| Objective:To evaluate the effect of Artemisia Capillaris Formula (ACF) on nonalcoholic fatty liver disease (NAFLD) in vivo and in vitro, and to explore the underlying mechanisms, as to provide theoretical basis for the clinical application of the drug.Method:In vivo study:72rats were randomly divided into control group (12rats) and model group (60rats). Control group of rats was used as control; model group of rats was fed a high-fat diet (HFD) ad libitum for8weeks. Confirmed by the early experimental observation model success after8-week, the rats fed with the HFD were randomly divided into5groups (12rats/group):the model group (model), polyene phosphatidylcholine-treated group (PP), ACF-treated groups (high-, middle-, low-dose). PP (0.076g/kg body weight/day), ACF high-dose (1.848g/kg body weight/day), ACF middle-dose (0.924g/kg body weight/day) and ACF low-dose (0.462g/kg body weight/day) were administered as previously described, while the control group and model group were orally treated with saline respectively. After30days, all rats were weighted and sacrificed; the liver weights were recorded to evaluate the liver index. Blood samples were collected for assays of AST, ALT, TG, TC, HDL-C and LDL-C. Livers were rapidly dissected. A part of each liver was cut and fixed in formaldehyde saline (4%) solution for HE staining and histological analysis; the rest was snap frozen in liquid nitrogen and stored at-80℃until use.In vitro study:OA/PA was used to construct the cell model of NAFLD, and following were treated with different concentrations of ACF. Oil red O staining was used to observe lipid deposition in cytoplasm; TC, TG content were determined by enzyme coupling colorimetric analysis; Real time-PCR analysis and western blot analysis were performed to detect the expression of miR-122and FASN.Result:In vivo study:(1) The liver index of rats in model group which were fed with HDF was significantly higher than the control group which were fed with a normal diet, while the treatment of ACF significantly decreased the liver index when compared with model rats (P<0.05).(2) The serum TC, TG and LDL-C levels were significantly increased in model group when compared with control group (P<0.05), and the HDL-C level was descreased (P<0.05); While the treatment of ACF significantly decreased the levels of TC, TG and LDL-C and increased the level of HDL-C (P<0.05).(3) HE staining showed that rats fed a control diet had normal liver histology, while rats fed with the HFD showed a high accumulation of fat and developed steatohepatitis, which was characterized by hepatocyte ballooning, scattered lobular inflammatory cell infiltration and inflammatory foci. Treatment of PP or ACF significantly reduced hepatic steatosis.(4) The PCR analysis showed that PP or ACF treatment markedly up-regulated the expression of miR-122and down-regulated its target gene FASN (P<0.05). Immunohistochemical assay confirmed that the expression of FASN was up-regualted in model group which was significantly down-regulated with the treatment of PP, ACF low-, middle-or high-dose ACF (P<0.05).In vitro study:Oil red O staining showed that OA/PA treatment increased intracellular lipid accumulation, and which was decreased with the treatment of ACF for24h; the amount of TC and TG were increased in model group which were significantly inhibited by the treatment of ACF. Moreover, Real time-PCR assays and western blot analysis showed that PP or ACF treatment markedly up-regulated the expression of miR-122and down-regulated the expression of its target gene FASN on both mRNA and protein level.Conclusion:ACF can significantly reduced hepatic steatosis and accumulation of lipid droplets in NAFLD model rats, suggesting that the ACF can be used to treat NAFLD, and by up-regulating the expression of miR-122and down-regulating the expression of its target gene FASN maybe the mechanisms. |
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