT2 and the contents of intracellular TC,CE and APOB100 in cell supernatant were significantly increased,as well as the expressions of ABCA1,ABCG5,ABCG8 and CYP27A1(P < 0.05 or P < 0.01),and SOD activity,MDA content and ROS level induced significantly(P < 0.05),indicating that 10 μM MβCD-CHO may alleviate NEFA mediated lipid deposition and reduce oxidative stress by promoting high NEFA mediated VLDL metabolism related gene expression and cholesterol transport.However,the results of 100 μM MβCD-CHO was contrary to 10 μM MβCD-CHO group,which meant 100 μM MβCD-CHO increased NEFA mediated lipid deposition and oxidative stress,decreased VLDL metabolism related gene expression and cholesterol transport.Compared with NEFA group,the expressions of SREBP-1c,FAS and ACC1 in SIM + NEFA group were significantly decreased,the expressions of SREBP-2c,HMGCR,MTP,ACAT2 and APOB100 were significantly increased,the contents of intracellular TC,CE and APOB100 in cell supernatant were significantly increased(P < 0.05 or P < 0.01),and the contents of TG and lipid droplets were decreased;The expression of ABCG5,ABCG8 and CYP27A1 increased significantly,but the expression of ABCA1 decreased significantly(P < 0.05 or P < 0.01);The significantly increased activity of SOD,the reduced content of MDA and the decreased level of ROS(P < 0.05 or P < 0.01),indicating that SIM can reduce the de novo synthesis of fatty acids mediated by high NEFA,reducing the oxidation of fatty acids and the cholesterol HDL transport mediated by ABCA1 to promote the cholesterol bile acid metabolism mediated by ABCG5.In U18666 A + NEFA group,m RNA expression levels of SREBP-2C,HMGCR,ACAT2,APOB100 were significantly increased,as well as intracellular TC,TG and lipid droplet content,while APOB100 content in cell supernatant was significantly decreased(P <0.05);The expression of ABCA1,ABCG5,ABCG8 and CYP27A1 decreased significantly(P <0.05 or P < 0.01);SOD activity decreased significantly,MDA content and ROS level increased significantly(P < 0.05),showing that U18666 A can aggravate NEFA mediated lipid deposition and oxidative stress by inhibiting intracellular cholesterol transport,metabolism,VLDL assembly and secretion.In conclusion,low concentration of cholesterol can effectively alleviate NEFA mediated lipid deposition occurred in cholesterol metabolism disorder of dairy cows under negative energy balance,promote the expression of VLDL metabolism related genes and cholesterol transport to reduce the oxidative stress of hepatocytes.