何首乌蒽醌类单体成分致HK-2细胞毒性研究  

Cytotoxicity of HK-2 induced by anthraquinones components of Polygonum multiflorum

作　　者：兰洁 文海若 黄芝瑛[2] 汪祺[1] 马双成[1] LAN Jie;WEN Hairuo;HUANG Zhiying;WANG Qi;MA Shuangcheng(National Center for Safety Evaluation of Drugs,National Institutes for Food and Drug Control,Beijing 100050,China;School of Pharmaceutical Sciences,Sun Yat-sen University,Guangzhou Guangdong 510006,China)

机构地区：[1]中国食品药品检定研究院国家药物安全评价监测中心,北京100050 [2]中山大学药学院,广东广州510006

出　　处：《中国药物警戒》2023年第6期616-622,628,共8页Chinese Journal of Pharmacovigilance

基　　金：国家科技重大专项重大新药创制(2018ZX09201017);国家自然科学基金资助项目(81973476)。

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、160μmol·L^(-1)的大黄素、芦荟大黄素、大黄酸和3.125、12.5、25、50、100、150、200μmol·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、100μmol·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200μmol·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100μmol·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100μmol·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、100μmol·L^(-1)的大黄素、芦荟大黄素和50、100μmol·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。Objective To determine the correlation between nephrotoxicity and five components of Polygonum multiflorum.Methods After HK-2 cells were treated with 2.5,10,20,40,80,120,160μmol·L^(-1) emodin,aloe-emodin,rhein and 3.125,12.5,25,50,100,150,200μmol·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 100μmol·L^(-1) emodin,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 200μmol·L^(-1) physcion and chrysophanol for 48 hours.48 hours after administration of 100μmol·L^(-1) emodin,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 100μmol·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 Polygon

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 

分 类 号：R994.1[医药卫生—毒理学] R917[医药卫生—药学]