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在棕榈酸诱导的 体外 模型中研究鸭嘴豆素D对非酒精性脂肪肝疾病的保护作用

Published: December 02, 2022
doi:
10.3791/64816
, , , , , , ,
1Chongqing Academy of Chinese Materia Medica, 2Bioengineering College of Chongqing University

Summary

该方案研究了桔霉素D在棕榈酸诱导的 体外 模型中对非酒精性脂肪性肝病的保护作用。

Abstract

非酒精性脂肪性肝病（NAFLD）的发病率在全球范围内以惊人的速度增加。 大鸭 嘴龙被广泛用作治疗各种疾病的传统民族医学，是一种典型的功能性食品，可以纳入日常饮食。研究表明，扁平紫芪D（PD）是 大花桫椤的主要活性成分之一，具有很高的生物利用度，可显着缓解NAFLD的进展，但其潜在机制尚不清楚。本研究旨在探讨PD在 体外对NAFLD的治疗效果。用300μM棕榈酸（PA）预处理AML-12细胞24小时，以在 体外模拟NAFLD。然后，细胞要么用PD处理，要么在24小时内不接受PD处理。采用2′，7′-二氯-二氢荧光素二乙酸酯（DCFH-DA）染色分析活性氧（ROS）水平，采用JC-1染色法测定线粒体膜电位。此外，通过蛋白质印迹分析细胞裂解物中LC3-II/LC3-I和p62/SQSTM1的蛋白表达水平。与对照组相比，PD发现PA处理组的ROS和线粒体膜电位水平显着降低。同时，与对照组相比，PA处理组PD升高了LC3-II/LC3-I水平，降低了p62/SQSTM1水平。结果表明，PD通过减少氧化应激和刺激自噬来改善体 外 NAFLD。该 体外 模型是研究PD在NAFLD中的作用的有用工具。

Introduction

Platycodon grandiflorus （PG），这是Platycodon grandiflorus（Jacq）的干根。A.DC.，用于传统中医（TCM）。主要产于我国东北、华北、华东、中部、西南地区1.PG组分包括三萜皂苷、多糖、黄酮类、多酚、聚乙二醇、挥发油和矿物质2。PG在亚洲被用作食品和草药的历史悠久。传统上，这种草药被用来制造治疗肺部疾病的药物。现代药理学也提供了PG治疗其他疾病疗效的证据。研究表明，PG对多种药物性肝损伤模型具有治疗作用。膳食补充PG或桔霉素提取物可以改善高脂肪饮食引起的肥胖及其相关的代谢疾病3，4，5。来自PG的多糖可用于治疗LPS/D-GalN引起的小鼠急性肝损伤6。此外，来自PG根部的皂苷可改善高脂肪饮食引起的非酒精性脂肪性肝炎（NASH）7。此外，铂族最重要的治疗成分之一桔霉素D（PD）可以增强人肝细胞癌（HepG2）细胞中低密度脂蛋白受体表达和低密度脂蛋白摄取8。此外，PD还可以诱导细胞凋亡并抑制HepG2细胞中的粘附，迁移和侵袭9，10。因此，本研究使用小鼠肝癌AML-12细胞进行体外模型构建，并进一步研究该模型中PD的药理作用和潜在机制。

术语非酒精性脂肪性肝病（NAFLD）是指一组肝脏疾病，包括单纯脂肪变性，NASH，肝硬化和肝细胞癌11。尽管NAFLD的发病机制尚不完全清楚，但从经典的“两击”理论到目前的“多次命中”理论，胰岛素抵抗被认为是NAFLD发病机制的核心12，13，14。研究表明，肝细胞中的胰岛素抵抗可能导致游离脂肪酸增加，游离脂肪酸形成沉淀在肝脏中的甘油三酯并导致肝脏变胖15，16.脂肪的积累可导致脂毒性、氧化应激诱导的线粒体功能障碍、内质网应激和炎性细胞因子释放，导致NAFLD17，18的发病机制和进展。此外，自噬也在NAFLD的发病机制中发挥作用，因为它参与调节细胞胰岛素敏感性，细胞脂质代谢，肝细胞损伤和先天免疫19，20，21。

