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目的建立高通量的高内涵分析(HCA)技术,探讨何首乌致肝损伤的可能物质及作用机制。方法人源肝癌细胞HepG2分别经不同质量浓度的何首乌不同极性提取物、何首乌主要单体成分二苯乙烯苷、芦荟大黄素、大黄素甲醚、儿茶素、大黄酚、大黄素、大黄酸、没食子酸孵育24 h后,利用Hoechst 33342等荧光探针对细胞进行染色,利用HCA技术检测并分析药物在不同浓度或不同孵育时间时对HepG2的细胞数目、细胞核形态、线粒体质量和线粒体膜电位的影响。结果何首乌醋酸乙酯提取物和二氯甲烷提取物在1 000μg/m L时对HepG2细胞增殖、形态和线粒体质量均有显著的影响,醋酸乙酯提取物还引起线粒体膜电位的明显下降。何首乌各单体成分在较低浓度(0.01、1μmol/L)时对细胞无显著影响;而在较高浓度(100μmol/L)时,芦荟大黄素、大黄素、大黄酸和没食子酸可引起细胞数目的明显下降,芦荟大黄素还引起细胞核的肿胀、导致细胞核面积增大。由大黄素和大黄酸的量效曲线可知,二者对细胞各指标的半数毒性浓度(TC50)值与文献报道基本一致。各成分不同孵育时间(24、48、72 h)对细胞数目和细胞核面积的影响未出现显著差异。结论蒽醌类成分芦荟大黄素、大黄素、大黄酸和没食子酸可能是何首乌致肝毒性的主要成分,由各单体对线粒体质量和线粒体膜电位的影响推测何首乌致肝毒性可能与线粒体介导的细胞凋亡有关。HCA技术适用于中药复杂体系的肝毒性评价。
Objective To establish a high-throughput, high-content analysis (HCA) technique to explore the possible substances and mechanism of liver damage caused by Polygonum multiflorum. Methods HepG2 human liver cancer cells were different concentrations of Polygonum multiflorum extract, the main monomer of Polygonum multiflorum stilbene glucoside, aloe-emodin, physcion, catechin, chrysophanol, emodin, rhein After 24 h incubation with gallic acid, the cells were stained with a fluorescent probe such as Hoechst 33342, and the cell number, nuclear morphology, mitochondrial mass and mitochondrial membrane potential of HepG2 cells were detected and analyzed by HCA assay at different concentrations or different incubation times Impact. Results HepG2 cells proliferation, morphology and mitochondrial mass were significantly affected by ethyl acetate extract and methylene chloride extract of HepG2 at 1 000 μg / mL. Ethyl acetate extract also caused obvious decrease of mitochondrial membrane potential. Polygonum multiflorum monomer had no significant effect on the cells at lower concentrations (0.01, 1μmol / L), while at higher concentration (100μmol / L), aloe-emodin, emodin, rhein and gallic acid could cause cells The number of significant decline in aloe emodin also caused swelling of the nucleus, resulting in increased nuclear area. The quantitative and efficient curves of emodin and rhein show that the half-value of TC50 value of each of the two indicators in cells is consistent with that reported in the literature. There was no significant difference in the number of cells and the nucleus area between different incubation time (24, 48 and 72 h). CONCLUSION Anthraquinones such as aloe-emodin, emodin, rhein and gallic acid may be the main components of hepatotoxicity of Polygonum multiflorum. It is concluded that the hepatotoxicity of Polygonum multiflorum may be mediated by mitochondria mediated by the influence of each monomer on mitochondrial mass and mitochondrial membrane potential Related to apoptosis. HCA technology is suitable for the evaluation of hepatotoxicity of traditional Chinese medicine complicated system.