Evaluation biochemical therapeutic effect of metformin, roziglitazon, N-Acetyl cysteine and etodalac in a model of nonalcoholic steatohepatitis in the rat

Non alcoholic Fatty Liver Disease (NAFLD) has recently gained increasing importance as the most common of all liver disorders.In Western countries it is recognized in up to 70% of autopsy series. (1) Non alcoholic Fatty Liver Disease (NAFLD) comprises a spectrum of chronic liver diseases,starting from a simple fatty liver which may progress to steatohepatitis,fibrosis and ultimately expand to liver cirrhosis.The link between obesity,insulin rezistance and Non alcoholic Fatty Liver Disease (NAFLD) and its more severe form called.Non-alcoholic steatohepatitis (NASH) remain as the major focus of investigation to enlighten the pathogenesis and the tretment of the disease . (1) Until other evidence based pharmocological therapies gain wide acceptance,diet and exercise remain the mainstay of therapeutic approach to Non-alcoholic steatohepatitis (NASH) . (1) Liver disease , is a reversible condition where large vacuoles of triglyceride fat accumulate in liver cells via the process of hepatosteatosis. Non-alcoholic steatohepatitis (NASH) is a liver disease characterized by macrovesicular steatosis, hepatocyte necrosis, inflammation, Mallory bodies, and fibrosis. (2) Whereas simple steatosis has a benign clinical course, non-alcoholic steatohepatitis is a subgroup with specific diagnostic features and a greater risk of progressive fibrosis, cirrhosis and end-stage liver disease. The two-hit theory best describes the progression from simple steatosis to non-alcoholic steatohepatitis, fibrosis and cirrhosis. These two hits consist of the accumulation of excessive hepatic fat due to insulin resistance and oxidative stress attributed to reactive oxygen species. (2)Although several predisposing factors, such as obesity, insulin resistance and type 2 diabetes, are related to the pathogenesis of non-alcoholic steatohepatitis, the exact mechanism of its progression to fibrosis and chronic liver disease is still unclear.(3) Male Sprague-Dawley rats weighing 300 ±50 gram from the Experimental Laboratory Animal Center,Dicle University, Diyarbakır were used.They were kept at a controlled temperature of 25 ± 1C° under Standard conditions (12 h dark: 12 h light cycle), fed with regular dry rat chow ad libitum, and had freely access to drinking water. Sprague-Dawley rats randomly divided into six groups (n=7 animals for each group) as following:Group 1: The control group (n=12) were fed with standard basal rat chow, until the end of the beginning of the studyGroup 2: High fat diet group (n=12)were fed with high fat diet until the end of the beginning of the study .Group 3: Metformin group (n=6) were fed with high fat diet the first four weeks. After sixteen weeks the animals treated by gavage with metformin (250 mg/kg/daily) end of the study.Group 4: Rosiglitazone group (n=6) were fed with high fat diet the first four weeks. After sixteen weeks the animals treated by gavage with rosiglitazon( 4 mg/kg/daily) end of the study. Group 5: N-acetylcysteine group (n=6) were fed with high fat diet the first four weeks. After sixteen weeks the animals treated by intraperitoneally with N-acetylcysteine (100 mg/kg/daily) end of the study. Group 6: Etodolac group (n=6) were fed with high fat diet the first four weeks. After sixteen weeks the animals treated by gavage with intraperitoneally etodolac (10 mg/kg/daily) end of the study.Standard diet was purchased from Elazığ Yem Sanayii A.Ş. (Elazığ, Türkiye) Non-alkolik steatohepatit (NASH) was brought with high fat diet (100 gram standart diet, 25 gram bütirik acid ) (4). These rats both a rat fed with standard diet and a rat fed with high fat diet examinated liver Histopathology end sixteen weeks,At the end of study, the rats were anaesthetized by ketamine (75 mg/kg) and xylazin HCl (15 mg/kg), the chest was opened and blood samples collected by cardiac puncture. Blood samples were centrifuged for 10 minutes at 1500 x g at 4 °C and the plasma was stored at ?80 °C until the analysis time.Liver cell death and inflammatory responses lead to the activation of stellat cellswhich play a pivotal role in hepatic fibrosis. Over time up to 20 percent of patients with NASH may develop cirrhosis .The livers were excised weighed and immediately frozen in liquid nitrogen , stored at ?80 °C .Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels were determined by use of Abbott Architec 16000 autoanalyser in Dicle University medical school center laboratory. MDA levels were measured with spectrofotometer(Shimadzu UV-160 A) according to the method of Ohkawa et al.The results were expressed as nmol/mg protein in tissue samples.Liver tissues of all group rats were histologically determined by using Masson?s trichrome and Hematoksilen-Eozin by light microscop in department of histology, Dicle University.COX-2, PPAR-? and PPAR-? receptors expression were detected by Western blotting in Department of Experimental Medicine Research İnstitute, İstanbul University. We obseved the study with high-fat diet may occur fatty liver and metformin and roziglitazonun are the best pharmacological agent for NASH treatment.We obseved N-acetylcysteine is a drug effective in the treatment of NASH. we investigated etodolac first the treatment of NASH. Etodolac were corrected values of statistically significant. Only ALT values did not improved. Etodolac were corrected high-density lipoprotein (HDL-C) values of statistically significant. Only triglycerides, cholesterol and low density lipoprotein (LDL-C) values did not improved.Etodolac; TNF-? levels in all groups with the control group was closest to fix only the MDA level was partial recovery.High fat diet induced a significant decrease of PPAR-? in liver lysates and only etodalac treatment partial prevented the high fat diet-induced PPAR-? degradation. On the contrary, PPAR-? expression was upregulated in liver from high fat diet fed animals, and etodalac treatment very little prevented its increase. High fat diet induced a significant increase receptör ekspretion of COX-2 and only etodalac treatment total prevented the high fat diet-induced induced a significant increase receptör ekspretion of COX-2.But etodolac in liver histology did not prevent steatosis and fibrosis.Key words: NAFLD, Metformin,Roziglitazone,N-acetylcysteine,Etodolac