| Objective: Nonalcoholic steatohepatitis(NASH) is a chronic liver disease that corrected with inheritance-environment -metabolism, which is a clinical pathologic syndrome that characterized by diffuse adipose infiltration and inflammation. NASH is becoming gradually more prevalent in younger people and its incidence is increasing with the improvement of people's living levels yearly. NASH is no longer regarded as a benign condition but a high risk of progression towards more serious illness, and is considered as one of important causes of steatohepatic cirrhosis at present. However, there is no widely accepted an approach to treating NASH. Cyclooxygenase(COX) is a key enzyme involved in the conversion of arachidonic acid to prostaglandins(PGs), COX-2 comprises two isozymes, constitutive COX-1 and inducible COX-2. COX-2 is an immediate early gene product, witch is normally absent in most cells, but is highly inducible in response to various stimuli such as mitogens, cytokines, inflammatory mediators and lipopolysaccharides et al. The induction of COX-2 results in high PG levels that participate in inflammation, immune response, and tumorigenesis et al. But the expression and significance of COX-2 in nonalcoholic steatohepatitis were poorly understood. In this study, the model of rats with NASH was established successfully by fat-rich diet to detect the expression of COX-2 in the liver of rats by RT-PCR and immunohistochemistry staining and explore the mechanism of COX-2 in NASH, and provide a potential way for preventing and treating patients with NASH.Methods: 16 healthy male Wistar rats were divided into two groups randomly. Normal group (8 rats) were fed with normal diet and model group (8 rats) were fed with fat-rich diet (10% lard, 2% cholesterol and 5% corn oil). All rats were sacrificed at the end of 16th week. Serum of rats was isolated and stored at -80℃for measuring TC,TG,ALT,AST and TNF-αby an auto-biochemical analyzer(Olympus AU 2700, Japan) and enzyme-linked immunosorbent assay (ELISA) respectively. Part of livers were fixed in 10% buffered formalin and stained with hematoxylin-eosin for routine examination, with SudanⅣfor steatosis. The expression of COX-2 mRNA and COX-2 protein in the livers was detected by means of RT-PCR and immunohistochemistry staining respectively.Results: 1. The examination of biochemical index of serum:The levels of serum TC, TG, ALT, AST and TNF-αof rats in model group (2.95±0.32,0.88±0.17,96.63±14.16,156.13±14.74,48.23±3.38) were significantly increased compared to that in normal group (1.80±0.22,0.51±0.16,39.50±12.19,71.25±14.40,1.74±1.30) (p<0.01).2. Routine pathologic examination: The normal rats livers were henna and bright. The livers of model rats were greasy and dim and focal degeneration of yellow and white can be seen in them. At the end of the 16th week, the normal hepatocytes arranged in radiation from the central veins under light microscope; all hepatocytes of the rats in model group presented moderate to severe steatosis with the inflammation and a bit necrosis of the interlobular and portal areas.3. The expression of COX-2 protein in the liver of rats: Under light microscope, there is no expression of COX-2 in the liver of rats in normal group. Many big dark brown particles were observed in the tissue of liver in model group. Five visual fields of four angels and a center in every piece of section were observed the quantitative expression of COX-2 protein by multifunctional pathological image analyzer(Beijing Aerospace University). Result: The expression of COX-2 protein in model rats (0.85±0.051) was obviously stronger that in the liver of normal rats (p<0.01).4. The expression of COX-2 mRNA in the liver of rats: the expression of COX-2 mRNA was obviously stronger in the liver of model rats (0.85±0.026) than that in the model rats, (p<0.01).5. The change of NF-κB binding activity in the livers of rats with NASH was detected by EMSA: the activity of NF-κB in model group (10.12±1.34) was increased significantly than that in normal group (1.58±1.23) (p<0.01).Conclusion:1 The model of rats with NASH could be established successfully by fat-rich diet. The level of TNF-αin serum of rats with NASH obviously increased, which may induce activation of NF-κB and lead to the expression of COX-2. At the same time COX-2 also induces cytokines such as TNF-αreleased. These factors all together involved in occurrence or progression of NASH at last.2 The expression of COX-2 in the liver of rats with NASH increased obviously. The high expression of COX-2 not only leads to lipid confused but also induces inflammatory mediators especially TNF-αreleased. In sum, COX-2 plays a pivotal role in the cascade reaction of adipose deposit-inflammatory mediators released-hepatocytes injuried, and if its expression inhibited, may it be acted as a key factor in the pathogensis of NASH. It will provide a new rational data for potential drugs treating patients with NASH. |
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