{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T21:03:01Z","timestamp":1792011781398,"version":"4.4.0"},"reference-count":54,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,10,19]],"date-time":"2023-10-19T00:00:00Z","timestamp":1697673600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100012273","name":"Lotte and John Hecht Memorial Foundation","doi-asserted-by":"publisher","award":["3693"],"award-info":[{"award-number":["3693"]}],"id":[{"id":"10.13039\/100012273","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Pharmacol."],"abstract":"<jats:p>\n                    <jats:bold>Objectives:<\/jats:bold>\n                    Non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH) and hepatocarcinoma is a serious and growing problem. However, the development of new therapies is severely hindered by a lack of high-throughput assays for drug testing.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Methods:<\/jats:bold>\n                    We have developed a simple transwell assay comprised of HepG2 hepatocytes, hepatic LX-2 stellate cells, and differentiated THP-1 cells. The cells were incubated with an activating mixture containing the NASH-associated risk factors, glucose, insulin, free fatty acids (FFAs), and lipopolysaccharide (LPS) for 72\u00a0h. We compared different combinations of culture conditions to obtain a model system that recapitulates the main features of NAFLD\/NASH, i.e., increased steatosis, reactive oxygen species (ROS), secretion of pro-inflammatory cytokines\/chemokines, and presence of fibrosis. To confirm the usefulness of the optimized model system, we screened for compounds that inhibit steatosis in the hepatocytes and evaluated the most effective compound in the triculture model system.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Results:<\/jats:bold>\n                    The activating mixture stimulated HepG2 cells in this triculture to accumulate more fat and produce higher levels of reactive oxygen species (ROS) than HepG2 cells in monocultures. As well, higher levels of inflammatory cytokines and chemokines (IL-8, IL-6, MIP-1\u03b1,\n                    <jats:italic>etc.<\/jats:italic>\n                    ) were produced in this triculture compared to monocultures. In addition, in all LX-2 monocultures and cocultures, exposure to the activating mixture increased markers of fibrosis. A major strength of our triculture system is that it makes possible the simultaneous monitoring of 4 main features of NASH, i.e., steatosis, oxidative stress, inflammation and fibrosis. Screening potential modulators that may reduce steatosis in HepG2 cells revealed the protective effects of the isoalkaloid, berberine. Tested using this novel triculture assay, treatment with 5\u00a0\u00b5M berberine decreased steatosis and ROS in HepG2 hepatocytes, reduced inflammatory cytokine production and inhibited collagen production from LX-2 cells.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Conclusion:<\/jats:bold>\n                    This simple triculture model recapitulates the main features of NAFLD\/NASH and should be useful for high-throughput preclinical drug discovery. In this model, berberine showed promising results in decreasing steatosis and ROS and protection against fibrosis.\n                  <\/jats:p>","DOI":"10.3389\/fphar.2023.1234300","type":"journal-article","created":{"date-parts":[[2023,10,19]],"date-time":"2023-10-19T03:17:01Z","timestamp":1697685421000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":21,"title":["Development of a novel human triculture model of non-alcoholic fatty liver disease and identification of berberine as ameliorating steatosis, oxidative stress and fibrosis"],"prefix":"10.3389","volume":"14","author":[{"given":"Hossein","family":"Rafiei","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle","family":"Yeung","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Kowalski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gerald","family":"Krystal","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ingrid","family":"Elisia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,10,19]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"586","DOI":"10.1016\/j.jhep.2010.01.003","article-title":"Palmitoleate attenuates palmitate-induced Bim and PUMA up-regulation and hepatocyte lipoapoptosis","volume":"52","author":"Akazawa","year":"2010","journal-title":"J. Hepatol."