| Objective:Nonalcoholic steatohepatitis(NASH) is an acquired metabolic disease, coming from simple fatty liver, it will develop to hepatic fibrosis even hepatic cirrhosis with continuing inflammation, which is one of the cause of cryptogenic cirrhosis. Inhibit kappa B kinase complex(IKKs) belong to the member of serine/ threonine(Ser/Thr) superfamily, composed of tris-subunit: bis-catalytic subunit of inhibit kappa B kinase beta(IKKβ) and inhibit kappa B kinase alpha(IKKα), mono-regulatory subunit of inhibit kappa B kinase gamma(IKKγ/NEMO). IKKβis essential to activate nuclear factor-kappa B(NF-κB),which regulated the expression of inflammation related gene and anti-apoptosis gene. Latest study indicated that IKKβexpressed in the tissues of liver, colon, lung and bone, having close relation with patho-process and diseases of inflammatory reaction, apoptosis, insulin resistance, fatty liver, acute injury of liver, but the pathogenesis is not clear. Zeng reported that insulin resistance and accrescent adipose tissue stearolysis are the main point of pathogenesis in nonalcoholic fatty liver disease(NAFLD). Rosiglitazone is thiazolidinediones euglycemic agent, which can elevate insulin sensibility and improve insulin resistance by activating peroxisome proliferator-activated receptor gamma (PPARγ) in target tissue. The purpose of this study is to investigate the effection of IKKβand rosiglitazone in NASH, in order to reveal the pathogenesis of NASH and provide a new idea for clinical treatment.Methods: 38 male depuratory grade Wistar rats which weighted 140±10g, were randomly divided into normal groups of 10 rats and fat-riched diet group of 28 rats after fed normally for a week. Rats were fed with standard diet or fat-riched diet (standard diet added by 10% lard, 2% cholesterol and 5% corn oil) individually in normal group and fat-riched diet group. At the end of 16th week, 2 rats in normal group and 4 rats in fat-riched diet group were sacrificed by bloodletting at femoral vein, the liver and serum were collected. The model of NASH was set up which was confirmed by histological examination. The other 8 rats in normal groups continued to fed with standard diet.The 24 rats in fat-riched diet group were randomly divided into: control group of NASH, treatment with high dose group(4mg/kg·d) and low dose group(1mg/kg·d), with 8 rats in each group. All rats were sacrificed at the end of 28th week, serum and three piece of liver were collected. One piece of liver for chemical and histological determination. The second piece was fixed in 10% formaldehyde for Hematoxylin-eosin(HE) staining and Masson staining to observe the inflammation, steatosis and fibrosis of liver.The third piece was fixed in 4% paraformaldehyde for immunohistochemical staining to examine the expression of IKKβ. The rest liver were frezzed quickly in liquid nitrogen, then in -80℃frig to detect the expression of IKKβmRNA in the liver by RT-PCR.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 TNF-αof rat. Hematoxylin-eosin staining to observe the general pathologic changes of liver, SudanⅣstaining for hepatocyte steatosis; Masson staining for fibrosis.Immunohistochemical staining(Power Envision method) of IKKβwas used in the specimens of liver. The expression of IKKβwas calculated by multifunctional pathological image analyzer(Beijing Aerospace University). Five areas were chosen randomly from the periphery and center 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.SAS10.0 was used to process all datas, and all datas were demonstrated as x±s, Homoscedasticity test was run before one-factor analysis of variance, Student-Newman-Keulsv test was applied for the comparison between groups.Simple correlation analysis were used for correlation analysis.Result:1 The common expressions of rats:Normal rats were active,with bright hair and the weight increased gradually.The weight of rats in model group increased remarkably,while the treatment group increased slowly.At the end of 28thweek,the liver of normal rats presented brunneus and bright.The liver of model rats presented light yellow or sand yellow ,the section of liver was greasy and dim without bright, while the liver in treat rats presented buff and bright.2 The examination of biochemical markers of serum: compared to the normal group, the level of serum TC, TG, ALT, AST and TNF-αof rats in model group were significantly increased(P<0.01),while those serum biochemical indexes in treat group with rosiglitazone were significantly decreased compared to the model group (P<0.01).3 Histopathological changes and immunohistochemical test of liver: the normal hepatocyte arranged in radiation from the central veins under light microscope.There are very little collagen in central veins and portal areas with Masson staining. At the end of 28th week, all hepatocytes of the rats in model group presented moderate to severe steatosis, with the inflammation of the interlobular and portal area. Fibrosis around the hepatic sinusoids and venofibrosis could also presented in some rats. Red granula could be found in hepatocyte cytoplasm with SudanⅣstaining, and there were inflammation in different levels. In treated group, inflammation and steatosis in hepatocytes decreased markedly without fibrosis. In normal group, the IKKβexpressed weakly in cytoplasm of the hepatocyte, and the positive parts were buff and the quantitive evaluation of the expression of IKKβin liver was 6.78±8.87. Comparing with normal liver, the IKKβexpressed mainly in nuclear with the positive parts stained buffy, and the quantitive evaluation was 47.93±11.72(P<0.01) in model group. The expression of IKKβin treated group was decreased markedly compared with that in model group,especially in high dose group(20.65±5.91 to 47.93±11.72 P<0.01)。4 Correlation analysis: The expression of IKKβmRNA in each groups showed positive correlation with the TNF-αlevel in serum (r=0.940 P<0.01,r=0.762 P<0.05,r=0.973 P<0.01,r=0.947 P<0.01).The expression of IKKβmRNA in model group and high dose treated group showed positive correlation with ALT and AST level in serum(r=0.772 P<0.05,r=0.810 P<0.05,r=0.720 P<0.05,r=0.842 P<0.01).Conclusion: 1 NASH model of rats could be established by fat-riched diet. The lipid metabolism were the important pathogenesis of NASH.2 The expression of IKKβand serum TNF-αlevel in model group increased remarkably compared with normal group, and which showed positive correalation with serum ALT and AST level, and no correlation with fibrosis. It indicate that IKKβmight promote the release of TNF-α, which resulted in vacious cycle of IKKβ-TNF-αby facilitated the development of insulin resistance.3 The expression of IKKβand serum TNF-αlevel in treated group decreased markedly compared with model group, and which showed positive correlation with serum ALT and AST level. It indicated that rosiglitazone may restrain the expression of IKKβ, reduce the produce and release of TNF-α, block the infernal circle of IKKβ-TNF-α, improve the insulin resistance. It provided theory to treat NASH with PPARγagonist in clinical practice. |
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