Transcriptomic and Proteomics Analysis of a Lipid-Loaded HepaRG Model for Steatosis Reveals Altered Regulation in Lipid and Xenobiotic Metabolism.

IF 1.8 4区医学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

Current drug metabolism Pub Date : 2025-09-09 DOI:10.2174/0113892002381234250727004847

Anitha Saravanakumar, Cassandra A Tierney, Wen He, Rohitash Jamwal, Benjamin Barlock, Xin Bush, Jillian G Johnson, David A Rodrigues, Fatemeh Akhlaghi

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引用次数: 0

Abstract

Introduction: Hepatic lipid accumulation (steatosis) is an early indicator of non-alcoholic fatty liver disease (NAFLD), preceding fibrosis and cirrhosis. Understanding its effects on drug-me-tabolizing enzymes (DMEs) and transporters is crucial for assessing potential alterations in drug dis-position among NAFLD patients. This study aimed to replicate steatosis in an in vitro HepaRG cell model and analyze its impact on DMEs and transporters.

Methods: Differentiated HepaRG cells were treated with a mixture of saturated (palmitate) and unsatu-rated (oleate) fatty acids (in a 1:2 ratio at 0.5 mM), complexed with BSA for 72 hours to induce lipid accumulation. Confirmation of steatosis was performed using Oil Red O staining and triglyceride (TG) quantification, while cell viability was assessed via the WST-1 assay. RNA sequencing and SWATH-MS proteomic analysis were employed to identify differentially expressed transcripts and proteins in lipid-loaded cells compared to controls.

Results: Lipid loading resulted in a ~6-fold increase in TG concentration without compromising cell viability. Transcriptomic analysis identified 393 differentially expressed transcripts (89 upregulated, 304 downregulated), while proteomic analysis detected 165 differentially expressed proteins (127 up-regulated, 38 downregulated). Notably, key mRNA transcripts related to transcription factors (NR1I2, HNF4α), phase 1 DMEs (CYP1A2, 2B6, 2C8, 2C9, 2C19, 3A4), phase 2 DMEs (UGT1A6, 2B7, SULT2A1, 1E1), and transporters (ABCC11, ABCG5, SLCO2B1, SLC10A1) exhibited significant downregulation.

Discussion: The observed alterations in DMEs and transporters suggest a potential shift in drug me-tabolism pathways under NAFLD conditions. Downregulation of transcription factors and metabolic enzymes could impact drug efficacy and toxicity, necessitating further research into the pharmacoki-netic implications.

Conclusion: The in vitro hepatic steatosis model demonstrated significant changes in the expression of clinically relevant DMEs and transporters. These findings highlight the importance of considering NAFLD-induced metabolic alterations when assessing drug disposition in affected patients.

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脂质负载HepaRG模型的转录组学和蛋白质组学分析揭示了脂质和外源代谢的调节改变。

肝脂质积累（脂肪变性）是非酒精性脂肪性肝病（NAFLD）的早期指标，是纤维化和肝硬化的前兆。了解其对药物代谢酶（DMEs）和转运体的影响对于评估NAFLD患者药物处置的潜在改变至关重要。本研究旨在在体外HepaRG细胞模型中复制脂肪变性，并分析其对DMEs和转运体的影响。方法：分化的HepaRG细胞用饱和（棕榈酸）和不饱和（油酸）脂肪酸的混合物（以1:2的比例在0.5 mM处）与BSA络合处理72小时，以诱导脂质积累。通过油红O染色和甘油三酯（TG）定量来确认脂肪变性，同时通过WST-1法评估细胞活力。采用RNA测序和SWATH-MS蛋白质组学分析鉴定脂质负载细胞中与对照组相比差异表达的转录本和蛋白质。结果：脂质负载导致TG浓度增加约6倍，但不影响细胞活力。转录组学分析鉴定出393个差异表达转录物（89个上调，304个下调），而蛋白质组学分析检测到165个差异表达蛋白（127个上调，38个下调）。值得注意的是，转录因子（NR1I2、HNF4α）、1期DMEs （CYP1A2、2B6、2C8、2C9、2C19、3A4）、2期DMEs （UGT1A6、2B7、SULT2A1、1E1）和转运体（ABCC11、ABCG5、SLCO2B1、SLC10A1）相关的关键mRNA转录物均出现显著下调。讨论：观察到的DMEs和转运体的改变提示NAFLD条件下药物代谢途径的潜在转变。转录因子和代谢酶的下调可能会影响药物的疗效和毒性，需要进一步研究药代动力学的意义。结论：体外肝脂肪变性模型临床相关DMEs及转运蛋白表达发生显著变化。这些发现强调了在评估受影响患者的药物配置时考虑nafld诱导的代谢改变的重要性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Current drug metabolism 医学-生化与分子生物学

CiteScore

4.30

自引率

4.30%

发文量

审稿时长

4-8 weeks

期刊介绍： Current Drug Metabolism aims to cover all the latest and outstanding developments in drug metabolism, pharmacokinetics, and drug disposition. The journal serves as an international forum for the publication of full-length/mini review, research articles and guest edited issues in drug metabolism. Current Drug Metabolism is an essential journal for academic, clinical, government and pharmaceutical scientists who wish to be kept informed and up-to-date with the most important developments. The journal covers the following general topic areas: pharmaceutics, pharmacokinetics, toxicology, and most importantly drug metabolism. More specifically, in vitro and in vivo drug metabolism of phase I and phase II enzymes or metabolic pathways; drug-drug interactions and enzyme kinetics; pharmacokinetics, pharmacokinetic-pharmacodynamic modeling, and toxicokinetics; interspecies differences in metabolism or pharmacokinetics, species scaling and extrapolations; drug transporters; target organ toxicity and interindividual variability in drug exposure-response; extrahepatic metabolism; bioactivation, reactive metabolites, and developments for the identification of drug metabolites. Preclinical and clinical reviews describing the drug metabolism and pharmacokinetics of marketed drugs or drug classes.