},{"key":"B2","doi-asserted-by":"publisher","first-page":"389","DOI":"10.1053\/j.gastro.2015.04.043","article-title":"Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease","volume":"149","author":"Angulo","year":"2015","journal-title":"Gastroenterology"},{"key":"B3","doi-asserted-by":"publisher","first-page":"1895","DOI":"10.1002\/hep.24290","article-title":"Induction of vesicular steatosis by amiodarone and tetracycline is associated with up\u2010regulation of lipogenic genes in heparg cells","volume":"53","author":"Anth\u00e9rieu","year":"2011","journal-title":"Hepatology"},{"key":"B4","doi-asserted-by":"publisher","first-page":"1753","DOI":"10.1016\/j.tiv.2015.07.010","article-title":"The interplay between hepatic stellate cells and hepatocytes in an in vitro model of NASH","volume":"29","author":"Barbero-Becerra","year":"2015","journal-title":"Toxicol Vitro"},{"key":"B5","doi-asserted-by":"publisher","first-page":"896","DOI":"10.1016\/j.jhep.2020.04.037","article-title":"Acetyl-CoA carboxylase inhibition disrupts metabolic reprogramming during hepatic stellate cell activation","volume":"73","author":"Bates","year":"2020","journal-title":"J. Hepatol."},{"key":"B6","doi-asserted-by":"publisher","first-page":"99","DOI":"10.1007\/s00018-018-2947-0","article-title":"Molecular pathways of nonalcoholic fatty liver disease development and progression","volume":"76","author":"Bessone","year":"2019","journal-title":"Cell Mol. Life Sci."},{"key":"B7","doi-asserted-by":"publisher","first-page":"524","DOI":"10.1016\/j.bbrc.2019.02.099","article-title":"NLRC4 inflammasome activation regulated by TNF-\u03b1 promotes inflammatory responses in nonalcoholic fatty liver disease","volume":"511","author":"Chen","year":"2019","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"B8","doi-asserted-by":"publisher","first-page":"373","DOI":"10.1053\/jhep.2002.30692","article-title":"NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome","volume":"35","author":"Chitturi","year":"2002","journal-title":"Hepatology"},{"key":"B9","doi-asserted-by":"publisher","first-page":"193","DOI":"10.1080\/17460441.2018.1410135","article-title":"Non-alcoholic fatty liver disease (NAFLD) models in drug discovery","volume":"13","author":"Cole","year":"2018","journal-title":"Expert Opin. drug Discov."},{"key":"B10","doi-asserted-by":"publisher","first-page":"32","DOI":"10.1159\/000097904","article-title":"Effect of berberine on monocyte chemotactic protein-1 and cytokine-induced neutrophil chemoattractant-1 expression in rat lipopolysaccharide-induced uveitis","volume":"39","author":"Cui","year":"2007","journal-title":"Ophthalmic Res."},{"key":"B11","doi-asserted-by":"publisher","first-page":"219","DOI":"10.1054\/clnu.2001.0529","article-title":"Plasma total and free fatty acids composition in human non-alcoholic steatohepatitis","volume":"21","author":"De Almeida","year":"2002","journal-title":"Clin. Nutr."},{"key":"B12","doi-asserted-by":"publisher","first-page":"1343","DOI":"10.1172\/JCI23621","article-title":"Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease","volume":"115","author":"Donnelly","year":"2005","journal-title":"J. Clin. investigation"},{"key":"B13","doi-asserted-by":"publisher","first-page":"e90954","DOI":"10.1172\/jci.insight.90954","article-title":"Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis","volume":"1","author":"Feaver","year":"2016","journal-title":"JCI insight"},{"key":"B14","doi-asserted-by":"publisher","first-page":"253","DOI":"10.3109\/19390211.2012.726702","article-title":"Aqueous extract of dried fruit of Berberis vulgaris L. in acne vulgaris, a clinical trial","volume":"9","author":"Fouladi","year":"2012","journal-title":"J. Diet. Suppl."