| Objective:This study aimed to establish model of nonalcoholic fatty liver disease in rats with high-fat diet, By detecting content of AdipoR-2 and IMD and MDA at different time points,the relationship between oxidative stress and the change of AdipoR-2 and IMD was investigated to further explore the pathogenesis in the pathogenesis of NAFLD.Methods:Forty healthy adult male Spague-Dawlay rats were divided into two groups randomly, normal control group (C group) 20 rats, the model group (F group) 20 rats. Each group was second,4th,8th,12th week team, five in each team. The normal control group was fed with the foundation diet, the model group was fed with high-fat diet (preparation of high fat diet:2% cholesterol,0.5% sodium cholate,5% sucrose,10% lard,82.5% foundation feed). Two groups drank water freely. Five rats of both control group and model group were killed at the time of second,4th,8th,12th weekends. Blood was collected by heart and determined the rat's blood lipid and serum aminopherase in each group in order to observe the progress of liver fat change in non-alcoholic fatty liver disease model.Then the liver was taken out rapidly to weigh, the liver index was calculated as follows:liver wet weight/body mass×100%. The liver tissue samples were taken to preparate paraffin sections. The liver tissue pathological changes were observed through the hematoxylin-eosin (HE) staining under light microscope and the degree of liver fibrosis was detected. The expression of protein of AdipoR-2 were examined by immunohistochemistry;IMD assayed by ELISA;MDA content was detected by thiobarbituric acid method. Measuring data was demonstrated with mean±standard deviation (x±S).With t test, the groups were compared with variance analysis. If there was homogeneity of variance, one-way ANOVA analysis was used,and if there was heterogeneity of variance H test was used. If it was count data Chi square test was used; Relationship between the two linear correlation was analysized;P<0.05 showed statistical significance. Result:All 40 rats were entered into the final analysis.(1) blood Serum biochemical index:With the model time expanding, ALT,AST and TG,TC in the model groups were obviously high compared with normal groups.(2)observation of pathology:①Gross observation of livenrat livers were brightly red in the normal groups by naked eye;liver in the model groups were yellow discolouration, greasiness and augmentation.②observation of histology:in the normal groups, hepatic lobule structure was clear and hepatic sinusoid fibers was porous.Hepatic tissue in the model groups showed moderate to hepatic cells adipose degeneration and inflammatory changes degree gradually aggravated was significant higher than in the control group (P<0.05).With time expanding,The fibrosis performance of a small number in the later period.③The model group rats'liver could be seen with varying degrees of lobular inflammation,especially in portal areas;At second,4th,8th,12th week in the model group rat's liver inflammation activity score were higher than in control group (P＜0.05). HE staining showed that at 8th week and 12th week in model group rat liver appeared a small quantity of fibrosis at perisinusoidal space,but not be seen at second to 4th week model rats'liver.(3)IMD content in normal rats was average 180.55pg/ml, no significant changes during the experiment;but in the model groups, along with the modeling time,was gradually decreased,there were significant differences compared with the normal groups (t=11.55, P<0.001);But there is no significant difference between serum IMD and organization IMD, both in the normal group and model group (P>0.05). IMD and liver steatosis,inflammatory activity was negatively correlated (r=-0.966,r=-0.971;P＜0.01). (4)In Normal groups, AdipoR-2 staining showed the light yellow scattered at the local,but the model group showed yellow-brown, and gradually deepen over time; compared with the normal, expression of AdipoR-2 was significantly increased(P<0.001).Correlation analysis showed AdipoR-2 and hepatic steatosis,inflammatory activity was positively correlated (r=0.924,r=0.897; P＜0.01). (5)The serum MDA content in the model rats increased with the feeding time expanding, in normal group mean was 1.99±0.83nmol/ml,whereas in the model group mean was 4.75±1.82nmol/ml, differences between them was significant (t=6.77,P<0.001).Correlation analysis showed AdipoR-2 protein expression was positively correlated with MDA (r=0.823,P<0.01);in serum and tissue expression of IMD was negatively correlated with MDA (r=-0.919, r=-0.902;P<0.01);MDA and hepatic steatosis,inflammation was positively correlated (r=0.92,r=0.938;P<0.01). Conclusion:(1)through the method of modeling high-fat diet, rat model of non-alcoholic fatty liver disease is successfully constructed at the 4th week,which reduces the modeling time and the cost of experiments.The model basically coincides with human NAFLD pathogenesis. (2) in the non-alcoholic fatty liver disease of rat model, degree of oxidative stress is closely related with the liver injury, and with steatosis, inflammation increasing, the product of oxidative stress in the body content is increasing. Oxidative stress, directly or indirectly leads to liver inflammation and fibrosis, and promote the progress of the disease NAFLD.(3)During the development of NAFLD, Oxidative stress level and IMD, adiponectin receptor-2 expression levels have a significant correlation; IMD in the pathogenesis of NAFLD has been depleted and adiponectin receptor-2 expression is compensatory increased, both of them participate in the regulation of oxidative stress during NAFLD. |
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