Establishment Of Hyperlipidemic Zebrafish Model And Antihyperlipidemic Mechanism Of Berbamine

With the improvement of living standards and changes in dietary, hyperlipidemia and its complications has become a major risk factor for human life and health. Hyperlipidemia is a kind of lipid metabolism disorders, which is associated with abnormal plasma total cholesterol(TC), triglycerides(TG), low-density lipoprotein cholesterol(LDL-c) and high-density lipoprotein cholesterol(HDL-c) levels. It suggested that the occurrence of hyperlipidemia is associated with atherosclerosis, coronary heart disease, hypertension, diabetes etc. Many factors contribute to the hyperlipidemia, such as gene, dietary, lifestyle and neurological factor. As for the treatment of hyperlipidemia, the mainly used drug in clinical is statins, such as simvastatin. Data showed that increased plasma TC and LDL-c levels are major factors for cardiovascular disease, while maintaining cholesterol balance is the key to the prevention and treatment of hyperlipidemia as well as its complications. The cholesterol homeostasis in the body is maintained in the following ways: down-regulating the expression of HMGCR to inhibit cholesterol synthesis; up-regulating LDLR to accelerate cholesterol uptake; up-regulating CYP7A1 to promote cholesterol conversion. Thus, HMGCR, LDLR and CYP7A1 are key targets to investigate the lipid-lowering effect of drugs.Current methods for lipid-lowering investigation include in vitro and in vivo experiments. In vitro methods contain spectrophotometry, thin layer chromatography, isotope labeling, HPLC and cell culture, etc, which are used for drug screening with the advantage of short period, convenience and low cost. However, their utilizations are limited due to fail to simulate the effect of the drug in the body. As for in vivo experiments, the most commonly used models are rodent model including rats, golden hamsters and guinea pigs. It starts with high-cholesterol and high-triglyceride diet followed by measuring serum TC and TG levels to determine whether the model have been established successfully, then oral administration of drugs and detect the lipid-lowering effect. But, it is time-consuming, expensive and also needs a large amount of drugs. Therefore, it is urgent to establish a fast and effective animal model for screening lipid-lowering drugs.As a new animal model, zebrafish is increasingly being used as a model of human diseases in recent years. Traditional Chinese medicine has a long history in treating human disease and a large number of studies have shown that it has a good effect in terms of lipid-lowering activity. This study focused on the establishment of hyperlipidemic zebrafish model and followed by the comparison of lipid-lowering effect of 54 traditional Chinese medicines. We also investigated the potential hypolipidemic effect and mechanism of berbamine from Rhizoma Coptidis(RC) on hyperlipidemic zebrafish.The main contents and results of this project are as follows:(1) Zebrafish was fed with diet containing 4% cholesterol, and serum TC level was detected at different times. The results showed that the plasma and liver lipid content in HC group were significantly increased after 20 days as compared with the NC group. Five positive drugs also showed hypolipidemic effect. In the model group, the expression of HMGCR and LDLR were inhibited while CYP7A1 a mRNA was up-regulated. The performance of hyperlipidemic zebrafish is similar to mammalian models.(2) To verify hyperlipidemic zebrafish model, we investigated the lipid-lowering effects of 54 traditional Chinese medicines and found that RC, Sophora japonica, Turmeric, Apocynum venetum, Platycodon grandiflorus, Red peony, Rosa laevigata significantly declined plasma TC and TG level.Green tea, Lctuc tiwnin Mxim, Aloe vera, Astragalus membranaceus, Hawthorn, Chinese yam and Salvia miltiorrhiza Bunge improved TC level. Lonicera japonica Thunb, Radix puerariae and pepper reduced plasma TG level.(3) Based on the above results, we further explored the antihyperlipidemic effect and mechanism of minor alkaloids berbamine(BBM) in RC. Research on zebrafish embryos found that 5 μg/m L, 10 μg/m L and 20 μg/m L BBM caused no significant difference in zebrafish embryonic development and cell death, and 20 μg/mL BBM can reduce heartbeat rate. The study of zebrafish larvae found that BBM can improve vascular cholesterol levels. The studies in adult zebrafish found that BBM can reduce plasma TC and LDL-c levels as well as fat accumulation in liver. The potential mechanism may be down-regulation of HMGCR and MTP transcription and up-regulation of LDLR and CYP7A1 a mRNA levels.In short, this model may represent a useful tool for high-throughput screening of lipid-lowering drugs and accelerate the development of lipid-lowering medicine. BBM has anti-hypercholesterolemic and hepatoprotective effects. The mechanism probably related to the up-regulation of cholesterol transport and bile acid synthesis as well as down-regulation of cholesterol synthesis and lipoprotein assembly or secretion.