马钱子对斑马鱼幼鱼肝脏毒性的初步研究

目的利用斑马鱼模型探讨马钱子的肝毒性机制。方法考察马钱子对受精后4天(day post fertilization,dpf)斑马鱼幼鱼死亡率的影响。此外,通过与对照组相比,以肝脏表型和肝脏功能、生理指标为评价标准确认马钱子的肝脏毒性,并利用荧光实时定量PCR(Q-RT-PCR)技术从转录水平对肝毒性相关的基因,如超氧化物歧化酶1基因(superoxide dismutase 1,sod1)、醌氧化还原酶1基因(nad(p)h quinone oxidoreductase 1,nqo1)、线粒体解偶联蛋白2基因(uncoupling protein 2,ucp2)、谷胱甘肽S-转移酶P 2基因(glutathione s-transferase p 2,gstp2)、B细胞淋巴瘤2基因(b-cell lymphoma-2,bcl-2)、骨髓细胞白血病-1B基因(myeloid cell leukemia-1b,mcl-1b)、BCL 2关联X蛋白基因(Bcl-2 associated X,apoptosis regulator,bax-2)、BH3互动死亡激动剂基因(recombinant BH3 interacting domain death agonist,bid)、脂肪酸合酶基因(fatty acid synthase,fasn)、3-羟基-3-甲基戊二酰辅酶A还原酶基因(3-hydroxy-3-methylglutaryl-coa reductase,hmgcra)、成纤维细胞活化蛋白α基因(fibroblast activation protein alpha,fap)、瘦素受体基因(leptin receptor,lepr)、视黄醇结合蛋白4基因(retinol binding protein 4,rbp4)、转化生长因子基因(transforming growth factor beta 1,tgf-β1)、转化生长因子β受体2基因(transforming growth factor beta receptor 2,tgf-βr2)基因的表达水平进行评价。采用SPSS24.0进行组间单因素方差分析和Dunnett’s t检验对不同处理组之间的数据进行差异分析。结果(1)在亚致死浓度(<76.523μg/mL)暴露条件下,马钱子能够引起斑马鱼幼鱼肝脏形态发生改变、引起细胞凋亡和脂质积累。(2)低中高浓度马钱子暴露24小时,超氧化物歧化酶(superoxide dismutase,SOD)相比对照组降低42.5%~67.3%、过氧化氢酶(catalase,CAT)活性降低22.1%~60.8%,同时丙二醛(malondialdehyde,MDA)含量相比对照组增加17%~105%、谷丙转氨酶(alanine aminotransferase,ALT)和谷草转氨酶(aspartate aminotransferase,AST)活性相比对照组分别增加30.49%~34.05%、13.42%~15.18%。(3)实时PCR结果显示,Sod1、Bcl-2、Gstp2、Nqo1、Bid、Bax-2、Hmgcra、Mcl-1b和Tgf-βr2的表达水平受到显著影响。结论马钱子对斑马鱼的肝毒性与氧化应激损伤和脂肪代谢失衡有关。

Preliminary study on the toxicity of scutellaria to the liver of zebrafish juveniles

Objective The hepatotoxicity mechanism of Nux vomica was studied in zebrafish larvae to provide reference for further study on the toxicity of Nux vomica.Methods The effect of nux vomica on mortality rate of 4day post fertilization(dpf)zebrafish larvae was assessed.Furthermore,the liver function,physiological parameters and expression level of sod1,nqo1,ucp2,gstp2,bcl-2,mcl-1b,bcl-2,bax-2,bid,fasn,hmgcra,fabp,lepr,rbp4,tgf-β1,tgf-βr2,were investigated.Results On the sub-lethal concentration,nux vomica resulted in the morphological alterations,cell apoptosis and lipids accumulation in the liver of larval zebrafish.Nux vomica exposure reduced superoxide dismutase,catalase activity while increased malondialdehyde content,hepatic alanine aminotransferase and aspartate aminotransferase activities.Real-time PCR analysis showed that expression level of sod1、bcl-2、gstp2、nqo1、bid、bax-2、hmgcra、mcl-1b and tgf-βr2 were affected significantly.Conclusion These findings displayed the effect of nux vomica on the hepatotoxicity in zebrafish,which was associated with the imbalances of oxidative emergency system and fat metabolism.