非酒精性脂肪性肝病细胞模型中胆固醇代谢紊乱机制

章玉婷; 王安慧; 杨晋妮; 林佳纯; 田媛; 董海娟; 张尊建; 宋瑞

doi:10.11665/j.issn.1000-5048.2023032401

非酒精性脂肪性肝病细胞模型中胆固醇代谢紊乱机制

1.
中国药科大学药物质量与安全预警教育部重点实验室，南京 210009
2.
中国药科大学公共实验室平台，南京 210009

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- 文章访问数: 115
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出版历程
- 收稿日期: 2023-03-23
- 修回日期: 2023-07-10
- 刊出日期: 2023-08-24

Mechanisms of cholesterol metabolism imbalance in a PA-induced non-alcoholic fatty liver disease cell model

1.
Key Laboratory of Drug Quality Control & Pharmacovigilance, China Pharmaceutical University, Ministry of Educational, Nanjing 210009
2.
The Public Laboratory Platform of China Pharmaceutical University, Nanjing 210009, China

摘要

摘要: 肝脏胆固醇代谢紊乱在非酒精性脂肪性肝病（NAFLD）的发生发展中具有重要作用。为揭示饱和脂肪酸诱导肝细胞胆固醇稳态失衡的分子机制，采用棕榈酸诱导HepG2细胞，通过油红O染色、试剂盒测定细胞内甘油三酯（TG）和胆固醇（TC）含量评估脂质累积；采用RT-qPCR和Western blot检测与胆固醇稳态相关基因和蛋白表达水平；LC-MS/MS检测细胞胆汁酸和线粒体氧甾醇水平。结果发现，棕榈酸处理后，细胞内脂滴明显累积且TG与TC含量显著升高（P < 0.000 1）；胆固醇合成和摄取相关基因表达无明显变化，但ABCG5和LXRα蛋白表达显著下调，表明胆固醇外排减少；负责胆汁酸替代合成的限速酶STARD1和CYP7B1基因表达显著增强，线粒体中胆固醇、27-羟基胆固醇（27-OHC）以及细胞中鹅去氧胆酸（CDCA）水平明显升高。本研究结果表明，棕榈酸刺激后可能通过抑制胆固醇外排和促进胆汁酸合成扰乱胆固醇稳态。
- 非酒精性脂肪性肝病 /
- 胆固醇代谢 /
- 氧甾醇 /
- 胆汁酸 /
- STARD1
Abstract: Liver cholesterol metabolism disorder plays an important role in the development of non-alcoholic fatty liver disease (NAFLD).In order to reveal the molecular mechanism of cholesterol homeostasis imbalance induced by saturated fatty acids, HepG2 cells were stimulated with palmitic acid (PA).Lipids accumulation was analyzed by Oil Red O staining, intracellular triglyceride and cholesterol quantification.The level of genes and proteins related to cholesterol homeostasis was measured by RT-qPCR and western blotting.Additionally, intracellular bile acids and mitochondrial oxysterols were detected by LC-MS/MS.The results demonstrated that intracellular lipids such as TG and TC were significantly increased in the model with PA stimulation.Although no significant difference was detected in genes related to cholesterol synthesis and uptake, the protein expression of ABCG5 and LXRα were significantly down-regulated, indicating a decrease in cholesterol efflux.Meanwhile, the gene expression of STARD1 and CYP7B1, which are responsible for bile acid alternative synthesis, were markedly enhanced, along with a significant increase of cholesterol and 27-OHC in mitochondria and CDCA in cells.These results suggested that PA overload may disrupt cholesterol homeostasis by inhibiting cholesterol efflux and promoting bile acids synthesis.
- non-alcoholic fatty liver disease(NAFLD) /
- cholesterol metabolism /
- oxysterols /
- bile acids /
- STARD1

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参考文献(37)

