基于计算毒理学的旱莲苷A致肾损伤机制

Mechanism of Nephrotoxicity Induced by Ecliptasaponin A Based on Computational Toxicology

摘要:

目的 基于中药毒性预警体系与基础毒理数据库系统（TCMTAS-BTD）预测中药保健食品中潜在肾损伤成分，并通过体内外实验筛选与验证，最后结合网络药理学进行毒性机制初探。方法 应用TCMTAS-BTD预测中药保健食品原料目录中的3 540个中药单体成分。利用细胞增殖与活性检测（CCK-8）和高内涵筛选技术对肾损伤概率值排名前5的化合物进行体外筛选。同时将ICR小鼠随机分为空白组、低剂量旱莲苷A（2.91 mg·kg^-1·d^-1）和高剂量旱莲苷A（29.1 mg·kg^-1·d^-1），每组10只，连续给药28 d。实验期间观察小鼠的一般情况，计算肾脏指数，检测血清肌酐（SCr）、血液尿素氮（BUN）与丙二醛（MDA）与超氧化物歧化酶（SOD）水平，观察肾组织的病理形态学变化，并利用网络药理学预测其致肾损伤的潜在通路，最后利用蛋白免疫印迹法（Western blot）对通路相关蛋白进行验证。结果 TCMTAS-BTD预测肾损伤概率排名前五分别为旱莲苷A、大黄酚、吴茱萸次碱、丹参酮Ⅰ和京尼平苷酸。CCK-8结果显示，10 μmol·L^-1旱莲苷A组、60 μmol·L^-1大黄酚组、40 μmol·L^-1吴茱萸次碱组和20 μmol·L^-1丹参酮Ⅰ对HK-2细胞活性产生明显影响（P<0.05，P<0.01）。高内涵分析结果显示，10 μmol·L^-1旱莲苷A、大黄酚、吴茱萸次碱和丹参酮Ⅰ细胞数目均明显减少（P<0.05，P<0.01）。同时高剂量旱莲苷A组小鼠出现行动迟缓、体质量增长缓慢（P<0.01），肾系数显著性增加（P<0.01），且SCr、BUN与MDA水平显著性升高（P<0.01），SOD显著性下降（P<0.01），组织病理学结果显示高剂量旱莲苷A组肾脏存在轻度的组织改变。同时网络药理学结果显示旱莲苷A致肾损伤的关键靶标主要富集于磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）信号通路、前列腺癌与脂质和动脉粥样硬化通路，Western blot结果显示验证高剂量旱莲苷A可上调PI3K与Akt的磷酸化水平（P<0.01）。结论 给药28 d时，29.1 mg·kg^-1旱莲苷A可对正常大鼠造成肾损伤，其机制可能与PI3K/Akt信号通路相关，提示超剂量长期服用墨旱莲一定程度上可能会增加药物不良反应的发生率。

Abstract:

Objective To predict the potential nephrotoxic components in traditional Chinese medicine health food products based on the Traditional Chinese Medicine Toxicity Alert System and Basic Toxicology Database （TCMTAS-BTD）， screen and validate the predicted components by cell and animal experiments， and decipher the mechanism of nephrotoxicity by network pharmacology.Method TCMTAS-BTD was utilized to predict the toxicity of 3 540 compounds found in the catalogue of traditional Chinese health food ingredients. In the cell experiment， the top 5 compounds with high toxicity probability were screened by measurement of cell proliferation and viability （CCK-8） and high-content screening. ICR mice were randomized into a control group， a low-dose （2.91 mg·kg^-1·d^-1） ecliptasaponin A， and a high-dose （29.1 mg·kg^-1·d^-1） ecliptasaponin A group， with 10 mice in each group， and treated continuously for 28 days. During the experiment， the general conditions of the rats were observed， and the kidney index was calculated. The levels of serum creatinine （SCr） and blood urea nitrogen （BUN） in the serum as well as the content of malondialdehyde （MDA） and superoxide dismutase （SOD） in the renal tissue were measured. The pathological changes of the kidney were observed. Network pharmacology was employed to predict the potential pathways of nephrotoxicity. Finally， the pathway-associated proteins were validated by Western blot.Result The top 5 compounds with high probability of nephrotoxicity were ecliptasaponin A， chrysophanol， rutaecarpine， tanshinoneⅠ， and geniposidic acid. In the cell experiment， CCK-8 results showed that 10 μmol·L^-1 ecliptasaponin A， 60 μmol·L^-1 chrysophanol， 40 μmol·L^-1 rutaecarpine， and 20 μmol·L^-1 tanshinone I altered the viability of HK-2 cells. High-content analysis showed that 10 μmol·L^-1 ecliptasaponin A， chrysophanol， rutaecarpine， and tanshinone Ⅰ reduced the cell number （P<0.05， P<0.01）. The animal experiment showed that the mice in the high-dose ecliptasaponin A group presented slow movement， slow weight gain （P<0.01）， increased kidney index （P<0.01）， elevated SCr， BUN， and MDA levels （P<0.01）， and lowered SOD level （P<0.01）. Mild histopathological changes were observed in the high-dose ecliptasaponin A group. The network pharmacology results showed that the key targets of nephrotoxicity induced by ecliptasaponin A were mainly enriched in the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway， prostatic cancer and lipid and atherosclerosis pathways. Western blot results verified that high dose of ecliptasaponin A raised the phosphorylation levels of PI3K and Akt （P<0.01）.Conclusion On day 28 of administration， 29.1 mg·kg^-1 ecliptasaponin A was found to induce renal injury in rats. The mechanism may be related with the PI3K/Akt signaling pathway， which implied that excessive and prolonged usage of Ecliptae Herba may increase the incidence of adverse drug reactions.