{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,2,21]],"date-time":"2025-02-21T14:09:22Z","timestamp":1740146962393,"version":"3.37.3"},"reference-count":49,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2024,4,17]],"date-time":"2024-04-17T00:00:00Z","timestamp":1713312000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001321","name":"National Research Foundation","doi-asserted-by":"publisher","award":["107104"],"award-info":[{"award-number":["107104"]}],"id":[{"id":"10.13039\/501100001321","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Harry Crossley Foundation Research Project Funding"},{"name":"South African Medical Research Council \u2013 Research Capacity Development","award":["awarded a bursary"],"award-info":[{"award-number":["awarded a bursary"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:sec id=\"sec001\"><jats:title>Background<\/jats:title><jats:p>Currently, it is acknowledged that vitamin E, insulin sensitizers and anti-diabetic drugs are used to manage non-alcoholic fatty liver disease (NAFLD), however, these therapeutic interventions harbour adverse side effects. Pioglitazone, an anti-diabetic drug, is currently the most effective therapy to manage NAFLD. The use of natural medicines is widely embraced due to the lack of evidence of their negative side effects. Rooibos has been previously shown to decrease inflammation and oxidative stress in experimental models of diabetes, however, this is yet to be explored in a setting of NAFLD. This study was aimed at investigating the effects of an aspalathin-rich green rooibos extract (Afriplex GRT<jats:sup>TM<\/jats:sup>) against markers of hepatic oxidative stress, inflammation and apoptosis in an<jats:italic>in vitro<\/jats:italic>model of NAFLD.<\/jats:p><\/jats:sec><jats:sec id=\"sec002\"><jats:title>Methods<\/jats:title><jats:p>Oleic acid [1 mM] was used to induce hepatic steatosis in C3A liver cells. Thereafter, the therapeutic effect of Afriplex GRT<jats:sup>TM<\/jats:sup>, with or without pioglitazone, was determined by assessing its impact on cell viability, changes in mitochondrial membrane potential, intracellular lipid accumulation and the expression of genes and proteins (<jats:italic>ChREBP<\/jats:italic>,<jats:italic>SREBF1<\/jats:italic>,<jats:italic>FASN<\/jats:italic>,<jats:italic>IRS1<\/jats:italic>,<jats:italic>SOD2<\/jats:italic>, Caspase-3, GSTZ1, IRS1 and TNF-\u03b1) that are associated with the development of NAFLD.<\/jats:p><\/jats:sec><jats:sec id=\"sec003\"><jats:title>Results<\/jats:title><jats:p>Key findings showed that Afriplex GRT<jats:sup>TM<\/jats:sup>added to the medium alone or combined with pioglitazone, could effectively block hepatic lipid accumulation without inducing cytotoxicity in C3A liver cells exposed oleic acid. This positive outcome was consistent with effective regulation of genes involved in insulin signaling, as well as carbohydrate and lipid metabolism (<jats:italic>IRS1<\/jats:italic>,<jats:italic>SREBF1<\/jats:italic>and<jats:italic>ChREBP<\/jats:italic>). Interestingly, in addition to reducing protein levels of an inflammatory marker (TNF-\u03b1), the Afriplex GRT<jats:sup>TM<\/jats:sup>could ameliorate oleic acid-induced hepatic steatotic damage by decreasing the protein expression of oxidative stress and apoptosis related markers such as GSTZ1 and caspase-3.<\/jats:p><\/jats:sec><jats:sec id=\"sec004\"><jats:title>Conclusion<\/jats:title><jats:p>Afriplex GRT<jats:sup>TM<\/jats:sup>reduced hepatic steatosis in oleic acid induced C3A liver cells by modulating<jats:italic>SREBF1<\/jats:italic>,<jats:italic>ChREBP<\/jats:italic>and<jats:italic>IRS-1<\/jats:italic>gene expression. The extract may also play a role in alleviating inflammation by reducing TNF-\u03b1 expression, suggesting that additional experiments are required for its development as a suitable therapeutic option against NAFLD. Importantly, further research is needed to explore its antioxidant role in this model.