Studies On The Pathological Evaluation, Selection Of Noninvasive Diagnostic Markers, Pathogenesis And Drug Intervention Of Nonalcoholic Fatty Liver Disease

ObjectiveLiver biopsy is considered the gold standard for diagnosing nonalcoholic liver fatty disease(NAFLD),but to date, there is no standard histological scoring system for NAFLD and the pathological features of NAFLD need to summarize. Due to the potential risk of liver biopsy, it is necessary to select the noninvasive markers to diagnosis or predict the NAFLD. The mechanism of NAFLD, especially the nonalcoholic steatohepatitis(NASH), remains unclear. As a result, there is no effective treatment for NASH. In this study, we summarize the pathological features of NAFLD in China; explore the corrective NAFLD histological scoring system; select the noninvasive diagnostic markers for NAFLD; study the pathogenesis of NAFLD and use drug to intervene NASH.MethodsThe specimens of liver needle biopsies from 130 cases patients with NAFLD were underwent histopathological analysis based on a histological scoring system for NAFLD designed by the Pathology Committee of NASH Clinical Research Network (NASH-CRN). The sera that matched the liver biopsies were collected and tested in laboratory. The results were statistically analyzed according to the pathologic results. Animal model of NASH induced by high-fat diet was established. Routine histological stain, histochemistrical stain, immunohistochemistrical stain and in situ hybridization stain combined with sera detection by enzyme linked immunosorbent assay (ELISA) and biochemistry test were used to determine the roles of adipocyte-derived hormone(leptin and adiponectin), cycloxygenase(COX), angiotensin II (AT-II), tumor necrosis factor alfa (TNF-α) and transforming growth factor beta 1 (TGF-β1) in the pathogenesis of NASH. The therapeutic effect of chitosan to NASH was also to be determined compare with vitamin E.Results1. Hepatic steatosis, lobular inflammation, hepatocyte ballooning and fibrosis were presented commonly in 130 cases NAFLD liver tissues. Furthermore, macrovesicular steatosis predominantly located in acinar zone 3 was the main histological feature of NAFLD, and lobular inflammation was usually mild. Hepatocyte ballooning was observed in 94.6 percent of all 130 cases tissues. Mild perisinusoidal fibrosis and periportal fibrosis were often observed in stage 1. According to the statistic analysis, hepatic steatosis was positively correlated with lobular inflammation, hepatocyte ballooning and fibrosis(r=0.587, 0.488, 0.374, respectively, all P value<0.01). The number of microgranulomas, lipogranulomas and apoptotic bodies increased following severity of steatosis, lobular inflammation and fibrosis. Meanwhile, the number of megamitochondria and glycogen nuclei was parallel to the degree of hepatocyte ballooning (P value all<0.01).2. The main abnormal serum markers of NAFLD included: overweight or obesity (according to the standard of BMI recommended by the World Health Organization), elevated aminotransferase, hypertriglyceridemia or hypercholesterolemia, increasedγ-glutamyltransferase (GGT) and high level of alkaline phosphatase(ALP). In addition, elevated serum phosphonium was presented in 14.6 percent of NAFLD patients. There were significant correlations between the main pathological changes and age, BMI, alanine aminotransferase(ALT), aspartate aminotransferaseherat(AST), heart rat, globulin, serum phosphonium, MB isoenzyme of creatine kinase, hypertension, ALP and lactate dehydrogenase(LDH), other than the levels of triglyceride and fasting serum glucose(P value all >0.05), according to the statistic analysis. Only BMI and AST had the obviously statistic significance(P value was <0.01 and <0.05, respectively) in the regression equation according to the Logistic regression results. The regression equation is NASH=0.365BMI+0.012AST-9.289.3. Classic changes of NASH, such as increased liver/body weight, mixed macrovesicle and microvesicle steatosis, hepatocyte ballooning, lobular inflammation, and proliferation of sinusoidal cells were presented in rat liver after feed with high-fat diet four weeks. The changes of the liver fibrosis were showed obviously after high-fat induced for twelve weeks. The serum levels of total cholesterol(TC), low-density lipoprotein(LDL), ALT, AST, ALP, and GGT were increased in NASH rat . But the levels of high density lipoprotein(HDL) and the ratio of albumin to globulin were decreased. The ELISA detection results showed that the levels of insulin, leptin, and TNF-αof NASH rat were increased significantly. The level of leptin, paralleled with the levels of insulin and TNF-α, increased gradually followed the NASH development and the liver fibrosis stage. In contrast, the level of adiponectin downregulated gradually followed the NASH model building time. The immunohistochemistry and in situ hybridization stain showed the proteins and mRNAs of leptin receptor (OB-Rb) and angiotensin II receptor 1 (ATR-1) were increasingly expressed significantly in NASH rat liver tissues and NASH patients liver tissues. The proteins and mRNAs of COX-1 and COX-2, which expressed very mildly or no expressed in the normal liver tissues, were increasingly expressed in significantly in NASH rat liver tissues and NASH patients liver tissues.4. After treatment with chitosan, the fatty enlarged liver reduced and the histological changes of NASH such as hepatocyte fatty degeneration, hepatocyte ballooning, lobular inflammation, perisinusoidal and perivenous fibrosis were definitely improved in experimental NASH rats. The ratio of albumin to globulin return to normal, and the elevated levels of TC, LDL, insulin and leptin were decreased. Chitosan was more effective than vitamine E in decreasing the levels of insulin and leptin and in increasing the level of adiponectin in NASH rat.Conclusions1. The role of hepatic steatosis, lobular inflammation, hepatocyte ballooning andfibrosis should be emphasized in pathological diagnosis and evaluation of NAFLD. It needs to further verify that if microgranulomas, lipogranulomas and apoptosis bodies could be used as histopathological markers for prediction of NAFLD.2. The noninvasive diagnostic markers associated with main pathological changes of NAFLD include: age, BMI, ALT, AST, heart rat, globulin, serum phosphonium, MB-CK, hypertension, ALP and LDH. We built elementarily a NASH diagnostic equation.3. We established successfully NASH model by feeding rat high-fat diet. The features of this NASH modle include fatty liver, inflammation, fibrosis, hyperlipodemia, hyperinsulin and biochemical evidences of NASH. Leptin resistance could occur in system and liver in NASH. Hyperleptinemia might play important roles in the development of inflammation and fibrosis of NASH. Adiponectin might be an inhibitor of TNF-αand could relieve the insulin resistance and leptin resistance. AT-II participates in the procedure of liver fatty degeneration and fibrosis combined with ATR-1 in liver. COX-1 and COX-2 play important roles in the pathogenesis of NASH4. Chitosan administration intervenes effectively in the development of NASH. The mechanism of chitosan exerts it's action may be mediated by influencing the secretion of adipokin.