HepaRG细胞3D培养和代谢组学的新补骨脂异黄酮肝毒性研究

Study on the hepatotoxicity of neobavaisoflavone based on 3D culture of heparg cells and metabolomics

关键词:  新补骨脂异黄酮  肝毒性  HepaRG细胞  3D培养  代谢组学  脂质代谢

Key words:  neobavaisoflavone  hepatotoxicity  HepaRG cells  3D-culture  metabolomics  lipid metabolism

摘要:

[目的] 基于HepaRG细胞3D培养和代谢组学技术，研究新补骨脂异黄酮（NBIF）的肝毒性，并探讨其可能的机制。[方法] HepaRG细胞3D培养至第4天时，NBIF 0、2.5、5.0、10.0 μmol/L给药，每天1次，连续5次。第1次和第5次给药24 h后，细胞计数试剂盒-8（CCK-8）法测定HepaRG细胞活力；第5次给药24 h后，流式细胞仪测定HepaRG细胞凋亡率，基于超高效液相色谱-串联飞行时间质谱（UPLC-Q-TOF-MS）技术并结合多元统计分析方法，对细胞上清代谢谱进行分析，筛选差异代谢物及相关代谢通路。[结果] 随着NBIF给药浓度的升高和给药次数的增加，HepaRG细胞肝细胞活力降低；与对照组（6.86%）比较，NBIF 5.0 μmol/L（11.56%）、10.0 μmol/L（28.44%）组HepaRG 细胞的早期凋亡率升高；与对照组比较，NBIF给药筛出17个差异代谢物，含量均下调，NBIF 2.5、5.0、10.0 μmol/L组分别涉及5、6和6条通路。[结论] 新补骨脂异黄酮可致3D培养HepaRG细胞活力降低、凋亡率升高，其毒性机制可能与苯丙氨酸、酪氨酸和色氨酸生物合成、苯丙氨酸的代谢、脂肪酸及不饱和脂肪酸的生物合成等脂质代谢途径紊乱有关。

Abstract:

[Objective] To investigate the hepatotoxicity of neobavaisoflavone（NBIF） and explore its potential mechanism based on 3D culture of HepaRG cells and metabolomics technology. [Methods] HepaRG cells were subjected to 3D culture for 4 days，followed by treatment with NBIF at concentrations of 0，2.5，5，and 10 μmol/L once daily for 5 consecutive days. At 24 h after the first and fifth administrations，the viability of HepaRG cells was determined by the CCK-8 assay. At 24 h after the fifth administration，the apoptosis rate of HepaRG cells was detected by flow cytometry. Ultra-performance liquid chromatography-tandem time-of-flight mass spectrometry（UPLC-Q-TOF-MS） combined with multivariate statistical analysis was employed to analyze the metabolic profiles of cell supernatants，so as to screen differential metabolites and related metabolic pathways. [Results] With the increase of NBIF concentration and administration frequency，the viability of 3D-cultured HepaRG cells decreased significantly. Compared with the control group（6.86%），the early apoptosis rates of HepaRG cells in the 5 μmol/L（11.56%） and 10 μmol/L（28.44%） NBIF groups were notably elevated. A total of 17 differential metabolites with downregulated levels were identified in the NBIF-treated groups compared with the control group. Moreover，5，6，and 6 metabolic pathways were involved in the 2.5，5 and 10 μmol/L NBIF groups，respectively. [Conclusion] NBIF can reduce the viability and increase the apoptosis rate of 3D-cultured HepaRG cells. Its hepatotoxic mechanism may be associated with the disorder of multiple metabolic pathways，including the biosynthesis of phenylalanine，tyrosine and tryptophan，phenylalanine metabolism，as well as the biosynthesis of fatty acids and unsaturated fatty acids.