[1]	Haas JT, Francque S, Staels B. Pathophysiology and mechanisms of nonalcoholic fatty liver disease[J]. Annu Rev Physiol, 2016, 78: 181-205.
[2]	Younossi ZM, Koenig AB, Abdelatif D, et al. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes[J]. Hepatology, 2016, 64(1): 73-84.
[3]	Deprince A, Haas JT, Staels B. Dysregulated lipid metabolism links NAFLD to cardiovascular disease[J]. Mol Metab, 2020, 42: 101092.
[4]	Li H, Yu XH, Ou X, et al. Hepatic cholesterol transport and its role in non-alcoholic fatty liver disease and atherosclerosis[J]. Prog Lipid Res, 2021, 83: 101109.
[5]	Huang DQ, El-Serag HB, Loomba R. Global epidemiology of NAFLD-related HCC: trends, predictions, risk factors and prevention[J]. Nat Rev Gastroenterol Hepatol, 2021, 18(4): 223-238.
[6]	Jiao TY, Ma YD, Guo XZ, et al. Bile acid and receptors: biology and drug discovery for nonalcoholic fatty liver disease[J]. Acta Pharmacol Sin, 2022, 43(5): 1103-1119.
[7]	Luo J, Yang HY, Song BL. Mechanisms and regulation of cholesterol homeostasis[J]. Nat Rev Mol Cell Biol, 2020, 21(4): 225-245.
[8]	Klaassen CD, Aleksunes LM. Xenobiotic, bile acid, and cholesterol transporters: function and regulation[J]. Pharmacol Rev, 2010, 62(1): 1-96.
[9]	Peng KP, Mo ZN, Tian GX. Serum lipid abnormalities and nonalcoholic fatty liver disease in adult males[J]. Am J Med Sci, 2017, 353(3): 236-241.
[10]	Puri P, Baillie RA, Wiest MM, et al. A lipidomic analysis of nonalcoholic fatty liver disease[J]. Hepatology,2007,46(4): 1081-1090.
[11]	van Rooyen DM, Gan LT, Yeh MM, et al. Pharmacological cholesterol lowering reverses fibrotic NASH in obese, diabetic mice with metabolic syndrome[J].J Hepatol,2013,59(1): 144-152.
[12]	Mendez-Sanchez N, Cruz-Ramon VC, Ramirez-Perez OL, et al. New aspects of lipotoxicity in nonalcoholic steatohepatitis[J]. Int J Mol Sci, 2018, 19(7): 2034.
[13]	Mota M, Banini BA, Cazanave SC, et al. Molecular mechanisms of lipotoxicity and glucotoxicity in nonalcoholic fatty liver disease[J]. Metabolism, 2016, 65(8): 1049-1061.
[14]	Arguello G, Balboa E, Arrese M, et al. Recent insights on the role of cholesterol in non-alcoholic fatty liver disease[J]. Biochim Biophys Acta, 2015, 1852(9): 1765-1778.
[15]	Wang XH, Tian Y, Guo ZJ, et al. Cholesterol metabolism and expression of its relevant genes in cultured steatotic hepatocytes[J]. J Dig Dis, 2009, 10(4): 310-314.
[16]	Oliveira AF, Cunha DA, Ladriere L, et al. In vitro use of free fatty acids bound to albumin: a comparison of protocols[J]. Biotechniques, 2015, 58(5): 228-233.
[17]	Alseekh S, Aharoni A, Brotman Y, et al. Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices[J]. Nat Methods, 2021, 18(7): 747-756.
[18]	Silva LP, Lorenzi PL, Purwaha P, et al. Measurement of DNA concentration as a normalization strategy for metabolomic data from adherent cell lines[J]. Anal Chem, 2013, 85(20): 9536-9542.
[19]	Minowa K, Rodriguez-Agudo D, Suzuki M, et al. Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease[J]. J Lipid Res, 2023, 64(5): 100363.
[20]	Borah K, Rickman OJ, Voutsina N, et al. A quantitative LC-MS/MS method for analysis of mitochondrial-specific oxysterol metabolism[J]. Redox Biol, 2020, 36: 101595.
[21]	Henkel AS, LeCuyer B, Olivares S, et al. Endoplasmic reticulum stress regulates hepatic bile acid metabolism in mice[J]. Cell Mol Gastroenterol Hepatol, 2017, 3(2): 261-271.
