In vitro assessment of aspalathin-enriched Rooibos (Aspalathus linearis) extract treatment in statin-induced hepatotoxicity

Millar, Danielle Ann (2019-04)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Rooibos (Aspalathus linearis) has been shown to have various health benefits including antidiabetic, lipid-lowering, and hepatoprotective properties. Although anecdotally Rooibos consumption is regarded as safe, recently, two case studies have associated chronic consumption of Rooibos with conventional prescription medications, such as atorvastatin, with hepatotoxicity. The cholesterol-lowering drugs, statins, act by competitively inhibiting hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in cholesterol synthesis. Although rare in occurrence, statins are potentially hepatotoxic. The safety of the concurrent use of Rooibos and statins thus needs to be elucidated. This study aims to investigate the interaction between the potential hepatoprotective effects of Rooibos and statin-induced hepatotoxicity using an in vitro C3A liver cell model. C3A liver cells, in both normal and hyperlipidaemic conditions, were exposed to atorvastatin (ATV; 10 μM and 25 μM) and Afriplex GRT™ (green Rooibos extract; 0.01 mg/mL and 0.1 mg/mL) and a combination thereof. Pre-treatment with palmitate (500 μM) for 24 hours was used to induce a hyperlipidaemic condition in vitro. The effects of the co-treatment on cell viability, oxidative stress, apoptosis, mitochondrial integrity and cellular ROS production were assessed in a C3A liver cell culture model. In addition, 3D culture was used to produce C3A liver spheroids, and the effect of the treatments in a chronic culture was then assessed. GRT was not cytotoxic at any of the concentrations tested, whereas ATV showed time- and concentration-dependent cytotoxicity in C3A cells. A significant increase in ROS production was observed in C3A cells exposed to 25 μM ATV and palmitate (353.10% ± 262.70 vs. 1431.00% ± 504.2). Similar results were seen following ATV and GRT combination therapy (845.00% ± 589.60 vs. 1493.00% ± 278.4). Under hyperlipidaemic conditions, ATV induced significant increases in apoptosis (19.50% ± 3.56 vs. 52.83% ± 7.14) which was not ameliorated by GRT co-treatment (13.83% ± 2.79). The results of the 3D culture showed complementary results. Treatment with palmitate and ATV was toxic to the spheroids and treatment with GRT was unable to attenuate this toxicity. A decrease in cellular ATP was found in the ATV and ATV+GRT treated spheroids (1.45 AU/μm3 ± 1.00, and 3.79 AU/μm3 ± 2.32, respectively, vs control, 161.02 AU/μm3 ± 55.26) as well as decreased glucose utilisation calculated from the increased remaining glucose in the treatment media (91.83% and 94.68%, respectively, vs control,100%). The culmination of experiments showed that ATV was hepatotoxic. This effect was exacerbated by exposing the cells to palmitate intended to mimic a hyperlipidaemic condition. GRT co-treatment did not show any modulating effects on ATV-induced hepatotoxicity under the acute or chronic conditions tested.

AFRIKAANSE OPSOMMING: Rooibos (Aspalathus linearis) besit verskeie gesondheidsvoordele insluitend antidiabetiese, lipiedverlagende eienskappe en bied beskerming teen lewertoksisiteit. Alhoewel die verbruik van Rooibos anekdoties as veilig beskou word, het twee gevallestudies onlangs hepatotoksisiteit, geassosieer met die gesamentlike gebruik van Rooibos met konvensionele kroniese voorskrifmedisyne, insluitend die statien, atorvastatien. Die cholesterolverlagende statiene, is mededingende inhibeerders van hidroksielmetielglutaryl-koënsiem A (HMG-CoA) reduktase, 'n tempo-beperkende ensiem in cholesterolsintese. Alhoewel dit selde voorkom, is statiene soms hepatotoksies. Die veiligheid van rooibos en statiene se gelyktydige gebruik moet dus getoets word. Hierdie studie het ten doel om die potensiële beskermings-interaksie tussen ‘n aspalatien-ryk groenrooibos ekstrak, Afriplex GRT™ (GRT) en statien-geïnduseerde hepatotoksisiteit in ‘n C3A lewerselkultuur model te ondersoek. C3A lewer selle, onder beide normale en hiperlipidaemiese toestande, is behandel met atorvastatien (ATV; 10 μM en 25 μM) en GRT (0.01 mg/mL en 0.1 mg/mL) asook 'n kombinasie daarvan. ‘n Voorbehandeling met palmitaat (500 μM) vir 24 uur is gebruik om 'n hiperlipidemiese toestand in vitro te veroorsaak. Die gevolge van die mede-behandeling op sel- lewensvatbaarheid, oksidatiewe stres, apoptose, mitochondriale membraan-integriteit en sellulêre ROS-produksie is in C3A-lewerselkultuur ondersoek. Daarbenewens is 3D selkultuur gebruik om C3A lewersferoïede te produseer, om die kroniese effek van die behandelings op lewer weefsel na te boots. GRT was nie sitotoksies by die konsentrasies wat getoets is nie, terwyl ATV tyd- en konsentrasie-afhanklike sitotoksisiteit in C3A-selle getoon het. 'n Beduidende toename in ROS-produksie is waargeneem in C3A-selle wat aan 25 μM ATV en palmitaat (353.10% ± 262.70 vs. 1431.00% ± 504.2) blootgestel was. Soortgelyke resultate is waargeneem met ATV- en GRT-kombinasie terapie (845.00% ± 589.60 teen 1493.00% ± 278.4). Onder hiperlipidemiese toestande het ATV beduidende toenames in apoptose (19.50% ± 3.56 teenoor 52.83% ± 7.14) veroorsaak wat nie deur GRT-mede-behandeling verbeter is nie (13.83% ± 2.79). Die resultate van die 3D-kultuur het komplementêre resultate getoon. 'n Afname in sellulêre ATP is gevind in die ATV- en ATV + GRT-lewersferoïede (1.45 AU/μm3 ± 1.00 en 3.79 AU/μm3 ± 2.32, onderskeidelik). Glukoseverbruik is ook verminder, soos bereken uit die oorblywende glukose in die behandelingsmedia (91.83% en 94.68%, onderskeidelik). Behandeling met palmitaat en ATV was toksies vir die lewersferoïede en behandeling met GRT kon nie hierdie toksisiteit ophef nie. Die resultate van die eksperimente het getoon dat ATV hepatotoksies was. Hierdie effek is vererger deur die selle aan palmitaat bloot te stel, wat gebruik was om 'n hiperlipidemiese toestand na te boots. Die GRT-medebehandeling was oneffektief om die selle teen die ATV-geïnduseerde hepatotoksisiteit onder die akute of kroniese toestande te beskerm.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105804
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