},{"key":"B15","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1152\/physrev.00013.2007","article-title":"Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver","volume":"88","author":"Friedman","year":"2008","journal-title":"Physiol. Rev."},{"key":"B16","doi-asserted-by":"publisher","first-page":"183","DOI":"10.1242\/dmm.018234","article-title":"In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis","volume":"8","author":"Garc\u00eda-Ruiz","year":"2015","journal-title":"Dis. Models Mech."},{"key":"B17","doi-asserted-by":"publisher","first-page":"425","DOI":"10.1146\/annurev-pathol-011110-130246","article-title":"Pathogenesis of liver fibrosis","volume":"6","author":"Hernandez-Gea","year":"2011","journal-title":"Annu. Rev. pathology Mech. Dis."},{"key":"B18","doi-asserted-by":"publisher","first-page":"E955","DOI":"10.1152\/ajpendo.90599.2008","article-title":"Berberine suppresses proinflammatory responses through AMPK activation in macrophages","volume":"296","author":"Jeong","year":"2009","journal-title":"Am. J. Physiology-Endocrinology Metabolism"},{"key":"B19","doi-asserted-by":"publisher","first-page":"230","DOI":"10.1002\/hep4.1150","article-title":"Role of monocyte chemoattractant protein-1 in liver fibrosis with transient myeloproliferative disorder in down syndrome","volume":"2","author":"Kobayashi","year":"2018","journal-title":"Hepatol. Commun."},{"key":"B20","doi-asserted-by":"publisher","first-page":"501","DOI":"10.3109\/10408444.2012.682115","article-title":"Organotypic liver culture models: meeting current challenges in toxicity testing","volume":"42","author":"LeCluyse","year":"2012","journal-title":"Crit. Rev. Toxicol."},{"key":"B21","doi-asserted-by":"publisher","first-page":"814871","DOI":"10.3389\/fphar.2022.814871","article-title":"Therapeutic effects of berberine on liver fibrosis are associated with lipid metabolism and intestinal flora","volume":"13","author":"Liu","year":"2022","journal-title":"Front. Pharmacol."},{"key":"B22","first-page":"538","article-title":"Cooperation of Ito cells and hepatocytes in the deposition of an extracellular matrix in vitro","volume":"143","author":"Loreal","year":"1993","journal-title":"Am. J. pathology"},{"key":"B23","doi-asserted-by":"publisher","first-page":"605","DOI":"10.1146\/annurev.pharmtox.45.120403.095906","article-title":"Hepatic fibrosis: molecular mechanisms and drug targets","volume":"45","author":"Lotersztajn","year":"2005","journal-title":"Annu. Rev. Pharmacol. Toxicol."},{"key":"B24","first-page":"2668","article-title":"Berberine prevents non-alcoholic steatohepatitis-derived hepatocellular carcinoma by inhibiting inflammation and angiogenesis in mice","volume":"11","author":"Luo","year":"2019","journal-title":"Am. J. Transl. Res."},{"key":"B25","doi-asserted-by":"publisher","first-page":"1124","DOI":"10.3390\/nu9101124","article-title":"Gut microbiota and nonalcoholic fatty liver disease: insights on mechanisms and therapy","volume":"9","author":"Ma","year":"2017","journal-title":"Nutrients"},{"key":"B26","doi-asserted-by":"publisher","first-page":"2222","DOI":"10.3390\/molecules26082222","article-title":"Analysis of monacolins and berberine in food supplements for lipid control: an overview of products sold on the Italian market","volume":"26","author":"Marcheluzzo","year":"2021","journal-title":"Molecules"},{"key":"B27","doi-asserted-by":"publisher","first-page":"5250","DOI":"10.2174\/13816128113199990344","article-title":"Pathophysiology of NASH: perspectives for a targeted treatment","volume":"19","author":"Marra","year":"2013","journal-title":"Curr. Pharm. Des."},{"key":"B28","doi-asserted-by":"publisher","first-page":"2057","DOI":"10.2337\/diacare.27.8.2057","article-title":"Approach to the pathogenesis and treatment of nonalcoholic steatohepatitis","volume":"27","author":"Medina","year":"2004","journal-title":"Diabetes Care"},{"key":"B29","doi-asserted-by":"publisher","first-page":"40","DOI":"10.1016\/j.taap.2015.12.020","article-title":"A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: application to acetaminophen","volume":"292","author":"Michaut","year":"2016","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"B30","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1016\/j.cotox.2019.03.001","article-title":"Human in vitro models of nonalcoholic fatty liver disease","volume":"16","author":"M\u00fcller","year":"2019","journal-title":"Curr. Opin. Toxicol."