已经建立了多种动物模型和细胞模型，为探索NAFLD22，23的发病机制和潜在治疗靶点提供了基础。然而，单个动物模型不能完全模拟NAFLD24的所有病理过程。动物之间的个体差异导致不同的病理特征。在NAFLD的 体外 研究中使用肝细胞系或原代肝细胞可确保实验条件下的最大一致性。肝脂代谢失调可导致NAFLD25中肝细胞脂滴积聚水平较高。油酸和棕榈油等游离脂肪酸已在 体外 模型中用于模拟由高脂肪饮食引起的NAFLD26，27。人肝母细胞瘤细胞系HepG2常用于 体外NAFLD模型的构建，但作为肿瘤细胞系，HepG2细胞的代谢与正常生理条件下的肝细胞代谢明显不同28。因此，使用原代肝细胞或小鼠原代肝细胞构建体 外 NAFLD模型进行药物筛选比使用肿瘤细胞系更有利。比较动物模型和 体 外肝细胞模型中药物效果和治疗靶点的协同检查，似乎利用小鼠肝细胞构建体 外 NAFLD模型具有更好的应用潜力。

进入肝脏的游离脂肪酸被氧化以产生能量或储存为甘油三酯。值得注意的是，游离脂肪酸具有一定的脂毒性，并可能诱导细胞功能障碍和细胞凋亡12。棕榈酸（PA）是人血浆中最丰富的饱和脂肪酸29。当非脂肪组织中的细胞长时间暴露于高浓度的PA时，这会刺激活性氧（ROS）的产生并引起氧化应激，脂质积累，甚至细胞凋亡30。因此，许多研究人员使用PA作为诱导剂来刺激肝细胞产生ROS，从而构建体外脂肪肝疾病模型并评估某些活性物质对细胞的保护作用31，32，33，34。本研究介绍了一种研究PD对PA诱导的NAFLD细胞模型的保护作用的方案。

Protocol

AML-12细胞（一种正常的小鼠肝细胞系）用于基于细胞的研究。细胞是从商业来源获得的（见 材料表）。 1. AML-12细胞的预处理以体外模拟NAFLD 将细胞维持在正常细胞培养基（DMEM 加含有 0.005 mg/mL 胰岛素、5 ng/mL 硒、0.005 mg/mL 转铁蛋白、40 ng/mL 地塞米松和 10% 胎牛血清 [FBS]，参见 材料表）中的细胞在 37 °C 下，在含有 5% CO2</…

Representative Results

细胞内的细胞内ROS用300 μM PA诱导AML-12细胞24 h，建立NAFLD细胞模型。随后，用PD处理细胞24小时。用DCFH-DA荧光探针标记细胞，并在荧光显微镜下观察ROS的产生。细胞内ROS的DCFH-DA染色结果如图 1所示。结果表明，PD可显著降低300 μMPA孵育细胞中的细胞内ROS水平（P < 0.01），表明PD可以降低细胞氧化应激。 细胞的线粒体膜电位…

Discussion

研究强调，NAFLD是一种临床病理综合征，从脂肪肝到NASH，可进展为肝硬化和肝癌51。高脂肪饮食和不活跃的生活方式是NAFLD的典型危险因素。非药物治疗和用于NAFLD治疗的药物治疗都已研究51，52，53。然而，NAFLD的发病机制尚未完全阐明。这里描述的方法涉及由PA刺激的AML-12细胞建立的NAFLD体外模型。我们…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了重庆市科委（cstc2020jxjl-jbky10002、jbky20200026、cstc2021jscx-dxwtBX0013、jbky20210029）和中国博士后科学基金（编号：2021MD703919）的资助。