<\/jats:p><\/jats:sec>","DOI":"10.1371\/journal.pone.0297572","type":"journal-article","created":{"date-parts":[[2024,4,17]],"date-time":"2024-04-17T17:50:17Z","timestamp":1713376217000},"page":"e0297572","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Afriplex GRTTM extract attenuates hepatic steatosis in an in vitro model of NAFLD"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0144-6664","authenticated-orcid":true,"given":"Kwazi","family":"Gabuza","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2066-3154","authenticated-orcid":true,"given":"Thendo I.","family":"Mabuda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Oelfah","family":"Patel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Noxolo","family":"Khuboni","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ruzayda","family":"van Aarde","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3439-9238","authenticated-orcid":true,"given":"Sylvia","family":"Riedel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nonhlakanipho F.","family":"Sangweni","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shantal","family":"Windvogel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rabia","family":"Johnson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6821-2120","authenticated-orcid":true,"given":"Christo J. 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Patel","year":"2019","journal-title":"Frontiers in pharmacology"},{"key":"pone.0297572.ref022","doi-asserted-by":"crossref","first-page":"1515","DOI":"10.3390\/molecules21111515","article-title":"Inhibitory Interactions of Aspalathus linearis (rooibos) extracts and compounds, aspalathin and Z-2-(\u03b2-D-glucopyranosyloxy)-3-phenylpropenoic acid, on cytochromes metabolizing hypoglycemic and hypolipidemic drugs.","volume":"21","author":"O Patel","year":"2016","journal-title":"Molecules."},{"key":"pone.0297572.ref023","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.phymed.2012.09.010","article-title":"Acute assessment of an aspalathin-enriched green rooibos (Aspalathus linearis) extract with hypoglycemic potential.","volume":"20","author":"CJF Muller","year":"2012","journal-title":"Phytomedicine."},{"key":"pone.0297572.ref024","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1080\/10408398.2016.1157568","article-title":"Potential of rooibos, its major C-glucosyl flavonoids, and Z-2-(beta-D-glucopyranosyloxy)-3-phenylpropenoic acid in prevention of metabolic syndrome.","volume":"58","author":"CJF Muller","year":"2018","journal-title":"Crit Rev Food Sci Nutr. 2016\/06\/16"},{"key":"pone.0297572.ref025","doi-asserted-by":"crossref","first-page":"646","DOI":"10.1016\/j.acthis.2019.05.008","article-title":"A histomorphometric study on the hepatoprotective effects of a green rooibos extract in a diet-induced obese rat model","volume":"121","author":"JI Layman","year":"2019","journal-title":"Acta histochemica"},{"key":"pone.0297572.ref026","first-page":"25","article-title":"In vitro models for non-alcoholic fatty liver disease: Emerging platforms and their applications.","author":"MJ Ramos","year":"2022","journal-title":"iScience"},{"key":"pone.0297572.ref027","doi-asserted-by":"crossref","first-page":"1920","DOI":"10.1016\/j.toxrep.2022.10.006","article-title":"In vitro and in vivo hepatotoxicity study of AfriplexTM GRT through an inflammatory response","volume":"9","author":"KB Gabuza","year":"2022","journal-title":"Toxicol Rep"},{"key":"pone.0297572.ref028","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/0022-1759(83)90303-4","article-title":"Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays","volume":"65","author":"T. Mosmann","year":"1983","journal-title":"Journal of immunological methods"},{"key":"pone.0297572.ref029","first-page":"9","article-title":"Analysis of the mitochondrial membrane potential using the cationic JC-1 dye as a sensitive fluorescent probe.","author":"F Sivandzade","year":"2019","journal-title":"Bio-protocol."},{"key":"pone.0297572.ref030","doi-asserted-by":"crossref","first-page":"922","DOI":"10.1002\/mnfr.201500656","article-title":"Aspalathin, a dihydrochalcone C\u2010glucoside, protects H9c2 cardiomyocytes against high glucose induced shifts in substrate preference and apoptosis","volume":"60","author":"R Johnson","year":"2016","journal-title":"Molecular nutrition & food research"},{"key":"pone.0297572.ref031","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.plipres.2008.08.001","article-title":"Recent insights into hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD).","volume":"48","author":"G Musso","year":"2009","journal-title":"Progress in lipid research"},{"key":"pone.0297572.ref032","doi-asserted-by":"crossref","first-page":"185","DOI":"10.3389\/fbioe.2018.00185","article-title":"Cytotoxic activity of fatty acids from antarctic macroalgae on the growth of human breast cancer cells","volume":"6","author":"BS Pacheco","year":"2018","journal-title":"Frontiers in bioengineering and biotechnology"},{"key":"pone.0297572.ref033","first-page":"1","article-title":"Anti-lipoapoptotic effects of Alisma orientalis extract on non-esterified fatty acid-induced HepG2 cells.","volume":"16","author":"H-S Jeong","year":"2016","journal-title":"BMC complementary and alternative medicine."