| Objective: Nonalcoholic steatohepatis(NASH) is one of the most common chronic liver diseases. It is frequently associated with many diseases such as obesity, hyperglycosemia, hyperlipidemia, hyperinsulinism and hypertensive disease. Resistin is an adipocytokine and hyper-resistinemia could induce insulin resistance. The potential mechanism is that resistin inhibits the phosphorylation of AMP-activated protein kinase (AMPK) and promotes inflammatory cytokines activation in the liver. The expression of resistin and AMPK in the liver, the level of serum resistin and and the correlation of them with the histological manifestations of liver, insulin resistance(IR), and tumor necrosis factorα(TNFα) with NASH were investigated, to reveal the effect of resistin on the pathogenesis of NASH and to provide theory basis for the prevention and therapy of this kind of disease.Methods: Forty male Wistar rats were randomly divided into 2 groups:control group (n = 10) and model group (n = 30). Control group were fed with standard diet and model group were fed with fat-riched diet (standard diet added by 10% lard, 2% cholesterol and 5% corn oil). Then the rats were sacrificed at 8th,12th and 16th weeks by bloodletting at femoral vein respectively.The model of NASH was set up which was confirmed by histological examination.The serum and specimens of liver were collected. The specimens of liver were divided into four parts. The one part was for chemical and histological determination. The second part was fixed in 10% formaldehyde for Hematoxylin-eosin (HE) staining and Masson staining. The third part was fixed in 4% paraformaldehyde for immunohistochemical staining to examine the expression of resistin. The rest liver were frezzed quickly in liquid nitrogen, then in -80℃frig to detect the activtion of AMPKαin the liver by Western blot.Serum cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) were measured using an Olympus AU 2700 auto-biochemical analyzer. Enzyme-linked immunosorbent assay (ELISA) was used to quantitative test serum TNFαand resistin of rat. SudanⅣstaining for hepatocyte steatosis; Hematoxylin-eosin staining to observe the general pathologic changes of liver, Masson staining for fibrosis; Immunohistochemical staining (Power Envision method) of resistin was used in the specimens of liver. The expression of resistin was calculated by multifunctional pathological image analyzer. Five areas were chosen randomly from the center and periphery of each section and to calculated the average area density under 20 power object lens (the percentage of positive area to statistical area), and take the average values. Protein levels of AMPKαand P-AMPKαwere measured by using Western blot respectively.SPSS 13.0 was used to analyse all data.Result:1. The general state of rats: Normal rats were all active and had good appetite with bright hair. The weight increased gradually. At the initial stage model rats had good appetite with bright hair, too and the weight increased more quickly than the normal rats. But after the 12th week their appetite and psychosis became worse and worse with no increasing of weight and their fur had no shine.2. Serum biochemistry, circulating TNFαand insulin levels of every group: The level of serum TC, TG, ALT, AST, TNFαand resistin in the 8wks, 12wks and 16wks of model rats respectively than that in normal control (P<0.01, P<0.05) and increased significantly with the time (P<0.01).3. Histopathological changes of liver: The normal rat livers were henna and bright, but the model rat livers were more and more greasy and dim with the time. Under light microscope the normal hepatocyte arranged in radiation from the central veins with HE staining. In the 8wks, 12wks, 16wks model rats, sections showed a gradual fat and inflammatory cells accumulation in liver cells upon high-fat feeding. There was very little collagen in central veins and portal areas in the normal and 8wks, 12wks model rats with Masson staining, but in the 16wks model rats, fibrosis presented around the hepatic sinusoids and venofibrosis. With SudanⅣstaining, no red granula could be found in hepatocyte cytoplasm of normal rats and the number of the red granula increased with the time .4. The expression of resistin in the rat liver tissue: Immunohistochemical staining was performed and showed that there was no expression of resistin in the liver of normal rats. Resistin expressed weakly around the central veins of the 8wks model rats. The positive signals were observed around the central veins more and more significantly with the time, and only a litte in the portal areas.5. The activtion of AMPKαin the liver: The expression of AMPKαprotein in rats and the phosphorylation of AMPK were detected by Western blot. The ratio of P-AMPKα/AMPKαin model rats was significantly decreased with the time (P<0.01, P<0.05), lower than in the normal rats (P<0.01, P<0.05). All those showed that progress of NASH was associated with the decreasing activation of AMPK.Conclusion:1 Rat model of NASH could be developed by feeding with gradually. IR and high-fat food played important role in the development of NASH.2 The positive signals of resistin were observed around the central veins more and more significantly with the time.3 The level of serum resistin and the expression of resistin in the rat liver tissue increased significantly with the time of feeding High-fat diet, which showed positive correlation with serum TNFαlevel and the degree of hepatic inflammation and fibrosis.4 The activtion of AMPKαin the liver decreased with the time which was negative correlation with the expression of resistin. It showed that resistin participate in the pathogenesis of NASH by reducing the the activtion of AMPK. |
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