Materials

5% BSA Blocking Buffer Solarbio, Beijing, China SW3015
AML12 (alpha mouse liver 12) cell line Procell Life Science&Technology Co., Ltd, China AML12
Beyo ECL Plus Beyotime, Shanghai, China P0018S
Bio-safety cabinet Esco Micro Pte Ltd, Singapore AC2-5S1 A2 
cellSens Olympus, Tokyo, Japan 1.8
Culture CO2 Incubator Esco Micro Pte Ltd, Singapore CCL-170B-8
Dexamethasone Beyotime, Shanghai, China ST125
Dimethyl sulfoxide Solarbio, Beijing, China D8371
DMEM/F12 Hyclone, Logan, UT, USA SH30023.01
Foetal Bovine Serum Hyclone, Tauranga, New Zealand SH30406.05
Graphpad software GraphPad Software Inc., San Diego, CA, USA 8.0
HRP Goat Anti-Mouse IgG (H+L) ABclonal, Wuhan, China AS003
Hydrophobic PVDF Transfer Membrane Merck, Darmstadt, Germany IPFL00010
Insulin, Transferrin, Selenium Solution, 100× Beyotime, Shanghai, China C0341
MAP LC3β Antibody Santa Cruz Biotechnology (Shanghai) Co., Ltd SC-376404
Mitochondrial Membrane Potential Assay Kit with JC-1 Solarbio, Beijing, China M8650
Olympus Inverted Microscope IX53 Olympus, Tokyo, Japan IX53
Palmitic Acid Sigma, Germany P0500
Penicillin-Streptomycin Solution (100x) Hyclone, Logan, UT, USA SV30010
Phenylmethanesulfonyl fluoride Beyotime, Shanghai, China ST506
Phosphate Buffered Solution Hyclone, Logan, UT, USA BL302A
Platycodin D Chengdu Must Bio-Technology Co., Ltd, China CSA: 58479-68-8
Protease inhibitor cocktail for general use, 100x Beyotime, Shanghai, China P1005
Protein Marker Solarbio, Beijing, China PR1910
Reactive Oxygen Species Assay Kit Solarbio, Beijing, China CA1410
RIPA Lysis Buffer Beyotime, Shanghai, China P0013E
SDS-PAGE Gel Quick Preparation Kit Beyotime, Shanghai, China P0012AC
SDS-PAGE Sample Loading Buffer, 5x Beyotime, Shanghai, China P0015
Sigma Centrifuge Sigma, Germany 3K15
SQSTM1/p62 Antibody Santa Cruz Biotechnology (Shanghai) Co., Ltd SC-28359
Tecan Infinite 200 PRO   Tecan Austria GmbH, Austria 1510002987
WB Transfer Buffer,10x Solarbio, Beijing, China D1060
β-Actin Mouse mAb ABclonal, Wuhan, China AC004
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References

