The Basic Research Of Low Molecular Weight Heparin Attenuating Non-Alcoholic Steatohepatitis Caused By High-Fat Diet In The Rat

Objective:To make and study the rat model of steatohepatitis and hepatic fibrosis induced by high-fat diet, in the period of nonalcoholic hepatitis, using low molecular weight heparin to intervene, and in order to supply the model of hepatic fibrosis for further study of low molecular weight heparin.Methods:Model establishment phase:Male Sprague-Dawley rats (N=60) were randomly divided into control group (n=30) and model group (n=30), rat in the control group were fed with common forage, and those in the model groups were given high-fat diet. Those rats were sacrificed at 4,8,12week during the experiment respectively. The serum-markers were measured. The degrees of hepatic steatosis, inflammation and fibrosis were observed with H-E staining and Masson staining. Moreover Masson staining was also performed in order to run semi-quantitative analysis with image analysis system. Low molecular weight heparin intervention stage:Male Sprague-Dawley rats (N=40) were randomly divided into control group (n=10) and model group(n=10). The model group was given high-fat diet for 8 weeks. The others were divided into two groups after 8 weeks high-fat diet:one was received low doses of low molecular weight heparin (50IU/kg/d) for 2 weeks; the other was received high doses of low molecular weight heparin (200IU/kg/d) for 2 weeks, in this period they were all fed with high-fat diet.Results:Model establishment phase:the two groups were all increased in size and weight. After 8 weeks high-fat diet, the index of hepatic (liver weight/body weight%) were increased obviously, serum cholesterol and triglyceride had significant statistical difference with control group at the 8th week and the 12th week. Aminotransferase in model groups all increasing in different times had statistical difference with control group at the 8th week and 12th week. After 4 weeks high-fat diet, in H-E staining we could observe that the structure of hepatic lobules were integrity, inflammatory cells infiltration was rare in portal area. Eight weeks, the rat liver tissue was disordered in structure using light microscope, the fatty liver cells is 1/3-2/3 in total liver cells, moreover piecemeal necrosis were found in liver tissue and in portal area mononuclear cell was infiltrated, inflammation scoring was markedly higher than those in normal controls group. After 12 weeks high-fat diet, the rat liver tissue achieved mild to severe steatosis, and liver cells turgescence, lobular inflammation was produced, inflammatory cells with mononuclear nucleus cell infiltrates primarily, as the center of central vein had necrotic strip outspread. After 12 weeks, fibrosis area was increased, hepatic lobule was disappeared, fibroplasia was increasing in perisinusoidal space which hardly full of it. Hyperplasia of fiber was formed rough note around the portal area and outspreaded to among hepatic lobule, inside of hepatic lobule, the central of hepatic lobule. Fibrosis smiquantitative Masson staining showed that the fibrosis area of model group enhanced greatly, compared with normal control group had statistically significant difference. Low molecular weight heparin intervention stage:After treatment of LMWH, the hepatic lobule was more completed in structure, liver cells regenerated significantly. Although it has fatty degeneration cells, the fatty degeneration and inflammatory cells was reduced compared with model group. In the improvement of fatty degeneration and inflammation, the high doses LMWH had a notable effect. After two weeks treatment, low doses of LMWH (50IU/kg/d) attenuating lipids deposits in the liver was weak, had no statistical difference with model group. However, compared with model group, the serum triglyceride and cholesterol has decreased remarkably by using high doses LMWH. The level of TNF-a in model group was increased compared with normal control group and had statistical difference. Compared with model group, after the treatment of LMWH, the level of serum TNF-a were all reduced, and showed a dose-dependent manner.Conclusions:1. The model group fatty degeneration at 8week achieved fatty liver disease diagnosis standard, and supplied the materials for the effect of low molecular weight heparin2. The rat model of non-alcoholic steatohepatitis/hepatic fibrosis has been established successfully by a high-fat diet after 12 weeks high fat diet.3. Low molecular weight-heparin has an excellent therapeutic effect for NAFLD by inhibiting chronic liver inflammation and reducing lipids deposits in the liver. The low molecular weight heparin may regulate blood fat level and decrease the level of TNF-a in serum.4. The effect of low molecular weight heparin is closely related to the dosage. Compared with low doses (50IU/kg/d) group, high doses (200IU/kg/d) of low molecular weight heparin could inhibit chronic liver inflammation and attenuate lipids deposits in the liver, significantly.