| Objective:In this study,we designed experiments to examine the effects of two different diets-very high fat diet(HFD)and moderately high fat plus cholesterol diet(HFC)-on wildtype(WT)and liver-specific FOXO1/3/4 triple knockout mice(LTKO),and study the mechanisms of action of Forkhead transcription factors(FOXOs)in the occurrence and development of nonalcoholic fatty liver disease(NAFLD).Methods:FOXO1/3/4 floxed mice(WT control)and liver-specific FOXOl/3/4 triple knockout mice(LTKO)were selected as the research objects.1?Two different diets--very high fat diet(HFD)and moderate high-fat diet plus cholesterol diet(HFC)together with regulator diet(Chow)were used to feed mice for 12 weeks,and the biological characteristics of mice at baseline and in different periods were obtained.2?The liver RNA was extracted by using TRI extraction reagents,cDNA was synthesized by using Invitrogen kit.Real-time PCR was used to quantify the mRNA level of FOXO1/3/4 gene.Western-Blot assay was used to measure the changes of Foxol and Foxo3 Protein.Foxo4 protein wasn't shown here because it's difficult to confirm in the condition of liver gene knockout.3?H&E staining and Sirius-red staining were used in the liver sections.Steatosis and fibrosis were observed under the microscope in order to investigate the phenotypic characteristics of different groups.4?Blood samples were collected to detect the level of various biochemical indexes,and triglyceride in the liver was detected after it was extracted from the liver.5?TRI reagent was used to extract RNA from liver,cDNA was synthesized by reverse transcription Invitrogen kit,and Real-time PCR was used to detect the mRNA expression level associated with liver fatty metabolism,inflammation and fibrosis.6?The protein of liver tissue was extracted and Western-Blot assay was used to measure the changes of Pdgfrb and Tgfb protein in liver.Immunohistochemical staining was used to detected the expression of MPO,and TIMP-l,a-SMA protein.7?Image-J software was used for quantitative analysis of fat accumulation of liver in H&E stained slices.The degree of inflammation and the density of immunohistochemical staining of MPO,and TIMP-1,a-SMA were analyzed.Results:1?FOXO1/3/4 gene knockout in the liver was confirmed by Real-time PCR and Western Blot.It was successful in all dietary groups.Because Foxo4 protein in liver tissue was very low,we did not show here.2?Food intake by kcal/day was not significantly difference among different dietary groups.The liver weight is increased significantly after gene knockout,and the ratio of liver to body weight was increased nearly 2-fold in the LTKO mice as compared to the WT mice under the HFC dietary condition3?In all dietary groups,fat accumulation of the liver is significantly increased in LTKO mice.The HFC mice showed the worst steatosis.Sirius red staining showed that FOXO1/3/4 deficiency further exacerbated the extent of fibrosis in all dietary groups,especially in those fed with HFC,which showed a remarkable increase in fibrosis.4?Serum triglycerides levels were significantly higher in the LTKO mice compared to the WT mice among all three dietary groups,but there was no significant effect by diet within the same genotype.Serum cholesterol levels were also elevated in the LTKO mice relative to the WT mice for the chow and HFC groups.Serum ALT levels were significantly affected by both FOXO1/3/4 deficiency and diets.The LTKO mice fed with the HFC diet had the highest level of serum ALT.5?The liver fat synthesis gene Srebpl levels were significantly increased in the liver of HFC-treated mice,but they did not reach a statistical significance.The expression of fatty acid oxidation gene Cptla was significantly decreased in the liver of mice HFC-treated mice,whereas Acox2 mRNA levels were decreased in the liver of HFD-treated mice as comared to Chow-WT mice Expression of Emrl gene in the WT livers was induced by either HFD and HFC diet,whereas it was further elevated in HFC-treated LTKO mouse liver.The hepatic Ccl2 mRNA levels were elevated in the LTKO mice compared to WT mice,but they did not reach a statistical significance.Fibrogenesis genes,Pdgfrb and Tgfb,were elevated in LTKO mice and were induced by HFC diet.Collal and Timpl were induced by HFD and HFC diet,and FOXO1/3/4 deficiency further increased its expression.6?Pdgfrb,Tgfb protein were significantly increased in LTKO mice.HFC Diet can increase their expressions.Inflammatory cell infiltration NASH marker MPO staining and fibrosis markers a-SMA and TIMP-1 were analyzed by immunohistochemistry of liver tissue section.The results show that the densities were higher in the LTKO mice among three diet groups compared to WT mice,HFD and HFC diets increased the expression,whereas it was severest in HFC diet.7?The results of Image-J software quantitative analysis showed that fat accumulation and inflammation(inflammatory cell infiltration and MPO)and fibrosis(a-SMA and TIMP-1)were increased in LTKO mice,HFD and HFC diet exacerbated the changes whereas they were severest in HFC dietConclusion:Both HFD and HFC diets induced severe hepatic steatosis in the LTKO mice as compared to WT controls.However,the HFC diet led to more severe liver injury and fibrosis.At the molecular level,hepatic FOXO1/3/4 deficiency triggered a significant increase in the expression of inflammatory and fibrotic genes including Emrl,Ccl2,Colal,Tgfb,Pdgfrb and Timp1.Thus,our data suggests that FOXO transcription factors play an important protective role against the diet-induced fatty liver disease. |
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