  1. Xunyan, X. Y., Fang, X. M. The effect of Platycodon grandiflorum and its historical change in the clinical application of Platycodonis radix. Zhonghua Yi Shi Za Shi. 51 (3), 167-176 (2021).
  2. Ma, X., et al. Platycodon grandiflorum extract: Chemical composition and whitening, antioxidant, and anti-inflammatory effects. RSC Advances. 11 (18), 10814-10826 (2021).
  3. Ke, W., et al. Dietary Platycodon grandiflorus attenuates hepatic insulin resistance and oxidative stress in high-fat-diet induced non-alcoholic fatty liver disease. Nutrients. 12 (2), 480 (2020).
  4. Kim, Y. J., et al. Platycodon grandiflorus root extract attenuates body fat mass, hepatic steatosis and insulin resistance through the interplay between the liver and adipose tissue. Nutrients. 8 (9), 532 (2016).
  5. Park, H. M., et al. Mass spectrometry-based metabolomic and lipidomic analyses of the effects of dietary Platycodon grandiflorum on liver and serum of obese mice under a high-fat diet. Nutrients. 9 (1), 71 (2017).
  6. Qi, C., et al. Platycodon grandiflorus polysaccharide with anti-apoptosis, anti-oxidant and anti-inflammatory activity against LPS/D-GalN induced acute liver injury in mice. Journal of Polymers and the Environment. 29 (12), 4088-4097 (2021).
  7. Choi, J. H., et al. Saponins from the roots of Platycodon grandiflorum ameliorate high fat diet-induced non-alcoholic steatohepatitis. Biomedicine & Pharmacotherapy. 86, 205-212 (2017).
  8. Choi, Y. J., et al. Platycodin D enhances LDLR expression and LDL uptake via down-regulation of IDOL mRNA in hepatic cells. Scientific Reports. 10, 19834 (2020).
  9. Li, T., et al. Platycodin D triggers autophagy through activation of extracellular signal-regulated kinase in hepatocellular carcinoma HepG2 cells. European Journal of Pharmacology. 749, 81-88 (2015).
  10. Lu, J. -. J., et al. Proteomic analysis of hepatocellular carcinoma HepG2 cells treated with platycodin D. Chinese Journal of Natural Medicines. 13 (9), 673-679 (2015).
  11. Neuschwander-Tetri, B. A. Therapeutic landscape for NAFLD in 2020. Gastroenterology. 158 (7), 1984-1998 (2020).
  12. Friedman, S. L., Neuschwander-Tetri, B. A., Rinella, M., Sanyal, A. J. Mechanisms of NAFLD development and therapeutic strategies. Nature Medicine. 24 (7), 908-922 (2018).
  13. Bessone, F., Razori, M. V., Roma, M. G. Molecular pathways of nonalcoholic fatty liver disease development and progression. Cellular and Molecular Life Sciences. 76 (1), 99-128 (2019).
  14. Buzzetti, E., Pinzani, M., Tsochatzis, E. A. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metabolism. 65 (8), 1038-1048 (2016).
  15. Watt, M. J., Miotto, P. M., De Nardo, W., Montgomery, M. K. The liver as an endocrine organ-Linking NAFLD and insulin resistance. Endocrine Reviews. 40 (5), 1367-1393 (2019).
  16. Khan, R. S., Bril, F., Cusi, K., Newsome, P. N. Modulation of insulin resistance in nonalcoholic fatty liver disease. Hepatology. 70 (2), 711-724 (2019).
  17. Karkucinska-Wieckowska, A., et al. Mitochondria, oxidative stress and nonalcoholic fatty liver disease: A complex relationship. European Journal of Clinical Investigation. 52 (3), 13622 (2022).
  18. Tilg, H., Adolph, T. E., Dudek, M., Knolle, P. Non-alcoholic fatty liver disease: The interplay between metabolism, microbes and immunity. Nature Metabolism. 3 (12), 1596-1607 (2021).
  19. Qian, H., et al. Autophagy in liver diseases: A review. Molecular Aspects of Medicine. 82, 100973 (2021).
  20. Du, J., Ji, Y., Qiao, L., Liu, Y., Lin, J. Cellular endo-lysosomal dysfunction in the pathogenesis of non-alcoholic fatty liver disease. Liver International. 40 (2), 271-280 (2020).
  21. Allaire, M., Rautou, P. E., Codogno, P., Lotersztajn, S. Autophagy in liver diseases: Time for translation. Journal of Hepatology. 70 (5), 985-998 (2019).