[22]	Brown GT, Kleiner DE. Histopathology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis[J]. Metabolism, 2016, 65(8): 1080-1086.
[23]	Gambino R, Bugianesi E, Rosso C, et al. Different serum free fatty acid profiles in NAFLD subjects and healthy controls after oral fat load[J]. Int J Mol Sci, 2016, 17(4): 479.
[24]	Gómez-Lechón MJ, Donato MT, Martínez-Romero A, et al. A human hepatocellular in vitro model to investigate steatosis[J]. Chem Biol Interact, 2007, 165(2): 106-116.
[25]	Moravcová A, ?ervinková Z, Ku?era O, et al. The effect of oleic and palmitic acid on induction of steatosis and cytotoxicity on rat hepatocytes in primary culture[J]. Physiol Res, 2015, 64(Suppl 5): S627-S636.
[26]	Zhao MG, Yang HM, Jiang CH, et al. Intervention effects of the triterpenoids from Cyclocaryapaliurus on free fatty acids-induced steatosis in HepG2 cells[J]. J China Pharm Univ (中国药科大学学报), 2018, 49(3): 333-340.
[27]	van Rooyen DM, Larter CZ, Haigh WG, et al. Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis[J]. Gastroenterology, 2011, 141(4): 1393-1403.
[28]	Aguilar-Olivos NE, Carrillo-Córdova D, Oria-Hernández J, et al. The nuclear receptor FXR, but not LXR, up-regulates bile acid transporter expression in non-alcoholic fatty liver disease[J]. Ann Hepatol, 2015, 14(4): 487-493.
[29]	Buccitelli C, Selbach M. mRNAs, proteins and the emerging principles of gene expression control[J]. Nat Rev Genet, 2020, 21(10): 630-644.
[30]	Jiao N, Baker SS, Chapa-Rodriguez A, et al. Suppressed hepatic bile acid signalling despite elevated production of primary and secondary bile acids in NAFLD[J]. Gut, 2018, 67(10): 1881-1891.
[31]	Conde de la Rosa L, Garcia-Ruiz C, Vallejo C, et al. STARD1 promotes NASH-driven HCC by sustaining the generation of bile acids through the alternative mitochondrial pathway[J]. J Hepatol, 2021, 74(6): 1429-1441.
[32]	Mouzaki M, Wang AY, Bandsma R, et al. Bile acids and dysbiosis in non-alcoholic fatty liver disease[J]. PLoS One, 2016, 11(5): e0151829.
[33]	Miyake JH, Wang SL, Davis RA. Bile acid induction of cytokine expression by macrophages correlates with repression of hepatic cholesterol 7alpha-hydroxylase[J]. J Biol Chem, 2000, 275(29): 21805-21808.
[34]	Montero J, Mari M, Colell A, et al. Cholesterol and peroxidized cardiolipin in mitochondrial membrane properties, permeabilization and cell death[J]. Biochim Biophys Acta, 2010, 1797(6/7): 1217-1224.
[35]	Garenc C, Julien P, Levy E. Oxysterols in biological systems: the gastrointestinal tract, liver, vascular wall and central nervous system[J]. Free Radic Res, 2010, 44(1): 47-73.
[36]	Kakiyama G, Minowa K, Rodriguez-Agudo D, et al. Coffee modulates insulin-hepatocyte nuclear factor-4α-Cyp7b1 pathway and reduces oxysterol-driven liver toxicity in a nonalcoholic fatty liver disease mouse model[J]. Am J Physiol Gastrointest Liver Physiol, 2022, 323(5): G488-G500.
[37]	Bieghs V, Hendrikx T, van Gorp PJ, et al. The cholesterol derivative 27-hydroxycholesterol reduces steatohepatitis in mice[J]. Gastroenterology, 2013, 144(1): 167-178.

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非酒精性脂肪性肝病细胞模型中胆固醇代谢紊乱机制

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出版历程

Mechanisms of cholesterol metabolism imbalance in a PA-induced non-alcoholic fatty liver disease cell model

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