Study On Clinical Characters Of Non-alcoholic Fatty Liver Disease And Preventive And Therapeutic Effects Of Melatonin

BackgroundNon-alcoholic fatty liver disease (NAFLD) is an increasingly recognized condition that may progress to end-stage liver disease and hepatocellular carcinoma, which ranges from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis. NAFLD is present in up to one-third of the general population and affects any age and ethnic group, making this liver disease the second leading cause of death in the general population. NAFLD has emerged as a substantial public health concern throughout the world. In China, NAFLD may soon be rivaling chronic hepatitis B as a cause of cirrhosis and become the commonest cause of chronic liver disease. To protect and treat NAFLD efficaciously can lower the incidence of end-stage liver disease and the mortality related to liver disease. However, there is lack of effective treatment for NAFLD at present. Therefore, it is of significance to explore the efficaciously pharmacological agents for NAFLD. Melatonin, a lipophilic indoleamine derived from tryptophan, is synthesized chiefly by the pineal gland but has recently been detected in many other tissues as well. It has a variety of important physiological and biochemical functions. It also has been shown to be a powerful antioxidant which scavenges reactive oxygen and reactive nitrogen species. The antioxidant properties of melatonin have been shown experimentally to protect against aging, cancer, liver injury, and atherosclerosis. Melatonin shows marked protective effects against liver injuries in experimental animals induced by a variety of agents and processes. However, there is not any report about preventive and therapeutic effect of melatonin on NAFLD until now. Therefore, we investigate the fatty liver disease incidence and its clinical characters, as well as establish rat model of NAFLD in order to investigate the effects of melatonin on NAFLD and its possible mechanism. Objective(1)To investigate non-alcoholic fatty liver disease incidence and its clinical characters.(2)To establish rat model of non-alcoholic fatty liver disease induced by high fat diet in order to observe the effects of melatonin on rat non-alcoholic fatty liver disease and investigate its possible mechanism.Methods(1)Questionnaire investigation were taken by middle-old employees in a certain enterprise between July and August 2006. It contains age, sex, history of drinking, past medical history, history of taking drugs and poisons, history of viral hepatitis. Authropometry indexes were measured on an empty stomach in early morning, including height, weight, Sbp, Dbp, BMI is calculated out by height and weight. Liver ultrasonic inspection was carried out, serum ALT, AST, TG, TC, VLDL, FPG were detected by automatic biochemistry analyzer. 78 NAFLD patients with intact clinical data and blood sample were choosed to become the NAFLD group. 78 age- and sex- matched normal subjects were choosed from all health examination subjects to become the normal control group, excluded fatty liver disease, hypertension, hyperlipemia, diabetes and other diseases, with case control study method. Fasting insulin were detected by RIA, HOMA-IRI were calculated by"IRI =(FINS×FPG)/22.5".(2)Fifty male Wistar rats were divided into five groups with ten for each randomly. The melatonin in low does, moderate does, high does group and model group were fed with high fat diet while the normal control group with common diet for 12 weeks. Melatonin ( contain≤1%dehydrated alcohol ) was intraperitoneally injected to the rats in various melatonin groups ( 2.5mg/kg, 5.0mg/kg and 10.0mg/kg , respectively ) and equal does of saline ( contain 1%dehydrated alcohol ) to control and model group once a day. After 12 weeks experiment, all rats were sacrificed. Histopathological changes in liver, liver humid weight and liver index were observed. The level of AST, ALT, glucose, insulin in serum; TG, TC, and FFA in serum and liver tissue homogenates; SOD,MDA and GSH-Px in liver tissue homogenates were measured by biochemical methods. IR was assessed by the homeostasis model assessment (HOMA) method using the formula"IRI=(FBG x FINS) /22.5". MTP activities in liver and small intestine microsome were observed by Fluorescence