Study On Nonalcoholic Fatty Liver Disease Induced By A High-fat Diet In Young And Adult Mice

Background and PurposeNonalcoholic fatty liver disease(NAFLD)is one of the most common chronic li-ver diseases,with an average prevalence of 26%worldwide.With the change of people' s diet and lifestyle,the incidence of NAFLD tends to be younger and it is more prevalent in populations with obesity or insulin resistance.NAFLD has become the most common cause of chronic liver disease in children and adolescents.Epidemiological studies have found that the incidence of NAFLD among moderately and severely obese adolescents in China is 30.14-49.85%.The widespread prevalence of metabolic diseases such as obesity,diabetes mellitus and NAFLD has brought severe challenges to people.High-fat diet(HFD)induced NAFLD mouse model has a close causal relationship with the progression of human disease,which is the most widely used model in pathological study of NAFLD.In this study,NAFLD mice models were established to detect the bodyweight,biochemical parameters and liver pathological changes,and to observe the development of NAFLD in young and adult mice induced by HFD.To explore the relationship between abnormal lipid metabolism and the early onset of fatty liver in young mice,gene array was used to detect the expression of lipid metabolism associated genes in mice liver.Furthermore,RT-PCR was used to detect the expression of inflammation-related genes,and to explore the potential mechanism of inflammation in NAFLD in young and adult mice,which provided theoretical basis and a research platform for the research and treatment of NAFLD.Methods1.Wild type(WT)C57BL/6J mice of 6-week-old were selected,and 20 females and 5 males.After 2 weeks of adaptive feeding,8-week-old female mice were mated with male mice.Male mice(3-week-old)were randomly divided into the following 3 groups:(1)HF-Y group(high fat young group):young mice were fed with HFD(40%fat,2%fructose,2%cholesterol)at 3 weeks of age;(2)HF-A group(high fat adult group):adult mice were fed with HFD after feeding normal chow diet(10%fat)until 8 weeks of age;(3)NC group(normal chow diet group)mice were fed with normal chow diet after weaning.2.The bodyweight of mice was monitored every week.The blood samples were obtained from hearts and the livers were collected at the time of sacrifice(HFD feeding for 2,4,12,16,20 and 25 weeks).Alalanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured by automatic biochemical analyzer.Total cholesterol(TC)were measured by liver cholesterol fluorometric assay kit and triglyceride(TG)were measured by triglyceride colorimetric assay kit.After fasting f-or 10 hours,intraperitoneal injection of glucose tolerance test(IPGTT)was performed,and blood was taken from tail vein for testing blood glucose.3.Livers were immediately removed,frozen and stored at-80 ?.Formalin-fixed liver were paraffin-embedded,sectioned,and stained with hematoxylin-eosin(HE).Immunohistochemical staining of CD45 antibody,F4/80 antibody and Collagen 1 alpha 1 antibody was used for evaluation of inflammation and fibrosis in mice liver.4.The expression of lipid metabolism associated genes in the liver of HF-Y group and HF-A group which fed HFD for 0 and 2 weeks was detected by gene array,including the mRNA expressions of ATP-binding cassette subfamily A member 1(ABCA1),ATP-binding cassette sub-family G member 1(ABCG1),Acetyl-CoA acetyltransferase,mitochondrial(ACAT1),Sterol O-acyltransferase 2(SOAT2),Very long-chain specific acyl-CoA dehydrogenase,mitochondrial(ACADVL),Perilipin-2(ADFP),Apolipoprotein E(APOE),Arachidonate 12-lipoxygenase,12S-type(ALOX12),Arachidonate 15-lipoxygenase(ALOX15),Arachidonate 5-lipoxygenase(ALOX5),Arachidonate 5-lipoxygenase-activating protein(ALOX5AP),Platelet glycoprotein 4(CD36),Sterol 26-hydroxylase,mitochondrial(CYP27A1),Fatty acid-binding protein,adipocyte(FABP4),Fatty acid-binding protein,epidermal(FABP5),Fatty acid desaturase 1(FADS1),Fatty acid desaturase 2(FADS2),Fatty acid desaturase 3(FADS3),Glycerol kinase(GYK),3-hydroxy-3methylglutaryl-coenzyme A reductase(HMGCR),HydroxymethylglutarylCoA synthase,cytoplasmic(HMGCS1),Tri functional enzyme subunit beta,mitochondrial(HADHB),Insulin-induced gene 1 protein(INSIG1),Interleukin-1 beta(IL-1?),Interleukin-6(IL-6),Low-density lipoprotein receptor(LDLR),Leukotriene A-4 hydrolase(LTA4H),Leukotriene C4 synthase(LTC4S),Lipoprotein lipase(LPL),Oxysterols receptor LXR-alpha(NR1H3),Cytosolic phospholipase A2(PLA2G4A),Peroxisome proliferatoractivated receptor