Effect of rooibos treatment on inflammatory and oxidative stress genes in in vitro and in vivo models of NAFLD
Date
2021-03
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Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Background: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) have been associated with the rise in metabolic diseases. Inflammation and oxidative stress are key mediators in NAFLD development. Current therapeutics used to treat NAFLD include insulin sensitisers and vitamin E. Afriplex GRT extract was previously showed to decrease inflammation and oxidative stress in cardiac cells. The study aimed to assess if Afriplex GRT extract could protect against hepatic oxidative stress, apoptosis and inflammation in in vitroand in vivoNAFLD models. Methods: Liver steatosis was induced in vitro by exposing HepG2/C3A human liver cells to 1 mM oleic acid for 24 h. Anti-steatotic effects of Afriplex GRT [1, 10 and 100 μg/mL] and/or Pioglitazone [30 μM] were determined in mono- and co-treatments for 24 hrs post induction of steatosis. The3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to determine cell metabolic activity and viability. An Oil red O assay was used to quantify the intracellular lipid content of the treated C3A cellsin vitro. Liver samples were obtained from a previous study, which used C57BL/6 mice with hepatosteatosis treated with or without Afriplex GRT [74 and 740 mg/kg] and pioglitazone [15 mg/kg] for ten weeks. Both in vitroand in vivomodels were analysed for the expression of genes and proteins associated with the development of NAFLD (AKT, AMPK-α, TNF-α, SOD2, IRS1, PPAR-α, SREBF1, ChREBP and FASN) using qRT-PCR and Western blot analysis. Results: Treatment with oleic acid experimentally induced steatosis in C3A cells (p < 0.01), as confirmed by Oil red O staining. Afriplex GRT treated cells showed a reduction in fat accumulation in a dose-dependent manner. Furthermore, co-treatment with pioglitazone was shown to be effective (p < 0.05). Genes involved in lipid metabolism (SREBF1, ChREBPand FASN) were suppressed by treatment with Afriplex GRT, contributing to reduced lipid content in both models. Afriplex GRT also induced a reduction in the expression of SOD2and the inflammatory cytokine (TNF-α). Conclusion: Afriplex GRT reduces hepatic steatosis in C3A cells and C57BL/6 mouse liver by modulating SREBF1, ChREBPand FASNgene expression. The extract also affected the expression of oxidative stress and inflammation related genes in both models, through the reduced gene expression of SOD2 and TNF-α, suggesting that Afriplex GRT has potential as a therapeutic for NAFLD.
AFRIKAANSE OPSOMMING: Agtergrond: Nie-alkoholiese vetterige lewersiekte (NAFLD) en nie-alkoholiese steatohepatitis (NASH) word geassosieer met die toename in metaboliese siektes. Inflammasie en oksidatiewe stres is van die hoof oorsake in die ontwikkeling van NAFLD. Huidige terapieë wat gebruik word vir die behandeling van NAFLD sluit insulien-sensitiseerders en vitamien E in. Voorheen is getoon dat Afriplex GRT inflammasie en oksidatiewe stres in hartselle verminder. Hierdie studie het ten doel gehad om te bepaal of Afriplex GRT ekstrak en pioglitazone, lewerselle kan beskerm teen oksidatiewe stress, apoptose en inflammasie in modelle van NAFLD. Metodes: Lewersteatose is in HepG2/C3A menslike lewerselle geïnduseer deur hulle met 1 mM oliesuur vir 24 uur te behandel. Anti-steatotiese effekte van Afriplex GRT [1, 10 en 100 μg/mL] en / of pioglitazone [30 μM] is bepaal 24 uur na steatose-induksie, beide as mono- en kombinasie behandelings. Lewermonsters afkomstig van 'n vorige studie waarin C57BL/6-muise vir tien weke lank behandel is met Afriplex GRT [74 en 740 mg/kg] en pioglitazone [15 mg/kg] is gebruik om die ekspressie van gene en proteïene wat verband hou met die ontwikkeling van NAFLD (AKT, AMPK-α, TNF-α, SOD2, IRS1, PPAR-α, SREBF1, ChREBP en FASN) deur qRT-PCR en Western blot te ondersoek. Resultate: In vitro veroorsaak oleïensuur steatose in C3A-selle (p <0.01). Afriplex GRT-behandelde selle het 'n konsentrasie-afhanklike afname van vet akkumulasie getoon. Verder is getoon dat die kombinasie van pioglitazone en Afriplex GRT (p <0.05) meer effektief was. Gene wat betrokke is by lipiedmetabolisme (SREBF1, ChREBPen FASN) was deur Afriplex GRT in beide modelle verlaag. Afriplex GRT het ook oksidatiewe stres (SOD2) en inflammasie (TNF-α) verminder. Gevolgtrekking: Afriplex GRT verminder hepatiese steatose in C3A-selle en in C57BL/6-muise se lewers deur die modulering van SREBF1, ChREBPen FASNgeenuitdrukking. Die ekstrak beskerm ook teen oksidatiewe stres en inflammasie deur die geenekspressie van SOD2en TNF-αte verlaag, wat daarop dui dat Afriplex GRT 'n terapeutiese potensiaal toon om NAFLD te behandel.
