The Role Of ABCG5/ABCG8 In Sitosterolemia Induced By Phytosterol In SD Rats

Research background: Sitosterolemia(STSL)is characterized by elevated levels and accumulation of plant sterols in plasma and tissues.Clinically,patients with younger age can present all-physical xanthoma,atherosclerosis,and hemolysis.The absence of ABCG5(ATP binding cassette transporter G5)or ABCG8(ATP binding cassette transporter G8)is thought to be closely related to its occurrence.ABCG5 and ABCG8 are members of the ABC transporter family.The main role of heterodimers is to promote cholesterol secretion and inhibit intestinal steroid absorption.This study aims to further explore the molecular mechanism of the development of sitosterolemia using the ABCG5/ABCG8-deficient SD rat model.Methods: 1.Identification of genotypes,obtaining ABCG5/ABCG8 knockout rats.2.Establish animal model of plant sterolemia: 5-weeks-old wild type(WT)and ABCG5/ABCG8 knockout(KO)rats were divided into standard feed group(LP)and high plant feed group(HP).The feeding cycle was 16 weeks,and the changes in body weight of the rats were recorded weekly.3.After 16 weeks of feeding,serum and liver,intestine,lung and other tissues were collected.Rat fecals were collected after fasting for 12 hours,and serum and liver sterol levels were measured.Tissues were embedded and stained to observe whether the tissues were damaged.4.Determination of phytosterols in serum by gas chromatography-mass spectrometry(GC-MS).5.Real-Time PCR was used to detect the expression of genes involved in bile acid metabolism pathway(CYP7A1,CYP7B1),liver enzymes related to cholesterol metabolism(HMGCR,NPC1L1,ABCG5,ABCG8,ACAT2).6.Western blotting was used to detect the expression of NPC1L1,ACAT2,CYP27A1,LXR-a and other related proteins in liver tissue.Result: 1.The results of GC-MS showed that the contents of serum ?-sitosterol,campesterol and stigmasterol in the phytosterol-fed group were 9.1,5.73,6.39 times of wild-type rats,and the number of blood platelets decreased and volume.The increase indicates that the sitosterolemic model was successfully established.2.After ABCG5/ABCG8 knockout,the body weight of the phytosterol-fed rats did not increase significantly,but the liver tissue enlargement,liver pyknosis,alveolar adhesion,lung bullae and other injuries were formed.3.Deletion of ABCG5/ABCG8 significantly reduced total cholesterol and serum free cholesterol in the liver.Phytosterol feeding reduced liver cholesterol content,but increased serum cholesterol levels;4.Deletion of ABCG5/ABCG8 significantly up-regulated the expression of genes LXR-a,CYP7A1,CYP7B1 and ABCB11 in liver tissues,and the expression of NPC1L1 in NPC1L1,HMGCR and small intestine in liver was inhibited.5.Phytosterol significantly inhibited the expression of genes LXR-?,CYP7A1,CYP7B1,ABCB11,ACAT2,HMGCR and NPC1L1 in liver tissues.Conclusion: The animal model of sitosterolemic was successfully established by using high-phytosterol diet to feed ABCG5/ABCG8-deficient rats.After ABCG5/ABCG8 knockout,the contents of the serum ?-sitosterol,campesterol and stigmasterol in the phytosterol-fed group were 9.1,5.73,6.39 times wild-type rats,and the body weight of the rats did not increase significantly,but the liver tissue tnlargement,hepatic pyknosis,alveolar adhesion,lung vesicle formation and other injuries were formed,and the serum cholesterol level was significantly increased.Tissue total cholesterol decreased;phytosterols down-regulated the expression of LXR-?,ACAT2,ABCG5,ABCG8,NPC1L1,HMGCR,CYP7A1,CYP7B1,CYP27A1,ABCB11 in liver tissue,significantly reduced the expression of ABCG5,ABCG8,NPC1L1 in small intestinal tissues,and inhibited the synthesis and secretion of bile acids in the liver tissue,the absorption and synthesis of cholesterol,and the synthesis of cholesterol esters lead to a decrease in total cholesterol in the liver,leading to accumulation of phytosterols in the body.Deletion of ABCG5/ABCG8 significantly up-regulated the expression of LXR-?,CYP7A1,CYP27A1,ABCB11 in liver tissue,promoted the synthesis and secretion of bile acid in liver tissue,significantly reduced the expression of HMGCR and NPC1L1 in liver tissue,and inhibited the absorption and synthesis of cholesterol in liver tissue.