背景:化合物/药物的风险,包括马兜铃酸在内的肾毒性仍然很高,是一个重大的公共卫生问题。因此,选择合理的动物模型对不同化学体系复杂的样品进行快速筛选和评价尤为重要。斑马鱼(Daniorerio)已用于研究化学诱导的肾毒性。然而,大多数已发表的文献都是针对单个成分或药物进行的,以及证实斑马鱼幼虫适用于评估复杂化学系统中马兜铃酸相关肾毒性的关键证据,如中医(TCM),是不够的。
方法:采用高效液相色谱法(HPLC)测定中药和中成药中马兜铃酸(AA)的含量。受精后4天的斑马鱼幼虫(dpf)用于评估各种样品的肾毒性,分别,根据肾脏的表型和组织学,和生化。转录组技术用于研究AA治疗后的相关信号通路和潜在机制。通过RT-PCR技术验证。
结果:结果表明,AAI的总量,AAII,不同样品的ALI范围为0.0004至0.1858g·g-1(%),包括马兜铃菌,Fibraurearecisa,细辛,万通金骨片,九味强火颗粒,和小青龙颗粒按降序排列。此外,与阴性/空白对照相比,表型的实质性变化,在接受亚致死浓度药物攻击的组中观察到肾功能的组织形态学和生化参数。转录组学结果显示PERK/ATF4/CHOP基因表达上调,ATM/Chk2/p53,Caspase/Bax/Bcl-2a,TGF/Smad/ERK,PI3K/Akt,马兜铃酸类似物诱导,与q-RT-PCR实验基本一致,强调与以前发表的文章类似的毒性反应与其他传统评估模型。
结论:稳定性,斑马鱼幼体模型在筛选和评价中药肾毒性中的准确性和可行性,确认和促进斑马鱼在评估具有复杂化学特征的样品的肾毒性中的适用性。
BACKGROUND: The risk of compounds/drugs, including aristolochic acid-induced nephrotoxicity remains high and is a significant public health concern. Therefore, it is particularly important to select reasonable animal models for rapid screening and evaluation of different samples with complex chemical systems. The zebrafish (Danio rerio) has been used to study chemical-induced renal toxicity. However, most of the published literature was performed on individual components or drugs, and the key evidence confirming the
applicability of zebrafish larvae for the evaluation of aristolochic acid-related nephrotoxicity in complex chemical systems, such as in traditional Chinese medicine (TCM), was insufficient.
METHODS: High-performance liquid chromatography (HPLC) was used to determine the content of aristolochic acid (AA) in herbs and Chinese patent medicines. The zebrafish larvae at 4 days post-fertilization (dpf) were used to evaluate the nephrotoxicity of various samples, respectively, based on the phenotype of the kidney and histological, and biochemical. Transcriptome technology was used to investigate the related signaling pathways and potential mechanisms after treatment with AA, which was verified by RT-PCR technology.
RESULTS: The results showed that the total amounts of AAI, AAII, and ALI ranged from 0.0004 to 0.1858 g·g-1( %) from different samples, including Aristolochia debilis, Fibraurea recisa, Asarum, Wantongjingu tablets, Jiuweiqianghuo granules, and Xiaoqinglong granules in descending order. Moreover, compared with the negative/blank control, substantial changes in phenotype, histomorphology and biochemical parameters of renal function were observed in the groups challenged with the sublethal concentration of drugs. The transcriptomics results showed the upregulation of most genes in PERK/ATF4/CHOP, ATM/Chk2/p53, Caspase/Bax/Bcl-2a, TGF/Smad/ERK, PI3K/Akt, induced by aristolochic acid analogues, which were essentially consistent with those of the q-RT-PCR experiments, highlighting the similar toxicity response to the previously published article with the other traditional evaluation model.
CONCLUSIONS: The stability, accuracy and feasibility of the zebrafish larval model in screening and evaluating the nephrotoxicity of TCM were validated for the first time on the AAs-related drugs in a unified manner, confirming and promoting the
applicability of zebrafish in assessing nephrotoxicity of samples with complex chemical character.