},{"key":"B31","first-page":"9","article-title":"Non alcoholic fatty liver disease and metabolic syndrome","volume":"13","author":"Paschos","year":"2009","journal-title":"Hippokratia"},{"key":"B32","doi-asserted-by":"publisher","first-page":"173","DOI":"10.1016\/j.jff.2018.02.033","article-title":"Protection by different classes of dietary polyphenols against palmitic acid-induced steatosis, nitro-oxidative stress and endoplasmic reticulum stress in HepG2 hepatocytes","volume":"44","author":"Rafiei","year":"2018","journal-title":"J. Funct. Foods"},{"key":"B33","doi-asserted-by":"publisher","first-page":"1600781","DOI":"10.1002\/mnfr.201600781","article-title":"Comparison of dietary polyphenols for protection against molecular mechanisms underlying nonalcoholic fatty liver disease in a cell model of steatosis","volume":"61","author":"Rafiei","year":"2017","journal-title":"Mol. Nutr. Food Res."},{"key":"B34","doi-asserted-by":"publisher","first-page":"541","DOI":"10.3390\/nu11030541","article-title":"Dietary polyphenols protect against oleic acid-induced steatosis in an in vitro model of NAFLD by modulating lipid metabolism and improving mitochondrial function","volume":"11","author":"Rafiei","year":"","journal-title":"Nutrients"},{"key":"B35","doi-asserted-by":"publisher","first-page":"6241","DOI":"10.1021\/acs.jafc.9b02367","article-title":"Phenolic breakdown products of cyanidin and quercetin contribute to protection against mitochondrial impairment and reactive oxygen species generation in an in vitro model of hepatocyte steatosis","volume":"67","author":"Rafiei","year":"","journal-title":"J. Agric. Food Chem."},{"key":"B36","doi-asserted-by":"publisher","first-page":"59","DOI":"10.1016\/j.freeradbiomed.2011.10.003","article-title":"Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis","volume":"52","author":"Rolo","year":"2012","journal-title":"Free Radic. Biol. Med."},{"key":"B37","doi-asserted-by":"publisher","first-page":"574832","DOI":"10.1155\/2015\/574832","article-title":"Nonalcoholic steatohepatitis: a search for factual animal models","volume":"2015","author":"Sanches","year":"2015","journal-title":"BioMed Res. Int."},{"key":"B38","doi-asserted-by":"publisher","first-page":"e25269","DOI":"10.1371\/journal.pone.0025269","article-title":"GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy","volume":"6","author":"Sharma","year":"2011","journal-title":"PLoS One"},{"key":"B39","doi-asserted-by":"publisher","first-page":"1018","DOI":"10.1111\/j.1478-3231.2005.01130.x","article-title":"High glucose stimulates hepatic stellate cells to proliferate and to produce collagen through free radical production and activation of mitogen-activated protein kinase","volume":"25","author":"Sugimoto","year":"2005","journal-title":"Liver Int."},{"key":"B40","doi-asserted-by":"publisher","first-page":"1533","DOI":"10.1248\/bpb.32.1533","article-title":"Berberine inhibits hepatic stellate cell proliferation and prevents experimental liver fibrosis","volume":"32","author":"Sun","year":"2009","journal-title":"Biol. Pharm. Bull."},{"key":"B41","doi-asserted-by":"publisher","first-page":"754","DOI":"10.1016\/j.gpb.2019.06.005","article-title":"Oleic acid and eicosapentaenoic acid reverse palmitic acid-induced insulin resistance in human HepG2 cells via the reactive oxygen species\/JUN pathway","volume":"19","author":"Sun","year":"2021","journal-title":"Genomics Proteomics Bioinforma."},{"key":"B42","doi-asserted-by":"publisher","first-page":"1901379","DOI":"10.1002\/adhm.201901379","article-title":"In vitro human liver model of nonalcoholic steatohepatitis by coculturing hepatocytes, endothelial cells, and Kupffer cells","volume":"8","author":"Suurmond","year":"2019","journal-title":"Adv. Healthc. Mater."