AFRIKAANSE OPSOMMING: Agtergrond: Nie-alkoholiese vetterige lewersiekte (NAFLD) en nie-alkoholiese steatohepatitis (NASH) word geassosieer met die toename in metaboliese siektes. Inflammasie en oksidatiewe stres is van die hoof oorsake in die ontwikkeling van NAFLD. Huidige terapieë wat gebruik word vir die behandeling van NAFLD sluit insulien-sensitiseerders en vitamien E in. Voorheen is getoon dat Afriplex GRT inflammasie en oksidatiewe stres in hartselle verminder. Hierdie studie het ten doel gehad om te bepaal of Afriplex GRT ekstrak en pioglitazone, lewerselle kan beskerm teen oksidatiewe stress, apoptose en inflammasie in modelle van NAFLD. Metodes: Lewersteatose is in HepG2/C3A menslike lewerselle geïnduseer deur hulle met 1 mM oliesuur vir 24 uur te behandel. Anti-steatotiese effekte van Afriplex GRT [1, 10 en 100 μg/mL] en / of pioglitazone [30 μM] is bepaal 24 uur na steatose-induksie, beide as mono- en kombinasie behandelings. Lewermonsters afkomstig van 'n vorige studie waarin C57BL/6-muise vir tien weke lank behandel is met Afriplex GRT [74 en 740 mg/kg] en pioglitazone [15 mg/kg] is gebruik om die ekspressie van gene en proteïene wat verband hou met die ontwikkeling van NAFLD (AKT, AMPK-α, TNF-α, SOD2, IRS1, PPAR-α, SREBF1, ChREBP en FASN) deur qRT-PCR en Western blot te ondersoek. Resultate: In vitro veroorsaak oleïensuur steatose in C3A-selle (p <0.01). Afriplex GRT-behandelde selle het 'n konsentrasie-afhanklike afname van vet akkumulasie getoon. Verder is getoon dat die kombinasie van pioglitazone en Afriplex GRT (p <0.05) meer effektief was. Gene wat betrokke is by lipiedmetabolisme (SREBF1, ChREBPen FASN) was deur Afriplex GRT in beide modelle verlaag. Afriplex GRT het ook oksidatiewe stres (SOD2) en inflammasie (TNF-α) verminder. Gevolgtrekking: Afriplex GRT verminder hepatiese steatose in C3A-selle en in C57BL/6-muise se lewers deur die modulering van SREBF1, ChREBPen FASNgeenuitdrukking. Die ekstrak beskerm ook teen oksidatiewe stres en inflammasie deur die geenekspressie van SOD2en TNF-αte verlaag, wat daarop dui dat Afriplex GRT 'n terapeutiese potensiaal toon om NAFLD te behandel.
Description
Thesis (MSc)--Stellenbosch University, 2021.
Keywords
Aspalathus linearis, Rooibos, Medicinal plants, Hepatic steatosis, Flavonoids, Oxidative stress, Lipid metabolism, Inflammation, UCTD