},{"key":"pone.0297572.ref034","doi-asserted-by":"crossref","first-page":"1109","DOI":"10.1016\/j.metabol.2016.05.003","article-title":"Non-alcoholic fatty liver disease and dyslipidemia: An update","volume":"65","author":"N Katsiki","year":"2016","journal-title":"Metabolism"},{"key":"pone.0297572.ref035","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1055\/s-0033-1358523","article-title":"Transcriptional control of hepatic lipid metabolism by SREBP and ChREBP.","author":"X Xu","year":"2013","journal-title":"Seminars in liver disease. NIH Public Access"},{"key":"pone.0297572.ref036","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1016\/j.bbadis.2016.11.029","article-title":"The transcription factor carbohydrate-response element-binding protein (ChREBP): A possible link between metabolic disease and cancer","volume":"1863","author":"K. Iizuka","year":"2017","journal-title":"Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease"},{"key":"pone.0297572.ref037","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/j.phymed.2013.08.011","article-title":"Effects of fermented rooibos (Aspalathus linearis) on adipocyte differentiation.","volume":"21","author":"M Sanderson","year":"2014","journal-title":"Phytomedicine"},{"key":"pone.0297572.ref038","doi-asserted-by":"crossref","first-page":"1131","DOI":"10.4161\/auto.25063","article-title":"Functions of autophagy in normal and diseased liver","volume":"9","author":"MJ Czaja","year":"2013","journal-title":"Autophagy"},{"key":"pone.0297572.ref039","doi-asserted-by":"crossref","first-page":"19353","DOI":"10.1074\/jbc.M201584200","article-title":"Absence of sterol regulatory element-binding protein-1 (srebp-1) ameliorates fatty livers but not obesity or insulin resistance inlep ob\/lep ob mice.","volume":"277","author":"N Yahagi","year":"2002","journal-title":"Journal of Biological Chemistry"},{"key":"pone.0297572.ref040","doi-asserted-by":"crossref","first-page":"1700028","DOI":"10.1002\/prca.201700028","article-title":"Alteration of liver peroxisomal and mitochondrial functionality in the NZO mouse model of metabolic syndrome","volume":"12","author":"B Knebel","year":"2018","journal-title":"PROTEOMICS\u2013Clinical Applications"},{"key":"pone.0297572.ref041","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1007\/s11892-009-0034-5","article-title":"The role of FOXO in the regulation of metabolism","volume":"9","author":"DN Gross","year":"2009","journal-title":"Current diabetes reports"},{"key":"pone.0297572.ref042","doi-asserted-by":"crossref","first-page":"1544","DOI":"10.1038\/s41366-018-0062-9","article-title":"Selective insulin resistance with differential expressions of IRS-1 and IRS-2 in human NAFLD livers.","volume":"42","author":"M Honma","year":"2018","journal-title":"International journal of obesity"},{"key":"pone.0297572.ref043","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1038\/414799a","article-title":"Insulin signalling and the regulation of glucose and lipid metabolism","volume":"414","author":"AR Saltiel","year":"2001","journal-title":"Nature"},{"key":"pone.0297572.ref044","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1016\/j.tem.2008.08.005","article-title":"Insulin resistance, inflammation, and non-alcoholic fatty liver disease","volume":"19","author":"H Tilg","year":"2008","journal-title":"Trends Endocrinol Metab. 2008\/10\/22"},{"key":"pone.0297572.ref045","doi-asserted-by":"crossref","first-page":"782","DOI":"10.1016\/j.jinsphys.2012.02.003","article-title":"The identification and oxidative stress response of a zeta class glutathione S-transferase (GSTZ1) gene from Apis cerana cerana.","volume":"58","author":"H Yan","year":"2012","journal-title":"Journal of insect 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Naik","year":"2013","journal-title":"Genomics"},{"key":"pone.0297572.ref049","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.orcp.2016.03.009","article-title":"Body mass index and the visceral adipose tissue expression of IL-6 and TNF-alpha are associated with the morphological severity of non-alcoholic fatty liver disease in individuals with class III obesity.","volume":"12","author":"ASB Jorge","year":"2018","journal-title":"Obesity Research & Clinical Practice."}],"container-title":["PLOS 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