Study On The Dynamic Role Of Cholesterol In The Occurrence And Development Of NASH Induced By High Fat And High Cholesterol

Objective:To investigate the dynamic role of cholesterol in the occurrence and development of non-alcoholic steatohepatitis(NASH)induced by high fat and high cholesterol.Methods:Animal level: There were 100 SD rats randomly divided into the normal diet(CON)group,20% fat(HFC0)group,20% fat+1% cholesterol(HFC1)group,20% fat+2% cholesterol(HFC2)group and 20% fat +5% cholesterol(HFC5)group,with 20 rats in each group.After 4,6,8 and12 weeks of feeding,5 rats were sacrificed in each group,and their abdominal aortic plasma and liver specimens were collected.Cell level: Normal human liver cells(HL-7702)were induced by oleic acid(OA)and low-density lipoprotein cholesterol(LDL-C)to construct the NASH model.The experiment was divided into CON group,OA group and OA+ LDL-C group,culture media and cells were collected after 5 days of cell induction.The collected animal plasma and medium were used to detect the activity of ALT and AST,the contents of total cholesterol(TC)and triglyceride(TG)were detected after liver and cells were collected.Hematoxylin-eosin(HE)staining was used to observe the steatosis,balloon-like degeneration and inflammatory response of the liver.The fibrous tissue deposition of liver was observed by Masson trichrome staining.The number and size of lipid droplets and the distribution and content of cholesterol in hepatocytes were observed by Oil red O staining and Filipin staining respectively.The protein expression levels of nSREBP-2,LDLR and HMGCR were detected by Western blot.Results:Animal level: After 12 weeks,plasma ALT,AST,liver TC and TG in HFC2 group and HFC5 group were significantly higher than those in CON group(P<0.05),hepatic steatosis,balloon like degeneration and fibrosis were obvious in HFC2 group and HFC5 group,and NASH induction was successful in HFC2 group and HFC5 group;On weekends from 4 to 8,LDLR content of HFC2 group and HFC5 group increased significantly compared with CON group(P < 0.05),at the end of the 12 th week,LDLR content in HFC5 group was significantly lower than that in CON group(P < 0.05).There was no significant difference in the expression of n-SREBP-2 in each group at the weekend of 4 to the weekend of 8,at the weekend of 12,the content of n-SREBP-2 in HFC2 group and HFC5 group increased significantly compared with CON group(P < 0.05).There was no significant difference in HMGCR protein expression in each group at the weekends of 4,6 and 12,and the HMGCR content in HFC5 group was significantly decreased compared with CON group at the weekend of 8(P < 0.05).Cell level: Compared with the CON group and the OA group,the intracellular cholesterol and lipid droplet contents in the OA+ LDL-C group increased significantly,the lipid droplet volume increased significantly,and the contents of n-SREBP-2 and HMGCR increased significantly(P<0.05),the content of LDLR in OA group and OA+ LDL-C group was significantly lower than that in CON group(P<0.05).Conclusion:(1)Cholesterol intake and duration were positively associated with NASH liver injury.(2)During the occurrence and development of NASH induced by high fat and high cholesterol,cholesterol can increase the expression of LDLR in liver cells,promote cholesterol to enter the liver,and inhibit the synthesis of endogenous cholesterol regulated by HMGCR.(3)With the increase of cholesterol accumulation in liver,LDLR expression in liver cells decreased,making intrahepatic cholesterol relatively insufficient,the feedback regulated the increase of n-SREBP-2 and HMGCR expression,thereby breaking the liver cholesterol metabolism homeostasis and promoting the occurrence and development of NASH.