| Background and objectiveHyperlipidemia is a common disorder of lipid metabolism characterized by abnormally elevated levels of one or more lipids or lipoproteins in the blood. In general, hyperlipidemia results from complex interactions between genetic, environmental and dietary factors. Among them, lipid metabolism disorder along with diet is one of the main causes of hyperlipidemia. Excessive absorption of lipids from diet or aberrant lipid metabolism in the body will lead in hyperlipidemia. Many epidemiology research and clinical trials have demonstrated that hyperlipidemia is one of the major risk factors of cardiovascular diseases (CVDs), including atherosclerosis, myocardial infarction and stroke. CVDs are the leading cause of death for Chinese in both urban area and rural area. Nowadays,41.09% of deaths in rural area and 41.52% of deaths in urban area are caused by CVDs in China. Likewise, approximately one sixth of the adults suffer from hypercholesterolemia in America. With an increasing incidence of CVDs, the prevention and treatment of hyperlipidemia present a primary challenge to the entire medical community. Many chemical drugs, such as fibrates and statins, are characterized by high liquid-lowering speed and good efficacy. However, they can not meet the demands for treatment due to the different hyperlipidemia patients, some potential adverse effects and great drug dependence. By comparison, natural products have minimal adverse effects and multiple targets for the prevention and cure of hyperlipidemia.Pedunculoside (PE), a pentacyclic triterpenoid compound, extracted from barks of Ilex rotunda Thunb.. Modern pharmacology studies have showed that PE possesses heat-clearing and detoxifying effects as well as alleviates pain and swelling. It is always applied to treat cold, tonsillitis, gastroenteritis and rheumatoid arthritis in Asia area. This study aims to evaluate the hypolipidemic activity of PE and analyzes its potential mechanism to provide experiment date for further study.MethodsPart one:We used a high fat diet to establish a model of hyperlipidemia, sought the experimental animal, components of high fat diet and the experimental period. To establish a rational, safe and effective animal model of hyperlipidemia.Part two:To generate the hyperlipidemia model, rats were administered a high-fat diet. In the test-drug groups, rats were administered PE at doses of 5,15,30 mg/kg/d. In the positive control group, rats were administered Sim at a dose of 10 mg/kg/d. In the control and model groups, rats were administered 0.5% CMC-Na aqueous solution. The experimental period was 7 weeks. During the experiment period, body weight and food consumption of rats were recorded every week. Dynamic serum lipid monitoring was performed from 3rd to 7th weeks and blood samples were collected after overnight fasting. At the end of the experimental period, all rats were sacrificed under anesthesia with urethane. Determined the levels of TG and TC in liver. The relative levels of SREBP-lc, FAS, PPAR-a, and SCD-1 gene transcripts in the liver were determined by RT-PCR. Frozen liver samples were used to perform hepatic lipid specific staining with Oil Red O staining. The organs including heart, liver, spleen, lung, kidney and epididymal adipose tissue were rapidly dissected and fixed in 10% formalin before being sectioned for histology analysis.Part three:We performed an MTT assay to determine the effect of PE on the cell viability of 3T3-L1 preadipocytes. The 3T3-L1 preadipocytes were induced to adipocytes with insulin, dexamethone, and IBMX. On day 8 after induction of differentiation, cells were stained with Oil Red O. And cellular TG contents were measured using a commercial TG assay kit.Part four:With different concentrations of PE treated cells, total protein of cells was extracted in each group, Western blot to detect the protein expression of CCAAT-enhancer-binding proteins-α (C/EBPa), Peroxisome proliferator-activated receptor-y (PPAR-y), Sterol regulatory element-binding protein-1 (SREBP-1) and the activation of AMPK.ResultsPart one:After 4-weeks of high-fat diet (66.5%basal diet,10% lard,10% yolk powder,10% sugar,0.5% bile salts and 3% cholesterol) feeding, serum levels of TC, TG and LDL-C in the model group were significantly higher than those in the control group (p<0.001). In addition, compared with the control group, HDL-C level was significantly decreased (p<0.001). Liver histopathological examination showed that the high-fat diet resulted in severe lipochondria infiltration in some parts of the liver, and balloon-like changes in a large number of cells. These data revealed that feeding high-fat diet for 4-weeks induced hyperlipidemia rat model.Part two:PE (5,15,30 mg/kg) administration significantly decreased serum TC, TG, LDL-C and increased HDL-C (p<0.001). PE also ameliorated pathological changes in liver tissue, as evidenced by decreases in the levels of TC and TG as well as suppression of hepatic lipid infiltration. PE 15,30 mg/kg significantly decreased the weights (p<0.05,p<0.01)and diameter (p<0.001) of epididymal adipose. Additionally, PE significantly modulated expression of peroxisome proliferator-activated receptor alpha (PPAR-a), sterol regulatory element-binding protein lc (SREBP-lc), fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD-1) in the liver (p<0.001). Moreover, no marked histological changes were observed in the organs from the PE treatment groups.Part three:In a concentration range of 25-100μmol/L, PE did not exhibit remarkable influence on proliferation of 3T3-L1 preadipocytes, it suggest that PE showed no cytoxicity to 3T3-L1 preadipocytes. PE 25,50, 100μmol/L significantly reduced the level of TG in differentiated 3T3-L1 adipocyte (p<0.001), in a dose-dependent manner. Oil Red O staining results were shown that PE significantly decreased the number of lipid droplets.Part four:Through Western Blot, we found PE 25,50, 100μmol/L markedly suppressed the protein expression of PPAR-y in differentiated 3T3-L1 adipocyte (p<0.01,p<0.001). PE 50, 100μmol/L significantly decreased the protein expression of C/EBPα and SREBP-1 (p<0.01, p<0.001). Moreover, PE 25,50, 100μol/L significantly increased the phosphorylation of AMPK(p<0.01,p<0.001).Conclusions(1) PE significantly decrease serum TC, TG, LDL-C and increased HDL-C, modulate the lipid metabolic disorder, and greatly alleviate the occurrence of liver steatosis in hyperlipidemia rats.(2) PE ameliorates high-fat diet induced hyperlipidemia in rats possibly by modulating the expression levels of genes involved in lipogenesis and fatty acid β-oxidation.(3) PE can significantly reduce the lipid content of differentiated 3T3-L1 adipocyte, which may be associated with down-regulated the expression of PPAR-y, C/EBPa, SREBP-1 and the activation of the AMPK signaling pathway. |
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