  22. Kanuri, G., Bergheim, I. In vitro and in vivo models of non-alcoholic fatty liver disease (NAFLD). International Journal of Molecular Sciences. 14 (6), 11963-11980 (2013).
  23. Lau, J. K., Zhang, X., Yu, J. Animal models of non-alcoholic fatty liver disease: Current perspectives and recent advances. The Journal of Pathology. 241 (1), 36-44 (2017).
  24. Reimer, K. C., Wree, A., Roderburg, C., Tacke, F. New drugs for NAFLD: Lessons from basic models to the clinic. Hepatology International. 14 (1), 8-23 (2020).
  25. Carpino, G., et al. Increased liver localization of lipopolysaccharides in human and experimental NAFLD. Hepatology. 72 (2), 470-485 (2020).
  26. Vergani, L. Fatty acids and effects on in vitro and in vivo models of liver steatosis. Current Medicinal Chemistry. 26 (19), 3439-3456 (2019).
  27. Scorletti, E., Carr, R. M. A new perspective on NAFLD: Focusing on lipid droplets. Journal of Hepatology. 76 (4), 934-945 (2022).
  28. Green, C. J., Pramfalk, C., Morten, K. J., Hodson, L. From whole body to cellular models of hepatic triglyceride metabolism: Man has got to know his limitations. American Journal of Physiology-Endocrinology and Metabolism. 308 (1), 1-20 (2015).
  29. Gambino, R., et al. Different serum free fatty acid profiles in NAFLD subjects and healthy controls after oral fat load. International Journal of Molecular Sciences. 17 (4), 479 (2016).
  30. Marra, F., Svegliati-Baroni, G. Lipotoxicity and the gut-liver axis in NASH pathogenesis. Journal of Hepatology. 68 (2), 280-295 (2018).
  31. Zhang, J., Zhang, H., Deng, X., Zhang, Y., Xu, K. Baicalin protects AML-12 cells from lipotoxicity via the suppression of ER stress and TXNIP/NLRP3 inflammasome activation. Chemico-Biological Interactions. 278, 189-196 (2017).
  32. Liang, Y., et al. γ-Linolenic acid prevents lipid metabolism disorder in palmitic acid-treated alpha mouse liver-12 cells by balancing autophagy and apoptosis via the LKB1-AMPK-mTOR pathway. Journal of Agricultural and Food Chemistry. 69 (29), 8257-8267 (2021).
  33. Peng, Z., et al. Nobiletin alleviates palmitic acid-induced NLRP3 inflammasome activation in a sirtuin 1dependent manner in AML12 cells. Molecular Medicine Reports. 18 (6), 5815-5822 (2018).
  34. Xu, T., et al. Ferulic acid alleviates lipotoxicity-induced hepatocellular death through the SIRT1-regulated autophagy pathway and independently of AMPK and Akt in AML-12 hepatocytes. Nutrition & Metabolism. 18 (1), 13 (2021).
  35. Aranda, A., et al. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay: A quantitative method for oxidative stress assessment of nanoparticle-treated cells. Toxicology in Vitro. 27 (2), 954-963 (2013).
  36. Eruslanov, E., Kusmartsev, S. Identification of ROS using oxidized DCFDA and flow-cytometry. Methods in Molecular Biology. 594, 57-72 (2010).
  37. Bankhead, P. . Analyzing Fluorescence Microscopy Images with ImageJ. , (2014).
  38. Wiesmann, V., et al. Review of free software tools for image analysis of fluorescence cell micrographs. Journal of Microscopy. 257 (1), 39-53 (2015).
  39. Lugli, E., Troiano, L., Cossarizza, A. Polychromatic analysis of mitochondrial membrane potential using JC-1. Current Protocols in Cytometry. , (2007).
  40. Sivandzade, F., Bhalerao, A., Cucullo, L. Analysis of the mitochondrial membrane potential using the cationic JC-1 dye as a sensitive fluorescent probe. Bio-protocol. 9 (1), 3128 (2019).
  41. Chazotte, B. Labeling mitochondria with JC-1. Cold Spring Harbor Protocols. 2011 (9), (2011).
  42. Walker, J. M. The bicinchoninic acid (BCA) assay for protein quantitation. The Protein Protocols Handbook. , 11-15 (2009).
  43. Goldman, A., Ursitti, J. A., Mozdzanowski, J., Speicher, D. W. Electroblotting from polyacrylamide gels. Current Protocols in Protein Science. 82, 1-16 (2015).
  44. Mozdzanowski, J., Speicher, D. W. Proteins from polyacrylamide gels onto PVDF membranes. Current Research in Protein Chemistry. , 87 (2012).
