| Objective:Male Wistar rat model of non-alcoholic fatty liver disease (NAFLD) was established by fat-riched diet, serum leptin concentration, the expression of leptin receptor in the liver and the correlation of them with the histological manifestations of liver were assayed to delineate the effect of leptin on the pathogenesis of NAFLD and to provide theory basis for prevention of the disease. Methods:40 male Wistar rats weighed 200g±15g were divided into two groups of 20 rats each randomly after fed normally for a week. Normal group fed with standard diet and fat-riched diet group fed with standard diet added by 10% lard and 2% cholesterol. All rats were sacrificed at the end of the 20th week. 5ml serum was isolated for serological tests and three pieces from right lobe of liver for histological examination. One piece went through quickly freezing slice for SudanⅣstaining to observe steatosis of liver. The second piece was fixed in 10% formaldehyde for Hematoxylin-eosin (HE) staining and Masson three-ply staining to observe the inflammation, steatosis and fibrosis of liver. The last piece was fixed in 4 % paraformaldehyde for immunohistochemical staining to examine the expression of leptin receptor. Serum fasting blood glucose (FBG), triglyceride (TG), free fatty acid (FFA) and fasting insulin (FINS) were measured using an Olympus AU 2700 auto –biochemical analyzer, colorimetric method and radio-immune assay respectively. Caculate insulin resistance index (IRI) according to HOMA model, IRI =(FINS×FBG)/22.5. Enzyme-linked immunosorbent assay (ELISA) was used for quantitative measurement of serum leptin of rat. Hematoxylin-eosin staining was for the general pathologic observation of liver; SudanⅣstaining for hepatocyte steatosis which was positive when the red granules were found in hepatocyte cytoplasm; Masson staining for fibrosis. Immunohistochemical staining (SP method) was used to measure the expression of leptin receptor of liver. The activity of inflammation was determined by scores from the inflammation of portal area (P), inflammation of interlobular (L), piecemeal necrosis (PN), bridging necrosis (BN). Every item scored 1 to 4 according to the degree of their pathologic changes. The formulation of score was P+L+2PN+2BN. Plus scores of all kinds of pathological changes got the total score. The percentage of steatosis, collagen and the expression of leptin receptor was calculated by multifunctional pathological image analyzer (Beijing Aerospace University). 5 areas were chosen randomly from the periphery and center of every section and the average area density (the percentage of positive area to statistical area) were calculated under 20 power object lens.SAS8.0 was used to process all data. Quantitative data were expressed as x ±s. T-test was for the comparison between two groups. Simple correlation and linear-regression analysis were used for correlation analysis. Result:1 The general manifestation of rats:Compared with normal group, the weight and liver index of the rats in model group were remarkably high(.P<0.01). At the end of 20th week, the liver of normal rats present mahogany and bright. But the liver of model rat present yellowish brown and was cohesive to adjacent tissue, the section of liver was greasy and dim. 2 Serological changes:The serum leptin, triglyceride, free fatty acid and insulin resistance index of model rats were higher than normal rats (p<0.01). 3 Pathologic changes:The hepatocytes of the model rats presented modest to severe big bubble steatosis. Red granules could be found in hepatocyte cytoplasm with SudanⅣstaining. There were different stages of inflammation and fibrosis in the livers of model rats. The quantitive evaluation of steatosis, inflammation, fibrosis were 40.57±7.77, 3.39±0.87, 4.10±0.55 respectively in model rats and 1.16±0.25, 0.65±0.28, 2.01±0.53 in normal rats. The indexes of model rats above were high comparing with normal rats (P<0.01, P<0.01, P<0.05 respectively). The leptin receptor expressed mainly in membrane of the hepatocyte. The positive parts were brownish yellow and the quantitive evaluation of the expression of leptin receptor in liver was 39.46±7.06 in normal rats. Comparing with normal liver, the color of positive cells was light and thequantitive evaluation was 14.52±4.63 (P<0.01). 4 Correlation analysis:In model rats the serum leptin concentration correlated with steatosis, inflammation positively, r = 0.80 (P<0.01), r = 0.68 (P<0.01), but not with fibrosis, r = 0.24 (P>0.05). The expression of leptin receptor of model rats livers correlated with serum leptin concentration, free fatty acid, insulin resistance indexes, steatosis, inflammation negatively, r = -0.83 (P<0.01), r = -0.71 (P<0.01), r = -0.65 (P<0.01), r = -0.83 (P<0.01), r = -0.87 (P<0.01), but not with fibrosis, r = -0.32 (P>0.05). Stepwise multiple regression analysis showed that the decrease of expression of leptin receptor in liver was an independent risk factor for steatosis. The regressive equation was Y=60.78-1.39X1. (Y: steatosis; X1: the expression of leptin receptor). The coefficient of determination (designated as R2) was 0.686. Conclusion:1 NAFLD model rats could be established by fat-riched diet. The lipid metabolism disorder and the disturbance of carbohydrate metabolism were the important pathogenesis of NAFLD. 2 The serum leptin concentration of model rat was higher significantly than normal rat and associated with steatosis, inflammation positively, not with fibrosis. It was shown that hyperleptinemia involved the pathogenesis of NAFLD and the leptin concentration reflected the degree of steatosis and inflammation. 3 The expression of leptin receptor in model group was decreased significantly, which impaired the effect of leptin on the metabolism regulation of liver and became one of important pathogenesis of leptin...
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