| With the improvement of human living standards, the proportion of hyperlipidemic population caused by dyslipidemia is gradually increased by years, and hyperlipidemia is an important risk factor in inducing atherosclerosis and coronary heart disease (CHD). One or more abnormalities of total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-c), high density lipoprotein-cholesterol (HDL-c) in plasma could cause hyperlipidemia. Modern medicine has proved that the development process of atherosclerosis and other cardiovascular diseases could be interfered by controlling the TC level and rising HDL-c level which leads to reducing the incidence of cardiovascular disease. Large number of documents had showed that Rhizoma Coptidis (RC) has the effects of antihyperlipidemic; But now, the study about antihyperlipidemic activity of RC focused on berberine (BBR), and the other alkaloids was rarely reported. Coptisine (Cop), of quaternary protoberberine alkaloid (QPA), is one of the main active ingredients with similar pharmacological activity with BBR in RC. Thus, on this dasis, the regulation effect of antihyperlipidemic and cholesterol metabolic mechanism of Cop on HepG2 cells and zebrafish were evaluated. Zebrafish, due to transparent of larvae and a unique advantage in the metabolism of lipids was selected as the subject of animal model.The following is the main contents and results of this project:(1) In this study, based on previous research of laboratory, the effects of Cop on some key genes about cholesterol metabolism in HepG2 cells induced by different fatty acid, cholesterol and 25-OH-cholesterol was evaluated. The results showed that Cop showed a good antihyperlipidemic activity which was similar to BBR and influenced the mRNA and protein expression of HMGR, LDLR, CYP7A1, improved the cholesterol metabolism imbalance because of the high fat and high cholesterol diet. On other hand, we found the regulation of Cop on HMGCR occurs synergistic effect with unsaturated fatty acids which proved that unsaturated fatty acids have the effect on controlling the level of cholesterol. An in vitro investigation showed that Cop has potential pharmacological activity of regulating cholesterol metabolism.(2) In this study the method of zebrafish high fat model was explored, and hypercholesterolemic zebrafish were induced successfully by feeding with high cholesterol food continuously which could be used for general study of the drug screening. And the regulation of antihyperlipidemic and cholesterol metabolic activity and molecular mechanisms of Cop was explored as this model.(3) In the initial study of exploration the regulating on antihyperlipidemic and cholesterol metabolism of Cop in hypercholesterolemic zebrafish, we found that high cholesterol diet (HCD) could induce the change of zebrafish morphological, also could increase the levels of TC, TG and LDL-c and decrease HDL-c. In study of Cholesterol accumulation in zebrafish larva, we found that Cop began to slow the accumulation of cholesterol in a dose dependent manner when administration of 5 days. In adult zebrafish, four months after the administration, Cop showed good regulating activity on blood lipids, especially at 20 mg/kg dose, TC, TG and LDL-c were reduced by 44.8%, 31.5% and 42.5%, HDL-c lift by 157%(P<0.01). In addition, Cop also exhibited the activity of decreasing the thickness of caudal vein (CV) in adult hypercholesterolemic zebrafish. It indicated that Cop has a good effect on the prevention of cardiovascular disease.(4) In this study, the mRNA expression of some key-related genes involved in cholesterol metabolism (including cholesterol synthesis, use, transfer and efflux pathway) were detected by real-time quantitative PCR (qRT-PCR). The results showed that, Cop could adjust multiple genes involved in cholesterol metabolism compared with hypercholesterolemic zebrafish model group.It significantly increase the mRNA expression of Ldlr, Abca1b, Abcgl, Aibp and Cyp7a1; However, it significantly increase the mRNA expression of Cetp, Hmgcr, Srebp2 and Cypllal, Cypl7al, Hsd3bl, Star.In summary, Cop has a potential effect of regulating blood lipids and cholesterol-lowering. It not only has excellent improving action of cholesterol metabolism disorders caused by high fat or high cholesterol diet, but also has great potential intervention action of atherosclerosis and other cardiovascular diseases. The mechanism of regulation on cholesterol metabolism might through multiple targets involved in cholesterol metabolism. However, whether Cop effects on these genes directly needs subsequent experiments to verify. And zebrafish hypercholesterolemic model shaped in this study, which provides a platform on subsequent experiments about drug screening of Rhizoma Coptidis alkaloids. |
PDF Full Text Request