| Aims 1. To establish a rat model of nonalcoholic steatohepatitis(NASH)with fibrosis by a high-fat diet. 2. To observe the activity of Kupffer cell in the process of NASH rat model and the sensitivity of liver injury to endotoxin in the NASH rat for determining the function of Kupffer cell in NASH.3. To explore the mechanism of fuction abnormality of Kupffer cell in NASH, including the role of intestine environment abnormality, gut-derived endotoxemia, and inceasing free fatty acid (FFA) and very low density lipoprotein (VLDL).Methods 1. SD rats were chronically fed with a high-fat diet consisted of nomal diet, 10% lard oil and 2% cholesterol. All rats sacrificed at the 4th, 8th, 12th, 16th and 24th week respectively. Body weight, liver weight, serum levels of lipid and aminotransferase were measured. The degrees of hepatosteatosis, inflammation, and fibrosis of livers were investigated by HE stain and VG stain. Expressions of type Ⅲ collagen, transfer growth factorβ1 (TGFβ1),and α-smooth muscle actin(α-SMA)in liver were deteced by immunohistochemistry. 2. Hepatic expressions of lysozym and CD14 antigen were observed by immunohistochemistry and expressions of TLR4 mRNA and TNFα mRNA were detected by RT-PCR at different stage. Serum levels of TNF-α,IL-1β and IL-6 were measured by ELISA. 3. At the 24th week, high-fat model rats were given low dosage of endotoxin (0.5mg/kg) by intraperitoneal injection. Survival rate for the next 24 hours was noted. Serum levels of glucose,aminotransferase, TNF-α, IL-1βand IL-6 were examined by ELISA. Liver tissues were observed by HE stain and VG stain.Serum levels of endotoxin in rat abdominal aorta and portal vein were detected. Rats in lactulose group were given lactulose orally after 8 weeks of high-fat diet feeding and sacrificed at the 16th week. Body weight, wet liver weight, serum levels of lipid and aminotransferase were detected. Hepatic pathology was4. investigated.5. The effect of palmitic acid, oleic acid and VLDL on the activiation of nuclear factor κB(NFκB)and the sensitivity of macrophage cell line RAW264.7 to endotoxin were observed by immunohistochemistry or radioimmunoassay.Results 1. The rats fed high-fat diet developed hepatocyte steatosis at the 4th weeks, fatty liver at the 8th week, steaohepatitis from the 12th week to the 24th week. Furthermore, hepatic perisinosodial fibrosis was observed at the 24th week.2. From the 4th week on after high-fat diet, rat hepatic exprssion of CD14 was up- regulated and Kupffer cells with positive stain of lysozym were activated, and these were aggravated gradually through the whole study. Rat hepatic exprssion of TLR4 mRNA was up-regulated from the 8th week, and was most intense at the 16th week and the 24th week. Expression of TNF-α mRNA in liver was up-regulated, but kept no changes from the 8th week to the 24th week. Serum levels of TNF-α increased from the 8th week , and IL-1β from the 16th week, but IL-6 was not increased.3. The survival rate of NASH rats challenged by LPS was significantly lower than LPS alone. Serum levels of ALT and AST were increased significantly after LPS injection while the serum levels of glucose was decreased significantly. In addition, serum levels of TNF-α,IL-1β,IL-6 were also increased significantly. Pathologically, the hepatic histological activity scores of inflammatory were increased significantly, and blood cells were stasis in cluster in hepatic sinus.4. Serum levels of endotoxin in abdominal aorta were increased significantly at the 24th week, and those in portal vein were not increased from the 4th week to the 16th week. However, the serum levels of endotoxin in portal vein were higher than those in abdominal aorta at the same time-point. Administration of lactulose orally could improve the rat serum levels of aminotransferase and hepatic inflammation significantly, but had no effect on the hepatocyte steatosis.5. Exposuring to palmitic acid, oleic acid or VLDL for 24 hours (especially palmitic acid), the... |
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