  45. Taylor, S. C., Posch, A. The design of a quantitative western blot experiment. Biomed Research International. 2014, 361590 (2014).
  46. Motulsky, H. J. Graphpad Statistics Guide. Options for multiple t tests. Graphpad. , (2020).
  47. Poltorak, A. Cell death: All roads lead to mitochondria. Current Biology. 32 (16), 891-894 (2022).
  48. Dadsena, S., Jenner, A., García-Sáez, A. J. Mitochondrial outer membrane permeabilization at the single molecule level. Cellular and Molecular Life Sciences. 78 (8), 3777-3790 (2021).
  49. Green, D. R., Kroemer, G. The pathophysiology of mitochondrial cell death. Science. 305 (5684), 626-629 (2004).
  50. Lange, N. F., Radu, P., Dufour, J. F. Prevention of NAFLD-associated HCC: Role of lifestyle and chemoprevention. Journal of Hepatology. 75 (5), 1217-1227 (2021).
  51. Liu, X., Zhang, Y., Ma, C., Lin, J., Du, J. Alternate-day fasting alleviates high fat diet induced non-alcoholic fatty liver disease through controlling PPARalpha/Fgf21 signaling. Molecular Biology Reports. 49 (4), 3113-3122 (2022).
  52. Romero-Gomez, M., Zelber-Sagi, S., Trenell, M. Treatment of NAFLD with diet, physical activity and exercise. Journal of Hepatology. 67 (4), 829-846 (2017).
  53. Mizushima, N., Levine, B. Autophagy in human diseases. New England Journal of Medicine. 383 (16), 1564-1576 (2020).
  54. Cui, B., Yu, J. M. Autophagy: A new pathway for traditional Chinese medicine. Journal of Asian Natural Products Research. 20 (1), 14-26 (2018).
  55. Law, B. Y., et al. New potential pharmacological functions of Chinese herbal medicines via regulation of autophagy. Molecules. 21 (3), 359 (2016).
  56. Zhou, H., et al. Research progress in use of traditional Chinese medicine monomer for treatment of non-alcoholic fatty liver disease. European Journal of Pharmacology. 898, 173976 (2021).
  57. Zhang, L., Yao, Z., Ji, G. Herbal extracts and natural products in alleviating non-alcoholic fatty liver disease via activating autophagy. Frontiers in Pharmacology. 9, 1459 (2018).
  58. Zhang, X., et al. C-X-C motif chemokine 10 impairs autophagy and autolysosome formation in non-alcoholic steatohepatitis. Theranostics. 7 (11), 2822-2836 (2017).
  59. Li, C. X., et al. Allyl isothiocyanate ameliorates lipid accumulation and inflammation in nonalcoholic fatty liver disease via the Sirt1/AMPK and NF-kappaB signaling pathways. World Journal of Gastroenterology. 25 (34), 5120-5133 (2019).
  60. Li, S., et al. Sirtuin 3 acts as a negative regulator of autophagy dictating hepatocyte susceptibility to lipotoxicity. Hepatology. 66 (3), 936-952 (2017).
  61. Farrell, G. C., Teoh, N. C., McCuskey, R. S. Hepatic microcirculation in fatty liver disease. The Anatomical Record. 291 (6), 684-692 (2008).
  62. Milner, E., et al. Emerging three-dimensional hepatic models in relation to traditional two-dimensional in vitro assays for evaluating drug metabolism and hepatoxicity. Medicine in Drug Discovery. 8, 100060 (2020).
  63. Zhang, X., Jiang, T., Chen, D., Wang, Q., Zhang, L. W. Three-dimensional liver models: State of the art and their application for hepatotoxicity evaluation. Critical Reviews in Toxicology. 50 (4), 279-309 (2020).
  64. Bilson, J., Sethi, J. K., Byrne, C. D. Non-alcoholic fatty liver disease: A multi-system disease influenced by ageing and sex, and affected by adipose tissue and intestinal function. Proceedings of the Nutrition Society. 81 (2), 146-161 (2022).

Tags

AML-12 CellsNon-alcoholic Fatty Liver Disease (NAFLD)Platycodin DPalmitic AcidCell ModelIn VitroOxidative StressMitochondrial Membrane PotentialMicroscopy
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Wen, X., Wang, J., Fan, J., Chu, R., Chen, Y., Xing, Y., Li, N., Wang, G. Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model. J. Vis. Exp. (190), e64816, doi:10.3791/64816 (2022).
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