{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,14]],"date-time":"2026-04-14T03:00:04Z","timestamp":1776135604569,"version":"3.50.1"},"reference-count":32,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2022,8,4]],"date-time":"2022-08-04T00:00:00Z","timestamp":1659571200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research","doi-asserted-by":"publisher","award":["21K06698"],"award-info":[{"award-number":["21K06698"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Japanese Ministry of Education, Culture, Sports Science, and Technology","award":["21K06698"],"award-info":[{"award-number":["21K06698"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DIC) frequently induce drug-induced liver injury (DILI). It is unclear whether macrophages such as M1 and M2 participate in NSAID-associated DILI; elucidating this relationship could lead to a better understanding of the detailed mechanism of DILI. We co-cultured human hepatoma HepG2 cells with M1 or M2 derived from human monocytic leukemia THP-1 cells to examine the roles of M1 and M2 in DIC-induced cytotoxicity. DIC was added to the direct or indirect co-cultures of HepG2 cells with M1 or M2 (HepG2\/M1 or HepG2\/M2, respectively) at cell ratios of (1:0, 1:0.1, 1:0.4, and 1:1). In both direct and indirect HepG2\/M2 co-cultures (1:0.4), there was lower lactate dehydrogenase release compared with HepG2\/M1 co-cultures. Other NSAIDs as well as DIC showed similar protective effects of DIC-induced cytotoxicity. There were only slight differences in mRNA levels of apoptosis- and endoplasmic reticulum stress-associated factors between M1 and M2 after DIC treatment, suggesting that other factors determined the protective effects of M2 on DIC-induced cytotoxicity. Levels of high mobility group box 1 (HMGB1) in the medium and the mRNA expression levels of HMGB1 receptors were different between M1 and M2 after DIC treatment. Increased HMGB1 concentrations and expression of toll-like receptor 2 mRNA in M1 were observed compared with M2 after DIC treatment. In conclusion, these results suggested that the HMGB1\/TLR2 signaling axis can be suppressed in M2 but not M1, leading to the different roles of M1 and M2 in NSAID-induced cytotoxicity.<\/jats:p>","DOI":"10.3390\/ijms23158660","type":"journal-article","created":{"date-parts":[[2022,8,4]],"date-time":"2022-08-04T21:52:48Z","timestamp":1659649968000},"page":"8660","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Diclofenac-Induced Cytotoxicity in Direct and Indirect Co-Culture of HepG2 Cells with Differentiated THP-1 Cells"],"prefix":"10.3390","volume":"23","author":[{"given":"Atsushi","family":"Kawase","sequence":"first","affiliation":[{"name":"Department of Pharmacy, Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashiosaka 577-8502, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ouka","family":"Takashima","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashiosaka 577-8502, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Satsuki","family":"Tanaka","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashiosaka 577-8502, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hiroaki","family":"Shimada","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashiosaka 577-8502, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masahiro","family":"Iwaki","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashiosaka 577-8502, Osaka, Japan"},{"name":"Pharmaceutical Research and Technology Institute, Kindai University, 3-4-1 Kowakae, Higashiosaka 577-8502, Osaka, Japan"},{"name":"Antiaging Center, Kindai University, 3-4-1 Kowakae, Higashiosaka 577-8502, Osaka, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,8,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1093\/toxsci\/kfq009","article-title":"Mechanisms of immune-mediated liver injury","volume":"115","author":"Adams","year":"2010","journal-title":"Toxicol. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1016\/S0041-008X(03)00368-5","article-title":"Diclofenac-induced liver injury: A paradigm of idiosyncratic drug toxicity","volume":"192","author":"Boelsterli","year":"2003","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/j.tiv.2015.11.020","article-title":"Mitochondrial toxicity of diclofenac and its metabolites via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria: Possible role in drug induced liver injury (DILI)","volume":"31","author":"Syed","year":"2016","journal-title":"Toxicol. In Vitro"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2071","DOI":"10.1021\/tx500288b","article-title":"Cytochrome P450-mediated bioactivation of mefenamic acid to quinoneimine intermediates and inactivation by human glutathione S-transferases","volume":"27","author":"Venkataraman","year":"2014","journal-title":"Chem. Res. Toxicol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"260","DOI":"10.1177\/1091581817700584","article-title":"Involvement of Reactive Metabolites of Diclofenac in Cytotoxicity in Sandwich-Cultured Rat Hepatocytes","volume":"36","author":"Kawase","year":"2017","journal-title":"Int. J. Toxicol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1080\/15376516.2018.1544384","article-title":"Involvement of diclofenac acyl-\u03b2-d-glucuronide in diclofenac-induced cytotoxicity in glutathione-depleted isolated murine hepatocytes co-cultured with peritoneal macrophages","volume":"29","author":"Kawase","year":"2019","journal-title":"Toxicol. Mech. Methods"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1488","DOI":"10.1016\/j.cgh.2018.04.049","article-title":"Development and Validation of a Test to Identify Drugs That Cause Idiosyncratic Drug-Induced Liver Injury","volume":"16","author":"Benesic","year":"2018","journal-title":"Clin. Gastroenterol. Hepatol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Danan, G., and Teschke, R. (2015). RUCAM in Drug and Herb Induced Liver Injury: The Update. Int. J. Mol. Sci., 17.","DOI":"10.3390\/ijms17010014"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"820","DOI":"10.1002\/hep.1840220320","article-title":"Diclofenac-associated hepatotoxicity: Analysis of 180 cases reported to the Food and Drug Administration as adverse reactions","volume":"22","author":"Banks","year":"1995","journal-title":"Hepatology"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1136\/bmj.284.6329.1605","article-title":"Diclofenac hepatitis","volume":"284","author":"Dunk","year":"1982","journal-title":"Br. Med. J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1327","DOI":"10.1124\/dmd.31.11.1327","article-title":"Studies on the chemical reactivity of diclofenac acyl glucuronide with glutathione: Identification of diclofenac-S-acyl-glutathione in rat bile","volume":"31","author":"Grillo","year":"2003","journal-title":"Drug Metab. Dispos."