苦参碱和氧化苦参碱体内外模型的肝毒性比较研究

Comparison of hepatotoxicity and toxic mechanisms of matrine and oxymatrine using in vivo and in vitro models

目的比较苦参碱(matrine,MT)和氧化苦参碱(oxymatrine,OMT)的肝毒性,以探索其毒性严重程度和特征,并初步阐明毒性机制。方法用对乙酰氨基酚(acetaminophen,APAP)、苦参碱和氧化苦参碱处理肝细胞,24 h后,检测IC50、肝细胞酶含量、病理形态、丙二醛(malondialdehyde,MDA)和谷胱甘肽(glutathione,GSH)含量,并检测凋亡率。用对乙酰氨基酚、苦参碱和氧化苦参碱处理斑马鱼,96 h后,检测LC50、肝细胞病理形态、丙二醛和谷胱甘肽含量,并检测凋亡率,同时检测氧化应激相关基因zgc:136383、凋亡相关基因EIF4EBP3和zgc:123120的表达。结果苦参碱和氧化苦参碱对肝细胞有毒性作用,苦参碱IC50为5.3 mmol/L,氧化苦参碱为>19mmol/L。苦参碱和氧化苦参碱处理肝细胞的谷丙转氨酶、碱性磷酸酶、谷草转氨酶和乳酸脱氢酶含量升高,肝细胞肿胀,丙二醛含量升高,谷胱甘肽含量降低,凋亡率增加(P<0.05)。苦参碱和氧化苦参碱对斑马鱼有毒性作用,苦参碱的LC50为0.41 mmol/L,氧化苦参碱为>3.8 mmol/L。苦参碱和氧化苦参碱处理的斑马鱼肝细胞呈现轻度至中度空泡化,丙二醛含量和凋亡率增加,谷胱甘肽含量降低(P<0.05)。苦参碱下调氧化应激相关基因zgc:136383(P<0.05),下调抗凋亡基因EIF4BP3,上调促基因zgc:123120(P<0.05)。结论体内外模型结果一致,苦参碱和氧化苦参碱具有肝毒性,其毒性特征相似,苦参碱的毒性大于氧化苦参碱。肝毒性机制与氧化应激和细胞凋亡有关:苦参碱下调基因zgc:136383减少脂质转运,并激活氧化应激反应;苦参碱上调促凋亡基因zgc:123120,下调抗凋亡基因EIF4EBP3并诱导肝细胞凋亡。

Objective To compare the hepatotoxicity of matrine（ MT） and oxymatrine（ OMT） and explore the severity and characteristics of their toxicity,and to preliminarily elucidate their toxic mechanisms. Methods Liver cell line LO-2 cells were treated with acetaminophen（ APAP）,matrine and oxymatrine for 24 h,and the IC50 values,the contents of enzymes in the liver cells,the pathological changes,the contents of malondialdehyde（ MDA） and glutathione（ GSH） and thecell apoptosis rate were detected. In addition,adult zebrafish were treated with APAP,matrine and oxymatrine for 96 h,and the LC50 values,the pathological morphology of the liver cells,the contents of MDA and GSH and the apoptosis rate were detected. Meanwhile, the expression of oxidative stress-related gene, zgc： 136383, and the apoptosis-related genes,EIF4 EBP3 and zgc： 123120,was also detected. Results Matrine and oxymatrine had toxic effects on liver cells in vitro.The IC50 value of matrine was 5. 3 mmol/L, and that of oxymatrine was〉 19 mmol/L. The contents of alanine aminotransferase（ ALT）,alkaline phosphatase（ ALP）,aspartate aminotransferase（ AST） and lactate dehydrogenase（ LDH）in the liver cells treated with matrine or oxymatrine were increased,and the cells appeared swollen,with an increase in the MDA level and a decrease in the GSH level. The cell apoptosis rate was also increased（ P〈0. 05）. Furthermore,matrine and oxymatrine had toxic effects on the zebrafish. The LC50 value of matrine was 0. 41 mmol/L,and that of oxymatrine was 〉3. 8 mmol/L. The hepatocytes of zebrafish treated with matrine and oxymatrine appeared vacuolization in a mild to moderate degree,with an increase of the MDA content and a decrease of the GSH content. The cell apoptosis rate was increased（ P〈0. 05 for all）. Expression of the oxidative stress-related gene zgc： 136383（ P〈0. 05） and the apoptosis-resistant gene EIF4 EBP3（ P〈0. 05） was down-regulated by matrine,but that of the apoptosis-promoting gene zgc： 123120 was upregulated（ P〈0. 05）. Conclusions Results of the experiments using liver cells in vitro are consistent with those using the in vivo zebrafish model. Matrine（ MT） and oxymatrine（ OMT） both have hepatotoxicity,with similar toxic characteristics,and the toxicity of matrine is greater than oxymatrine. The mechanism of their hepatotoxicity is related with oxidative stress and cell apoptosis. Matrine reduces lipid transportation and activates oxidative stress reactions through down-regulation of gene zgc： 136383. In addition,matrine induces apoptosis in the liver cells via up-regulation of the apoptosis-promoting gene zgc： 123120 and down-regulation of the apoptosis-resistant gene EIF4 EBP3.