delta(PPARD),Peroxisome proliferatoractivated receptor gamma(PPARG),Prostaglandin G/H synthase 2(PTGS2),Monocarboxylate transporter 7(SLC16A6),Long-chain fatty acid transport protein 1(SLC27A1),Long-chain fatty acid transport protein 3(SLC27A3),Sterol regulatory element-binding protein 1(SREBF1),Sterol regulatory element-binding protein 2(SREBF2),SCAR-related lipid transfer protein 4(STARD4),Acyl-CoA desaturase 1(SCD1),Thromboxane-A synthase(TBXAS?),Tumor necrosis factor(TNF)and Mitochondrial uncoupling protein 2(UCP2).RT-PCR was used to verify the expression of specific lipid metabolism associated genes in mice liver.5.The expression of inflammation genes in the liver of HF-Y group and HF-A group which fed HFD for 16 and 20 weeks was detected by RT-PCR,including the mRNA expressions of Interleukin-6(IL-6),Tumor necrosis factor-a(TNF-a),Interferon-?(INF-?)and Interferon-?(INF-?).Results1.In the first 4 weeks of HFD,the growth rate of weight in HF-Y group was higher than that in HF-A group;after 4 weeks,the growth rate of weight in HF-Y group decreased,which was nearly the same as that in HF-A group.From 3 to 13 weeks old,the weight of HF-Y group was higher than that of HF-A group.From 13 to 25 weeks old,the weight of HF-A group was higher than that of HF-Y group.The weight of HF-Y group and HF-A group was higher than that of NC group when they were the same age(P<0.05).2.Compared with HF-A group,ALT increased significantly in HF-Y group after 2 weeks of HFD(P<0.05).At 4 weeks,ALT in HF-Y group was lower than that in 2 weeks,which was nearly the same as that in HF-A group.Then,ALT increased in both groups gradually.At 25 weeks,ALT in HF-A group was significantly higher than that in HF-Y group(P<0.05).There was no significant difference in AST between HF-A group and HF-Y group in the first 12 weeks of HFD(P>0.05).At 16 weeks,AST in HF-A group was significantly higher than that of HF-Y group(P<0.05).After that,compared with HF-Y group,AST increased in HF-A group,but the difference was not statistically significant(P>0.05).3.After 2 weeks of HFD,TG was significantly higher in HF-Y group than that in HF-A group(P<0.05).At 4 weeks,compared with HF-A group,TG increased in HF-Y group,but the difference was not statistically significant(P>0.05).There was no significant difference in TG between HF-Y group and HF-A group from 12 weeks to the end of the experiment(P>0.05).Also,there was no significant difference in TC between HF-A group and HF-Y group in the first 16 weeks of HFD(P>0.05).At 20 weeks,TC was significantly higher than that in 16 weeks in both groups.Compared with HF-Y group,TC in HF-A group increased,but the difference was not statistically significant(P>0.05).4.After 20 weeks of HFD,the results of IPGTT showed that the blood glucose in HF-Y group and HF-A group was significantly higher than that in NC group(P<0.05).Compared with NC group,the area under curve(AUC)in HF-Y group and HF-A group increased significantly(P<0.05).Compared with HF-Y group,AUC in HF-A group tended to increase,but the difference was not statistically significant(P>0.05)5.After 2 weeks of HFD,hepatic steatosis was obvious in HF-Y group.At 4 weeks,hepatic fat infiltration was improved in HF-Y group,which was consistent with the degree of hepatic steatosis in HF-A group.At 16 and 20 weeks,diffuse lipid changes were observed in both groups.Compared with HF-Y group,severe macrovesicular steatosis,inflammatory infiltration and fibrosis were observed in HF-A group.6.The results of lipid metabolism associated gene expression showed that compared with the HF-A group,the mRNA expression of LPL,FABP5,UCP2 and TBXAS1 increased,while ALOX12 decreased in HF-Y group(P<0.05).7.The results of inflammation gene expression showed that compared with the HF-A group,the mRNA expression of TNF-a decreased in HF-Y group at 16 weeks(P<0.05).And the mRNA expression of IL-6 and TNF-a decreased in HF-Y group at 20 weeks(P<0.05).Conclusion1.A rapid weight gain and fatty liver occurred in the young mice soon after HFD feeding,but they became more tolerant to chronic lipid stress later.Changes in the adult mice were not as obvious as in the young mice in the early stage of HFD.In the end,the adult mice developed more severe injuries than the young mice.2.Lipid metabolism associated genes such as LPL,FABP5,UCP2,TBXAS1 and ALOX12 may be involved in the early onset of NAFLD in the young mice.3.The slow disease progression of NAFLD in young mice may be related to the decreased expression of inflammation genes such as IL-6 and TNF-?.