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1093\/toxsci\/61.1.49","article-title":"Characterization of rat and human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of diclofenac","volume":"61","author":"King","year":"2001","journal-title":"Toxicol. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"706","DOI":"10.1124\/dmd.104.003095","article-title":"Detection of a novel reactive metabolite of diclofenac: Evidence for CYP2C9-mediated bioactivation via arene oxides","volume":"33","author":"Yan","year":"2005","journal-title":"Drug Metab. Dispos."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2816","DOI":"10.1021\/jm030891w","article-title":"Syntheses and characterization of the acyl glucuronide and hydroxy metabolites of diclofenac","volume":"47","author":"Kenny","year":"2004","journal-title":"J. Med. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1006\/taap.1993.1097","article-title":"Diclofenac covalent protein binding is dependent on acyl glucuronide formation and is inversely related to P450-mediated acute cell injury in cultured rat hepatocytes","volume":"120","author":"Boelsterli","year":"1993","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Genin, M., Clement, F., Fattaccioli, A., Raes, M., and Michiels, C. (2015). M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer, 15.","DOI":"10.1186\/s12885-015-1546-9"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1124\/dmd.32.3.359","article-title":"Kupffer cell-mediated IL-2 suppression of CYP3A activity in human hepatocytes","volume":"32","author":"Sunman","year":"2004","journal-title":"Drug Metab. Dispos."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1186\/s13045-020-00920-3","article-title":"High mobility group box 1 (HMGB1): A pivotal regulator of hematopoietic malignancies","volume":"13","author":"Yuan","year":"2020","journal-title":"J. Hematol. Oncol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"888","DOI":"10.1124\/dmd.116.069575","article-title":"Zomepirac Acyl Glucuronide Is Responsible for Zomepirac-Induced Acute Kidney Injury in Mice","volume":"44","author":"Iwamura","year":"2016","journal-title":"Drug Metab. Dispos."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/j.cbi.2017.11.011","article-title":"Neutrophil depletion protects against zomepirac-induced acute kidney injury in mice","volume":"279","author":"Yamashita","year":"2018","journal-title":"Chem. Biol. Interact."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1038\/nri978","article-title":"Alternative activation of macrophages","volume":"3","author":"Gordon","year":"2003","journal-title":"Nat. Rev. Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1016\/j.it.2004.09.015","article-title":"The chemokine system in diverse forms of macrophage activation and polarization","volume":"25","author":"Mantovani","year":"2004","journal-title":"Trends Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"667","DOI":"10.3892\/ijo.2014.2785","article-title":"The HMGB1 protein sensitizes colon carcinoma cells to cell death triggered by pro-apoptotic agents","volume":"46","author":"Zhang","year":"2015","journal-title":"Int. J. Oncol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"C917","DOI":"10.1152\/ajpcell.00401.2005","article-title":"High mobility group box 1 protein interacts with multiple Toll-like receptors","volume":"290","author":"Park","year":"2006","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"7370","DOI":"10.1074\/jbc.M306793200","article-title":"Involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein","volume":"279","author":"Park","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"21884","DOI":"10.1038\/srep21884","article-title":"HMGB1 Facilitated Macrophage Reprogramming towards a Proinflammatory M1-like Phenotype in Experimental Autoimmune Myocarditis Development","volume":"6","author":"Su","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Teschke, R., Eickhoff, A., Brown, A.C., Neuman, M.G., and Schulze, J. (2019). Diagnostic Biomarkers in Liver Injury by Drugs, Herbs, and Alcohol: Tricky Dilemma after EMA Correctly and Officially Retracted Letter of Support. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21010212"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1038\/s41586-021-04025-w","article-title":"SLC25A39 is necessary for mitochondrial glutathione import in mammalian cells","volume":"599","author":"Wang","year":"2021","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1592","DOI":"10.1016\/j.tiv.2007.06.017","article-title":"Phase II enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells","volume":"21","author":"Westerink","year":"2007","journal-title":"Toxicol. In Vitro"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1124\/jpet.121.000790","article-title":"Protein Kinase N Family Negatively Regulates Constitutive Androstane Receptor-Mediated Transcriptional Induction of Cytochrome P450 2b10 in the Livers of Mice","volume":"379","author":"Kawase","year":"2021","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Ito, A., Nasako, H., Akizuki, R., Takashina, Y., Eguchi, H., Matsunaga, T., Yoshino, Y., Endo, S., and Ikari, A. (2021). Elevation of Chemosensitivity of Lung Adenocarcinoma A549 Spheroid Cells by Claudin-2 Knockdown through Activation of Glucose Transport and Inhibition of Nrf2 Signal. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22126582"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Akasaka, J., Naruse, K., Sado, T., Uchiyama, T., Makino, M., Yamauchi, A., Ota, H., Sakuramoto-Tsuchida, S., Itaya-Hironaka, A., and Takasawa, S. (2019). Involvement of Receptor for Advanced Glycation Endproducts in Hypertensive Disorders of Pregnancy. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20215462"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/15\/8660\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:03:57Z","timestamp":1760141037000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/15\/8660"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,8,4]]},"references-count":32,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2022,8]]}},"alternative-id":["ijms23158660"],"URL":"https:\/\/doi.org\/10.3390\/ijms23158660","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,8,4]]}}}