Establishment of a zebrafish model of alcohol-induced acute hepatic steatosis
目的建立一种简单易行、重复性好的急性酒精性脂肪肝斑马鱼模型。方法选取野生型斑马鱼288条,随机均分为对照组和2%、2.5%、3%乙醇处理组,于胚胎受精后5 d给予相应体积浓度比乙醇处理。观察斑马鱼一般生存率和表型变化,用整体油红染色评判脂肪肝发生率,石蜡切片观察肝组织病理形态。结果 2%、2.5%和3%乙醇处理组斑马鱼32 h内生存率分别为100%、(91.21±1.61)%、0%。3%乙醇处理24 h即出现畸形。整体油红染色显示2%乙醇处理组斑马鱼在处理32 h后脂肪肝发生率显著高于对照组(P=0.002)。病理切片可见2%乙醇处理32 h后斑马鱼肝细胞肿胀,肝内脂滴沉积增多,脂肪肝形成。结论利用2%乙醇处理胚胎受精后5 d的幼鱼32 h成功地建立急性酒精性脂肪肝斑马鱼模型,为进一步开展急性酒精性脂肪肝的发病机制研究和药物筛选奠定实验基础。Abstract:
Objective To establish a zebrafish model of hepatic steatosis induced by acute alcohol exposure as a model system that will be useful for investigations of disease pathogenesis and high-throughput screenings.Methods At 5 days post fertilization (dpf) , 288 juvenile zebrafish (wild-type strain AB) with normal liver development were selected and randomly divided into four groups (n=72 each) for culturing in water and Hank′s balanced salt solution alone (controls) or supplemented with ethanol at 2.0%, 2.5% and 3.0% concentrations.At 4, 24 and 32 h of culturing, the physical activity (swimming, circling) was observed, the survival rate was recorded, and the morphology was assessed by whole-mount and liver section microscopy.Oil Red O staining was used to determine the percent of steatosis and pathological features were assessed by hematoxylin-eosin staining.Differences between two groups were assessed by independent samples t-test.Results Starting at 24 h of ethanol exposure, the survival rates showed a decreasing trend that corresponded to increased alcohol concentration, so that by 32 h of exposure there were remarkable differences among the three alcohol-treated groups: 2.0% ethanol: 100% survival;2.5% ethanol: (91.21±1.61) % survival;and 3.0% ethanol: 0% survival.Abnormalities in liver morphology and physical activity were also observed at the 24 h time point and showed concentration-dependence: 2.0% ethanol: normal morphology, increased activity, abnormal circling;2.5% ethanol: some deformities;and 3.0% ethanol: bent spinal cord, pericardial and yolk sac edema, stationary behavior.Significant steatosis was observed in the 2.0% ethanol group at 32 h of exposure, but not at 24 h, as evidenced by the percent of steatosis[ (71.25±0.15) % vs.controls: (31.25±0.05) %, P=0.002] and pathological features (swollen size, lipid droplet-induced nucleus displacement) .Conclusion A model of acute alcohol-induced hepatic steatosis was successfully established in 5 dpf zebrafish by treating with 2.0% alcohol for 32 h.This model may represent a useful tool for investigating the disease pathogenesis and performing high-throughput screenings of potentially therapeutic drugs.
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