何首乌蒽醌类单体成分致HK-2细胞毒性研究

doi:10.19803/j.1672-8629.20230138

摘要/Abstract

摘要： 目的采用人肾皮质近曲小管上皮细胞系HK-2细胞明确何首乌中主要蒽醌类成分大黄素、芦荟大黄素、大黄酸、大黄素甲醚和大黄酚与肾毒性的相关性。方法 HK-2细胞经2.5、10、20、40、80、120、160 μmol·L^-1的大黄素、芦荟大黄素、大黄酸和3.125、12.5、25、50、100、150、200 μmol·L^-1的大黄素甲醚和大黄酚作用 24、48 h后,采用细胞计数试剂盒法（CCK-8）测定这些单体对HK-2细胞存活率的影响。HK-2细胞经6.25、12.5、25、50、100 μmol·L^-1的大黄素、芦荟大黄素、大黄酸、大黄素甲醚和大黄酚作用 24、48 h后,收集细胞培养液检测乳酸脱氢酶（LDH）释放水平;采用酶联免疫吸附法（ELISA）检测肾损伤分子-1（Kim-1）蛋白表达水平;以JC-10荧光探针检测线粒体膜电位（MMP）的变化;并以流式检测法测定5种蒽醌类化合物对细胞凋亡率的影响。结果大黄素、芦荟大黄素、大黄酸和大黄酚致 HK-2 细胞存活率随给药时间和给药浓度的增加而降低,与对照组相比差异具有统计学意义（P<0.05）,而大黄素甲醚和大黄酚 200 μmol·L^-1给药 48 h 后细胞存活率仍大于 75%。大黄素、芦荟大黄素和大黄素甲醚100 μmol·L^-1给药48 h后,LDH释放水平相较于对照组显著升高（P<0.01）。5种何首乌蒽醌类单体成分对HK-2细胞给药后细胞上清中Kim-1水平未呈现出随浓度增加而升高的趋势,仅在100 μmol·L^-1给药后,Kim-1水平较对照组显著上升（P<0.05）。5种何首乌蒽醌类单体成分给药后,HK-2细胞的线粒体膜电位均随处理浓度的增大和给药时间的延长而下降,但仅25、50、100 μmol·L^-1的大黄素、芦荟大黄素和50、100 μmol·L^-1的大黄酸给药48 h后检测到显著的细胞凋亡（P<0.05）。结论大黄素、芦荟大黄素和大黄酸是导致何首乌肾毒性的潜在蒽醌类成分。何首乌中其他成分的肾毒性风险有待进一步研究,提高安全合理用药水平。

关键词: HK-2细胞, 何首乌, 蒽醌类单体成分, 细胞毒性, 乳酸脱氢酶, 肾损伤分子-1, 线粒体膜电位, 安全性

Abstract: Objective To determine the correlation between nephrotoxicity and five components of Polygonum multiflorum. Methods After HK-2 cells were treated with 2.5, 10, 20, 40, 80, 120, 160 μmol·L^-1emodin, aloe-emodin, rhein and 3.125, 12.5, 25, 50, 100, 150, 200 μmol·L^-1 physcion and chrysophanol for 24 and 48 hours, the effects of these monomers on viability of HK-2 cells were determined with the cell counting kit. After HK-2 cells were treated with 6.25, 12.5, 25, 50 and 100 μmol·L^-1emodin, aloe-emodin, rhein, physcion and chrysophanol for 24 and 48 hours, the cell culture medium was collected to detect the release level of lactate dehydrogenase(LDH). The expression level of Kim-1 protein was detected via Elisa assay, the changes of the mitochondrial membrane potential (MMP) were detected by the JC-10 fluorescence probe, and the effects of the five anthraquinones components on apoptosis were measured by flow cytometry. Results The viability of HK-2 cells decreased with the increased concentration and administration time of emodin, aloe-emodin, rhein and chrysophanol significantly (P<0.05), but remained more than 75% after administration of 200 μmol·L^-1 physcion and chrysophanol for 48 hours. 48 hours after administration of 100 μmol·L^-1emodin, aloe-emodin and physcion, the release level of LDH was significantly higher than in the control group (P<0.01). After administration of the five anthraquinone monomers in Polygonum multiflorum, the level of Kim-1 in the HK-2 cell supernatant didn't increase with the concentration, but increased significantly compared with the control group after 100 μmol·L^-1 administration (P<0.05). The mitochondrial membrane potential of HK-2 cells decreased with the concentration of treatment and duration of administration after the administration of the five anthraquinone components in Polygonum multiflorum. Conclusion Emodin, aloe-emodin and rhein are considered the potential anthraquinone components that causes nephrotoxicity in Polygonum multiflorum, and the nephrotoxicity risk of other components in Polygonum multiflorum needs to be studied.

Key words: HK-2 cells, Polygonum multiflorum, anthraquinones components, cytotoxicity, LDH, Kim-1, mitochondrial membrane potential, safety

中图分类号:

R994.1
R917

兰洁, 文海若, 黄芝瑛, 汪祺, 马双成. 何首乌蒽醌类单体成分致HK-2细胞毒性研究[J]. 中国药物警戒, 2023, 20(6): 616-622.

LAN Jie, WEN Hairuo, HUANG Zhiying, WANG Qi, MA Shuangcheng. Cytotoxicity of HK-2 induced by anthraquinones components of Polygonum multiflorum[J]. Chinese Journal of Pharmacovigilance, 2023, 20(6): 616-622.

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