酮康唑对原代培养大鼠肝细胞的毒性作用及其机制

Study on ketoconazole-induced hepatotoxicity and its mechanisms in a primary cultured rat hepatocytes system

目的观察酮康唑(ketoconazole,KCZ)对原代培养大鼠肝细胞的毒性效应,探讨其可能的毒性机制。方法采用胶原酶二步灌流法分离制备雄性SD大鼠肝细胞,进行原代培养。量效关系研究中KCZ设56、75、94、113和188μmol/L,染毒时间4 h;时效关系研究中KCZ设188μmol/L,染毒时间分别为0.5、1、2和4 h;同时设溶剂对照。检测染毒后肝细胞活力、胞内LDH泄漏情况以及巯基状态的变化。结果(1)量效关系研究中,随着KCZ剂量的升高,肝细胞活力逐渐下降,培养液中LDH活力逐渐增高,肝细胞内巯基也呈下降趋势,上述指标均有明确的剂量-反应关系(γLDH=0.906,P<0.01);(2)时效关系研究中,KCZ在188μmol/L剂量下染毒后肝细胞活力呈时间依赖性下降;培养液中LDH活力随染毒时间延长逐渐增高;细胞内巯基出现相应性下降。结论KCZ可以导致肝细胞活力下降,胞内LDH泄漏,巯基含量减少,并有明确的量效和时效关系,提示酮康唑对肝细胞的毒作用可能与细胞内巯基状态改变有关。

Objective To observe the effects of ketoeonazote on cell livability, lactate dehydrogenawe（ LDH ） leakage and the contents of sulfhydryl groups in a primary cultured hepatocytes system of Sprague-Dawley rat and to investigate the potential hepatoxic mechanisms of ketoconazole. Methods Hepatocytes were isolated from male adult Sprague-Dawley rats by the two-step perfusion using collagenase and cultured in William＇s E culture medium with 10% fetal serum. Rat hepatocytes were administered with different doses （0,56, 75, 94, 113 and 188 μmnol/L） of ketoconazole for 4 h or with 188 μmol/L for different time （0, 1, 2 and 4 h） to investigate dose-effect and time-effect relationships by measuring the leakage of the LDH into the medium; by assessing mitochondrial reduction of 3- （4,5-dimethythiazol-2yl）-2,5-diphenyl tetrazolium bromide （MTT） and lysosomal uptake of neutral red （NR）;and by ssessing the content of total sulfhydryl and non-protein binding sulfhydryl. Results 1. cell livability and the content of sulfhydryl groups declined and LDH leakage increased significantly with the increase of the administered dose. 2. cell livability and the content of sulfhydryl groups declined and LDH leakage increased significantly with the prolongation of the administered dose. Conclusion These results demonstrate that the hepatoxicity of ketoconazole is related with the alterations of the contents of sulfhydryl groupsintra-cellular.