},{"key":"B43","doi-asserted-by":"publisher","first-page":"20704","DOI":"10.3390\/ijms141020704","article-title":"Multiple hits, including oxidative stress, as pathogenesis and treatment target in non-alcoholic steatohepatitis (NASH)","volume":"14","author":"Takaki","year":"2013","journal-title":"Int. J. Mol. Sci."},{"key":"B44","doi-asserted-by":"publisher","first-page":"3593951","DOI":"10.1155\/2016\/3593951","article-title":"The therapeutic effect of berberine in the treatment of nonalcoholic fatty liver disease: a meta-analysis","volume":"2016","author":"Wei","year":"2016","journal-title":"Evid. Based Complement. Altern. Med."},{"key":"B45","doi-asserted-by":"publisher","first-page":"142","DOI":"10.1136\/gut.2004.042127","article-title":"Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis","volume":"54","author":"Xu","year":"2005","journal-title":"Gut"},{"key":"B46","doi-asserted-by":"publisher","first-page":"e0134172","DOI":"10.1371\/journal.pone.0134172","article-title":"Efficacy of berberine in patients with non-alcoholic fatty liver disease","volume":"10","author":"Yan","year":"2015","journal-title":"PLoS One"},{"key":"B47","doi-asserted-by":"publisher","first-page":"374","DOI":"10.1038\/s41420-021-00768-7","article-title":"Berberine alleviates liver fibrosis through inducing ferrous redox to activate ROS-mediated hepatic stellate cells ferroptosis","volume":"7","author":"Yi","year":"2021","journal-title":"Cell death Discov."},{"key":"B48","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1038\/nrgastro.2017.109","article-title":"Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention","volume":"15","author":"Younossi","year":"2018","journal-title":"Nat. Rev. Gastroenterology hepatology"},{"key":"B49","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1002\/hep.28431","article-title":"Global epidemiology of nonalcoholic fatty liver disease\u2014meta\u2010analytic assessment of prevalence, incidence, and outcomes","volume":"64","author":"Younossi","year":"2016","journal-title":"Hepatology"},{"key":"B50","doi-asserted-by":"publisher","first-page":"172618","DOI":"10.1016\/j.ejphar.2019.172618","article-title":"Pinolenic acid ameliorates oleic acid-induced lipogenesis and oxidative stress via AMPK\/SIRT1 signaling pathway in HepG2 cells","volume":"861","author":"Zhang","year":"2019","journal-title":"Eur. J. Pharmacol."},{"key":"B51","doi-asserted-by":"publisher","first-page":"20848","DOI":"10.1038\/srep20848","article-title":"Berberine prevents progression from hepatic steatosis to steatohepatitis and fibrosis by reducing endoplasmic reticulum stress","volume":"6","author":"Zhang","year":"2016","journal-title":"Sci. Rep."},{"key":"B52","doi-asserted-by":"publisher","first-page":"1169","DOI":"10.1248\/bpb.31.1169","article-title":"Chronic effects of berberine on blood, liver glucolipid metabolism and liver PPARs expression in diabetic hyperlipidemic rats","volume":"31","author":"Zhou","year":"2008","journal-title":"Biol. Pharm. Bull."},{"key":"B53","doi-asserted-by":"publisher","first-page":"1336","DOI":"10.3390\/molecules21101336","article-title":"The potential mechanisms of berberine in the treatment of nonalcoholic fatty liver disease","volume":"21","author":"Zhu","year":"2016","journal-title":"Molecules"},{"key":"B54","doi-asserted-by":"publisher","first-page":"221","DOI":"10.1016\/j.ejphar.2004.07.027","article-title":"Recent pharmacological studies on natural products in China","volume":"500","author":"Zu Zhu","year":"2004","journal-title":"Eur. J. Pharmacol."}],"container-title":["Frontiers in Pharmacology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphar.2023.1234300\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,10,19]],"date-time":"2023-10-19T03:17:08Z","timestamp":1697685428000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphar.2023.1234300\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,10,19]]},"references-count":54,"alternative-id":["10.3389\/fphar.2023.1234300"],"URL":"https:\/\/doi.org\/10.3389\/fphar.2023.1234300","relation":{},"ISSN":["1663-9812"],"issn-type":[{"value":"1663-9812","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,10,19]]},"article-number":"1234300"}}