头孢唑林及其杂质诱导的斑马鱼胃肠道毒性反应比较分析

Comparative analysis of gastrointestinal toxicity induced by cefazolin and its impurities in zebrafish

目的本研究利用药源性斑马鱼胃肠道毒性反应模型,比较分析头孢唑林及其杂质F和杂质A诱导的胃肠道毒性反应及其机制。方法荧光显微镜下活体观测头孢唑林、杂质F和杂质A对斑马鱼幼鱼胃肠道排空过程的影响,评价其毒性反应,比较分析3种化合物结构与毒性关系;利用RNA-seq技术分析3种化合物胃肠道毒性反应机制的差异;利用分子对接模拟分析3种化合物与胆汁盐外排蛋白(BSEP)蛋白结合亲和力。结果杂质F胃肠排空能力明显大于头孢唑林,杂质A的胃肠排空能力明显小于头孢唑林,MMTD结构是头孢唑林导致胃肠道毒性反应的主要毒性功能基团;RNA-seq分析发现杂质F处理组分别与头孢唑林和杂质A处理组相比较具有45个共同呈显著差异的基因,主要显著富集在矿物质吸收通路和胆汁分泌通路中,其中abcb11b基因的表达与胃肠道毒性反应呈明显相关;分子对接结果显示杂质F与斑马鱼BSEP的亲和力高于头孢唑林,头孢唑林的结合亲和力高于杂质A。结论临床中头孢唑林钠的胃肠毒性反应可能与产品中杂质F有关,通过药物和杂质与BSEP蛋白结合的亲和力可以评估头孢唑林杂质引起的胃肠道毒性反应的差异,可为头孢唑林钠的生产工艺评估和质量控制提供理论依据。

Objective This study employed a zebrafish model to comprehensively assess and compare the gastrointestinal toxicity induced by cefazolin,impurity F,and impurity A,shedding light on the underlying mechanisms.Methods Real-time observation of zebrafish larvae's gastrointestinal emptying under a fluorescence microscope allowed the evaluation of toxicity responses to cefazolin,impurity F,and impurity A.Structural relationships among the three compounds were analyzed.Genome expression patterns associated with gastrointestinal toxicity reactions were elucidated by means of RNA-seq technology.Molecular docking assessed the binding affinity between the compounds and the bile salt export pump(BSEP)protein.Results Impurity F exhibited significantly higher gastrointestinal emptying capacity compared to cefazolin,while impurity A demonstrated notably lower emptying capacity than cefazolin.The MMTD structure emerged as the primary toxic functional group responsible for cefazolin-induced gastrointestinal toxicity.RNA-seq analysis unveiled 45 significantly differentially expressed genes shared between the impurity F group and the cefazolin and impurity A groups.These genes were predominantly enriched in the mineral absorption and bile secretion pathways,with the expression of the abcb11b gene closely linked to gastrointestinal toxicity reactions.Molecular docking results indicated that impurity F had a stronger affinity for zebrafish BSEP than cefazolin,while cefazolin exhibited a higher binding affinity compared to impurity A.Conclusion Clinical gastrointestinal toxicity reactions attributed to cefazolin sodium may be associated with impurity F in the product.The assessment of the binding affinity of the drug and the impurities to the BSEP protein offered insights into variations in gastrointestinal toxicity reactions caused by the impurities in cefazolin sodium.This study provided a theoretical foundation for evaluating the production process and quality control of cefazolin sodium.