| Background and aim: Nonalcoholic fatty liver disease(NAFLD)is currently the most common chronic liver disease.It is defined as a type of liver disease without excessive drinking or other causes of liver disease(including autoimmune,drug-induced or viral hepatitis)),a clinicopathological syndrome characterized by steatosis and fat accumulation in more than 5% of liver parenchymal cells.It is estimated that the global prevalence of NAFLD is 25%,and it continues to rise globally.This increase has caused widespread concern because NAFLD is usually a progressive disease that may be associated with major complications such as liver cirrhosis,hepatocellular carcinoma.In view of the fact that there are currently no approved drug interventions for the treatment of NAFLD,the focus has been shifted to the study of the pathogenesis of NAFLD.In this study,we established a mouse model of non-alcoholic fatty liver,explore whether lanosterol synthase has a protective effect on non-alcoholic fatty liver,and prelimina ry explore the possible molecular mechanism,in order to clarify the role of lanosterol synthase in non-alcoholic fatty liver disease.Methods:(1)MCD feed diet was used to induce mouse model with NAFLD.40 SPF male 8-week-old mice with wild type(Wid e type,Wt),LSS knockout mice(LSS + /-,KO),body weight(24±2)g,randomly divided into four groups and given separately Normal diet control(NC)or methionine-choline-deficient(MCD),the four groups are named N-wt,N-ko,M-wt,and M-ko groups,10 animals in each group.Feed the mice continuously for 2 weeks,weigh the mice every day,and monitor the dietary data dynamically.During the experiment,observe the general condition of the mice.After the experiment,the mice were anesthetized and weighed;serum was collected;liver was taken and weighed to calculate liver index;serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),serum and liver homogenize the triglycerides(TG)and total cholesterol(TC)were measured,and conduct pathological analysis of the liver.(2)Hematoxylin-eosin staining,oil red O staining and electron microscopy were used to detect live cell structure and the lipid droplets in the liver of mice;Sirius red staining was used to detect liver fibrosis;mass spectrometry metabonomics methods were used to detect lipid compotents in liver tissues.gene chip method to detect the genes of differnce expression in liver;western blot(WB)method to detect the expression of fatty acid oxidation and bile acid synthesis related proteins,and the expression of SREBP2 protein and p AKT signaling pathway related proteins.Results:(1)General situation of mice: during the feeding process of MCD,the weight of the normal control group mice continued to increase,and there was no significant difference between the N-ko and N-wt groups.The body weight of the experimental group mice gradually decreased,and there was no significant difference between the Mko and M-wt mouse groups.(2)The results of mouse liver tissue staining showed that the liver of the N-wt group and the N-ko group has normal tissue structure,and the hepatocytes were arranged in a cord with the central vein as the center;oil red staining showed no obvious lipid droplets exist.The lipid droplets in the M-wt group were large,with obvious space-occupying,cell necrosis and inflammatory cell infiltration.while the lipid droplets in the M-ko group were significantly smaller,with no significant spaceoccupying,and no obvious cell necrosis and inflammatory cell infiltration.The Nash score of the M-wt group was significantly higher than that of the M-ko group(p<0.05).The sirius red staining showed no obvious fibrotic changes in four groups.(3)The results of serum transaminase showed that there was no significant difference in serum ALT and AST between the N-wt group and the N-ko group;the level of ALT and AST in the serum of the M-wt grou was higher than the one in M-ko group.(4)Serum and liver lipid analysis results showed that the serum TG of the N-wt group was higher than that of the N-ko group(p<0.05),and there was no significant difference in the levels of CH,HDL-C,and LDL-C;the M-wt group the serum TG level was significantly lower than the M-ko group(p<0.05)and the serum HDL level has the opposite change.The liver TG level of the N-wt group was lower than that of the N-ko group(p<0.05);the liver TG and TC levels of the M-wt group were significantly higher than those of the Mko group(p<0.01).(5)Transcriptome analysis of mouse liver found that endoplasmic reticulum protein processing,sterol anabolism,peroxisomes,circadian rhythm,glutathione metabolism,apoptosis and bile acid metabolism are different between the N-wt group and the N-ko group;comparison between the N-wt group and the M-wt group found that there are differences in oxidative phosphorylation,sterol hormone synthesis and metabolism,peroxisomes,glutathione metabolism,PPAR signal pathway,cholesterol metabolism,fatty acid metabolism,primary bile acid synthesis met abolism and other processes;there are differences between N-ko group and M-ko group in retinol metabolism,fatty acid degradation,cholesterol metabolism,glutathione metabolism,PPAR signaling;comparison between the M-wt group and the M-ko group found that there are differences in processes such as oxidative phosphorylation,fatty acid degeneration,peroxisomes,PPAR signaling pathway,primary bile acid synthesis and p53 signaling pathway.(6)Real-time PCR analysis showed that the expression levels of 14 genes related to fatty acid degenaration in the M-ko group were significantly up-regulated compared with the M-wt group;the expression of 9 genes related to bile acid synthesis in the M-ko group were significantly increased compared with the M-wt group.(7)The results of liver metabolome analysis found that the main lipid components of N-wt and N-ko livers were not significantly different between the two groups;the Percentage and absolute contents of TG and CHE in the M-wt group were significantly higher than those in the M-ko group,while the PC The result of PE is the opposite.(8)Analysis of the results of liver electron microscopy revealed that the mitochondrial structure of hepatocytes in the N-wt group and the N-ko group was intact,and there was no significant difference between the two groups;the mitochondrial damage of the liver cells in the M-wt group was more serious than that in the M-ko group;The lipid droplets in liver cells of the M-wt group was lager than that of the Mko group.(9)Western-blotting results found that the expression levels of ACSL1,CPT1 B protein related to fatty acid oxidation and CYP27A1,SLC27A5 protein related to bile acid synthesis were significantly higher in the M-ko group than in the M-wt group;The content of the active form of SREBP2(SREBP2 55KD)in the M-wt group was significantly lower than that of the M-ko group and the content of the inactive form(SREBP2 126KD)in the M-ko group was significantly lower than that of the M-wt group;The content of p AKT was significantly lower than that of the M-wt group;while the content of p AKT was significantly higher than that of the M-ko group.Conclusion: LSS gene heterozygous knockout protects MCD-induced non-alcoholic fatty liver disease(NAFLD)in C57 mice,and its protective mechanism is partly dependent on promoting fatty acid β-oxidation and promoting the conversion of cholesterol to bile acids. |
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