The Role Of ABCG5/G8 Deficiency In High Fat Diet-induced Hepatic Steatosis In SD Rats

Background and Aims:Non-alcoholic fatty liver disease(NAFLD)is one of the most common cause of chronic liver diseases in the world.The progressive development of NAFLD can be divided at least three different stages-simple fatty liver(SFL),non-acoholic steatohepatitis(NASH)and their related cirrhosis.Steatosis refers to the abnormal accumulation of lipid droplets in the cells other than adipocytes.Severe hepatic steatosis is called fatty liver(FL),often companying with inflammation,hepatomegaly and abnormal liver function,and can be further developed into NASH or cirrhosis.Due to sedentary life style and cholesterol/fat-rich dietary,the incidence of hepatic steatosis increased significantly.ABCG5 and ABCG8 are members of the ATP-binding cassette(ABC)transporter family and are critical for hepato-enteral cholesterol secretion under physiological condition to maintain cholesterol homeostasis.It has been reported that abnormal cholesterol metabolism and free cholesterol accumulation in liver tissue is closely related to the occurrence and development of NAFLD.The role of ABCG5 and ABCG8 on sterol metabolism and NAFLD formation remains unclear in rats.This study aimed to explore the role and mechanism of ABCG5 and ABCG8 in hepatic steatosis induced by high-fat diet in SD rats.Methods:1.Systemic knockout rats of ABCG5 and ABCG8 were generated with CRISPR/Cas9 targeting technology,and confirmed by RT-PCR.2.Eight-weeks-old wild-type(WT)and ABCG5/G8 knockout(KO)rats were divided into two groups:(1)normal diet(ND)group;(2)high-fat diet(HFD)group and fed for 14 weeks.The body weight was observed and amount of food intake were recorded by TSE Phenotype Master.3.The livers were harvested for HE staining and the measurement of lipid contents.The animal serum were collected for the measurement of liver function and lipid contents.4.The mRNA expression of ABCG5,ABCG8,CYP7A1,CYP27A1,NPC1L1 and CD36 were analyzed by RT-PCR.Western blots were used to detect the expression of the proteins related to cholesterol and fatty acid metabolism such as HMGCR,NPC1L1,CD36,SREBP1,ACC,and FASN.Results:1.ABCG5 and ABCG8 are highly expressed in the liver and small intestine tissues in SD rats and not detected in heart,spleen,lung and kidney tissues.2.There were no detectable ABCG5/G8 expression in the liver and small intestine in ABCG5/G8 KO rats.3.After 14 weeks of high-fat diet,the body weight gain or hepatic lipid/cholesterol accumulation induced by high-fat diets were significantly reduced in ABCG5/G8 KO rats;and serum cholesterol levels also decreased significantly;the two groups of glucose tolerance(IPGTT)and transaminases were in the normal range.There were no significant inflammatory cell infiltration in both groups.It was suggested that pathological changes of NAFLD in the experimental rats were in the fatty phase of hepatic steatosis.4.Hepatic expressions of ABCG5,ABCG8,SREBP1,ACC,and FASN were significantly reduced after high-fat diet.Deletion of ABCG5/G8 significantly increased the expression of CYP27A1 in the liver,and significantly inhibited the expression of HMGCR,NPC1L1,and CD36 in high-fat-mediated liver comparing to WT control group.In the mean time,the expression of NPC1L1 and CD36 were significantly inhibited in the small intestine in ABCG5/G8 KO rats.Conclusion:The body weight gain or hepatic lipid/cholesterol accumulation induced by high-fat diets were significantly reduced in ABCG5/G8 KO rats.Deletion of ABCG5/G8 enhanced the expression of CYP27A1 in liver and significantly inhibited the expression of HMGCR,NPC1L1,and CD36 in liver induced by high-fat diet,there by the reduced biosynthesis and uptake of cholesterol and fatty acid in the liver and promoted excretion of cholesterol in the bile might contribute to the protective role of ABCG5/G8 decifiency under high-fat dieting condition.