| Obesity is a “modern civilized disease” caused by excessive fat accumulation.Nowadays,it has become one of the important factors endangering human health.Obesity is often accompanied by insulin resistance and chronic inflammation,which further leads to metabolic syndrome,and of which nonalcoholic fatty liver disease(NAFLD)is the liver manifestation.With the development of human society and the rapid growth of obese population,NAFLD has become the most widespread chronic liver disease in most regions of the world.At present,the research on the pathological mechanism of NAFLD is extensive,but the more comprehensive study on diet-induced NAFLD is still lacking.In this study,the NAFLD model was established by imitating human eating habits and induced by high-fat and high-fructose syrup diet,and the pathological mechanism was discussed by using multi-group methods.The main results are given as follows:(1)The NAFLD model of SD rats was successfully established by feeding with high fat diet(D12492)with 60 % fat energy and 12.5 % high fructose corn syrup(45%glucose + 55%fructose)drinking water for 13 weeks.In comparison with the control group(Cont),the body weight and adiposity of the high-fat and high-fructose syrup diet group(HFF)were significantly increased.The cell size of epididymal and inguinal white adipose tissue in HFF group was bigger than Cont.The area under the blood glucose curve of OGTT was significantly increased.The liver wet weight increased significantly.There were a large number of lipid droplets in the liver tissue and the level of TG,TC and ALT were increased significantly.The factor of oxidative stress including MDA,GSH-Px and total SOD were changed.The levels of inflammatory factors such as IL-6 and IL-1β in liver tissue increased significantly,indicating that consuming of high-fat and high-fructose corn syrup diet for 13 weeks could cause non-alcoholic fatty liver in rats.(2)Non-targeted metabolic analysis of liver tissue and intestinal contents of SD rats was carried out by GC-MS,and 36 and 51 differential metabolites were screened respectively between the two groups,indicating that high-fat and high-fructose corn syrup diet influenced the metabolic pattern of liver and intestinal in rats.By using IPA analysis,it was found that the function and pathway of differential metabolites in liver tissue and intestinal contents of the two groups were mainly related to glucose and lipid metabolism(Akt-PI3 K pathway)and inflammation(NF-κB pathway).(3)GC-MS was used to detect the short-chain fatty acids in the feces of rats.The results showed that the level of short-chain fatty acids of the HFF group were significantly lower than that of the Cont group.The 16 S rDNA sequencing technique was used to detect the intestinal microflora.The results showed that the diversity of intestinal microbial decreased,the relative abundance of Bacteroidetes and Bacteroides increased,and the relative abundance of Firmicutes decreased in HFF group.The relative abundance of Bacteroides,Blautia and Parasutterella increased significantly,while the abundance of Prevotellaceae-Ga6A1-group,Roseburia and LachnospiraceaeNK4A136-group decreased significantly at the genus level.(4)A total of 473 differential genes(217 up-regulated genes and 256 down-regulated genes)were screened by transcriptional analysis of rat liver tissue by RNA-seq technique.According to the pathway enrichment analysis of differential genes,it was found that lipid metabolism and inflammatory signal pathway were the main related pathways,including cholesterol,fatty acid biosynthesis pathway,fatty acid metabolism pathway,glyceride metabolism pathway and TNF-α signal pathway.According to the statistics of the differential genes in the main pathways,it was found that the expressions of Akt2,mTOR,SREBP2,Tm7sf2,FASN,ACC,ACSL1,CPT1/2 and other genes related to liver lipid metabolism were significantly down-regulated in rat liver.(5)Based on the results of multi-omics,it was suggested that high-fat and high-fructose syrup diet might cause lipid metabolism disorder and inflammation in liver by affecting Akt-SREBP pathway,ACSL1-CPT1a-CPT2 pathway and LPS-TLR4 pathway.Q-PCR,WB and ELISA were used to determine the key factors in the pathway,such as Akt,p-Akt,SREBP-1,SREBP-2,ACSL1,CPT1,CPT2,LPS,TLR4 and so on.In the model group,the Akt-SREBP and ACSL1-CPT1a-CPT2 pathways in liver tissue were inhibited,and the levels of LPS and TLR4 were significantly increased.To sum up,in this study,GC-MS non-targeted metabolome,16 S rDNA genome and transcriptome were used to analyze the pathological mechanism of NAFLD rats induced by high-fat and high-fructose corn syrup diet.It was suggested that long-term high-fat and high-fructose corn syrup diet reduced lipid synthesis,inhibited fatty acid degradation,and induced inflammation by changing intestinal flora,increasing liver LPS levels and activating TLR4,finally promoted the process of NAFLD. |