Lifetime and Steady State Spectrometer, the level of MTP protein in liver and small intestine were determined by western blot. The expression of CYP2E1 in liver was observed by immunohistochemical method and western blot.Results(1)453 fatty liver disease were detected in 1277 subjects, the prevalence is 35.47%(453/1277). In the total fatty liver disease patients, male is 236, account for 52.10% while female is 217, account for 47.90%. The male prevalence is obviously higher than female (P<0.01). The maximal fatty liver disease prevalence group is between 51~60 in all patients, account for 45.34%. The maximal fatty liver prevalence group is between 51~60 in male, account for 55.29%, while the maximal fatty liver prevalence group is between 61~70 in female, account for 44.68%. The fatty liver disease prevalence in male is obviously higher than female in subjects who's age≤60(P<0.01). According to etiological factor, nonalcoholic fatty liver disease patients account for 75.72%, alcoholic liver disease patients account for 13.25%, hepatitis fatty liver patients account for 5.30% and other reason caused fatty liver patients account for 5.73% in 453 fatty liver disease patients. In NAFLD group, those who accompany with two or more than two symptoms in metabolism syndrome, such as hypertension, obesity, hyperlipidemia and T2DM, account for 53 (67.95%, 53/78). And there are 26 NAFLD patients accompany with metabolism syndrome (33.33%, 26/78). Compared with the normal control group, BMI, Sbp, Dbp, ALT, AST, TG, TC, VLDL, FBG, FINS, IRI in NAFLD group is significantly increased(P<0.01). (2) NAFLD rat model was successfully established with high fat diet for twelve weeks. Compared with control group, the model group developed moderate - serious steatosis and focus infiltration of inflammatory cells and punctiform necrosis in hepatic lobules, with significant increasing liver humid weight, liver index, level of serum AST, ALT, TC, FFA, FPG(P<0.05)and TC,TG , MDA and FFA in liver (P<0.05), simultaneously with decreasing hepatic SOD , GSH-Px (P<0.05). The expression and protein level of CYP2E1 in liver was also increased. MTP activities and protein level in liver and small intestine microsome were reduced(P<0.05, P<0.001). Compared with model group, hepatic steatosis , integration of histological activity index (P<0.05), liver index and liver humid weight(P<0.05), MDA level in liver of high dose of MEL group was improved, simultaneously with increasing of SOD and GSH-PX in liver in moderate and high does of MEL group(P<0.05,P<0.01). The level of serum AST, ALT, hepatic TC,TG in high does of MEL group and the level of hepatic TC in moderate does of MEL group were lower significantly than model group(P<0.05). Compared with model group, serum FFA level in melatonin-treated groups and hepatic FFA level in a moderate or high dose of MEL group was reduced( P<0.05, P<0.01 respectively). Expression of CYP2E1 in liver of rats treated with moderate or high dose of MEL and protein level of CYP 2E1 in liver of rats treated with high does of MEL was decreased (P<0.05 ). MTP activities in small intestine microsome in high dose of MEL group , MTP protein level in liver and small intestine microsome in high dose of MEL group were higher than that in model group, respectively(P<0.05,P<0.05, P<0.01).Conclusions(1)The fatty liver disease prevalence is 35.47% in this health examination crowd, with nonalcoholic fatty liver disease accounting for 75.72%. NAFLD is closely correlated with obesity, hyperlipidemia, diabetes and metabolic syndrome.(2)Rat NAFLD induced by chronic feeding of an high fat diet is associated with the decrease of MTP activities and protein level in liver and small intestine microsome.(3)Melatonin in a dose dependent manner significantly improved liver morphology, serum transaminase,lipid and antioxidase activity in liver, depressed lipid peroxidation,expression of hepatic CYP2E1 and up-regulated MTP activities and protein level of liver and small intestine microsome in NAFLD rats induced by high fat diet, presenting the markedly protective and therapeutic effect against NAFLD. Melatonin may ameliorate oxidative stress,inhibit lipid peroxidation and interfere in lipid metabolism in liver and small intestine to protect rats treated with high fat diet against NAFLD.