The Metabonomics Study On Nonalcoholic Fatty Liver Disease In Rats Interfered By EGCG

Recently, with the development improvement of living standards and changes of people’s living standards diet structures, More and more high-fat and high-cholesterol diet makingthe incidence of non-alcoholic fatty liver disease (NAFLD) is constantly increased, and thereby the risk of illness rapidly,whichhas become one of the three major liver diseases that have a hazard to human health. The pathological process of NAFLD pathological is complex, involving the body of multi-angle and, multi-level physiological and biochemical processes, and thereby.Tthe exact relative mechanism is still not yet clear. So far, we have not an ideal drug for the treatment of non-alcoholic fatty liver yet. The major clinical use of lipid regulating agent of its treatment.However, these drugs will increase the deposition of lipids in the liver, easy to increase the liver injury.Therefore, the security of such drugs also need further evaluation.Therefore,We trying to find an efficient, safe and natural products,from which plant resources, to improve the treatment of non-alcoholic fatty liver disease and metabolic syndrome has become a hot topic of research in recent yearsSo far, there is still no ideal drug for NAFLD treatment. The blood lipid regulating drugs have primarily been used for the clinical treatment, however, they will increase the deposition of lipids in the liver and thus may cause and aggravate liver damage. In this context, it is very important in terms of practical significance and social value to find efficient, safe and natural products from the plant resources for improving the NAFLD treatment.EGCG is a catechin monomer with polyhydricly phenol hydroxyl groupsdiverse phenolic hydroxyl catechins, which was isolated from green tea leaves., It is the highest content of catechin monomer compounds, which accounts for80%of the total catechins. Accordingly, it is the mainly active ingredients of green tea for healthcare functions and pand harmacological functions. Some studies have shown that EGCG containing has many biological activities such as antioxidant, hypoglycemic, cardiovascular protection, anti-fibrotic, anti-tumor, etc biological activity. In addition, there are a large number of studies have also shown that polyphenols could protect improve the liver from damage, reduce the fat and lose weight. But However, the relatively action mechanism was it rarely reported mechanism of action. Existing research results are made only based on the analyses by a singleseveral small molecules of biochemical index, genes or proteins levels, making it difficult to fully understand the role and means of catechins and or EGCG on the NAFLD, thereby it is also difficult to explain this complexly pathogenic and preventiveon mechanism.Metabolomics is the globalcharacteristics of through complex, dynamic changes to identify and resolve discipline.To clarify the complex system of life has a very important significance for the study of physiological and pathological state. Therefore, this study base on the high fat diet-induced liver lesions in SD rats, establish of the non-alcoholic fatty liver model.By EGCG intervention in the rat model and use of metabolomics technology research the therapeutic effect of EGCG on fat-induced fatty liver disease.From a biological system point of view, a comprehensive study for EGCG on of NAFLD lipid metabolism in rats.Efficient use of the resources of tea, to promote tea consumption market and provide scientific basis for the development of efficient, safe treatment drugs on non-alcoholic fatty liver disease and metabolic syndrome.Metabolomics has the characteristics of the whole study of the physiological and pathological state by systematicly determinating complex and dynamic changes in the researched objects, which has extremely important significance for the illustration of the complicated life phenomena. In our project, the well NAFLD rat model was established with the high fat diet and EGCG will be employed as the protective materials for NAFLD. Using metabolomics technology, the effects and targets of EGCG on NAFLD will be analyzed in the system biology levels. Then, the corresponding reaction signal pathway and molecular mechanism were further elucidated. Altogether, this study will provide some scientific bases for the high-value utility of tea resources, the increase of tea consumption, and the development of efficient and safe drugs in the NAFLD and metabolic syndrome treatment.Main research results as follows:1.The pharmacodynamic study of EGCG effecting on NAFLD rats. Feeding the rats with high-fat diet result in liver disease in SD rats,and construct the NAFLD modern. Then Fed rats model with a solution containing EGCG5weeks.At the end of the9th week, all rats were executed to detect their biochemical indexes of blood and liver,and then make pathological section of rats’livers. Result shows that:Compared with HCG,TC TG,LDL-C,HDL-C,ALT,AST,GLU and MDA in their blood in MCG all increased,but SOD decreased.After EGCG intervention,Compared with MCG group, except a slight increase of LDL-C in blood of EIG, Other indexes all trend to the direction of the HCG.Compared with HCG, the body weight,liver wet weight, liver index and liver-spleen ratio of MCG all were increased and the TC, TG, MDA of liver homogenates were increased,while SOD was decreased. After EGCG intervention, Compared with MCG, all indexes were reversed decline in the liver homogenates of the EIG.The liver slices with optical microscope test showed that the degree of hepatic steatosis and fat particles accumulation significantly reduced in EIG.2.The study of urine metabonomics in NAFLD rats after EGCG intervention. We established a analysis method of urine metabonomics based on UPLC-Q-TOF,and used it to evaluate different pretreatment methods,then filtered the best pretreatment method:400μL urine+400μL cold acetonitrile+400μL cold methanol, Vortex blending, icebath5min, then supernatant with14000rpm on4degrees Celsius, collecting supernatant at the end.After pretreatment of all samples,all the data is continuously acquired.And then run molecular feature extraction from original data, two-dimensional data matrix exportion,filter noise, alignment, matching peaks standardized and normalized etc data pretreatment.Then divide all data into three groups,carry out frequency filtering, significant analysis,fold change filter,then through ID Browser function of the MPP search database for potential biomarkers. Finally returned to the MassHunter software verify the truth of potential compound difference.Verified real difference biomarkers are as follows:succinic acid, alpha-ketoglutaric acid, hippuric acid, gluconic acid, caprylic acid, xanthine, tryptophan,flavin mononucleotide, AMP, betaine, coenzyme R.Finally, according to the differences biomarkers explort the possible pathways.3. The study of liver metabonomics in NAFLD rats after EGCG intervention.Firstly, we established a analysis method of liver homogenate metabonomics based on UPLC-Q-TOF, and used it to evaluate different pretreatment methods,then filtered the best pretreatment method:Acetonitrile, distilled water=8:2(V/V) as the extraction solvent. Tissue homogenizer on ice before the liver tissue is crushed, and homogenize sufficiently in a glass tube,then put the slurry into the ultrasound extraction10min. Finally,we put the extraction centrifuged at14000rpm for5min under4℃, collect the supernatant.And it was diluted to containing30%acetonitrile with evaporated water.All samples were treatment by the pretreatment method, and continuously get of all data.After Multicompartmental statistical analysis raw data,the result shows: Found a total of103different metabolites, of which26compounds in the metabolic pathway. The variation of difference compounds in metabolic pathways show that: Exogenous glycerides and cholesterol increased, endogenously synthesis of TG also increased in hepatocytes of MCG.At the same time,the function of fatty acid beta-oxidation weakened in the mitochondrial. It result in the deposition of fat in liver cells, and finally lead to fatty liver.After EGCG intervention, many endogenous metabolites in the pathway reversely decline.The results showed that:EGCG could inhibit endogenous TG synthesis, enhance the beta-oxidation of fatty acids, which can reduce the deposition of fatty liver, thereby therapy NAFLD.