The effects of Rooibos (Aspalathus linearis) supplementation on the pancreas, liver, and kidney of male Wistar rats following antiretroviral treatment

Pereira, Daniella Lagoa (2017-12-05)

Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Anatomy and Histology

Thesis (MSc)--Stellenbosch University, 2017

Thesis

ENGLISH ABSTRACT : The health burden of human immunodeficiency virus (HIV) has improved with the introduction of antiretroviral therapy (ART). Near perfect adherence to ART is needed to ensure the maximum benefits of the drugs. Adherence is compromised by side effects and adverse drug reactions (ADRs) which may, in part, be due to drug induced oxidative stress. Rooibos (Aspalathus linearis) is a shrub-like bush native to South Africa and is cultivated primarily as an herbal tea. Rooibos contains various polyphenols that add to its overall antioxidant capabilities. Aspalathin, the most abundant polyphenol present in rooibos, has shown potent antioxidant capabilities in vitro, providing an attractive model for therapy. The following study questions whether the histological observable effects of ART can be attenuated with the introduction of rooibos tea in vivo. The study aimed to assess the effects of combination ART, and rooibos tea on the histology of the pancreas, liver, and kidney in a HIV-negative rat model. Rats (n=40) were subcatogorised into four groups namely: control (C), control ART (C-ART), control rooibos (C-R), and experimental (ART+R) groups. The C-ART and ART+R groups were administered a dose of combination ART consisting of 200 mg Emtricitabine (EMT), 600 mg Efavirenz (FTC) and 300 mg Tenofovir (TDF). A 2% (w/v) rooibos tea solution was administered ad libitum to C+R and ART+R groups. Samples of rooibos tea were analysed using liquid chromatography-mass spectrometry techniques to determine whether rats received the same concentrations of polyphenols throughout the investigation. Samples from the pancreas, liver, and kidney underwent standard histological processing. Routine haematoxylin and eosin stains and appropriate immunohistochemical and tinctorial techniques were conducted. Detailed morphometric measurements on pancreatic islet size, number and percentage of α and β-cells were performed. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were evaluated to test liver function. Changes in size of the glomerular corpuscle, glomeruli and proximal convoluted tubules (PCT) in the kidney were measured. Organ architecture, cellular structure and associated pathology were analysed in each organ, scored, and confirmed by a histopathologist. The size and number of pancreatic islets and the percentage of α and β-cells did not vary significantly from the control group. Serum AST and ALT levels did not vary significantly from controls indicating normal liver function. Glomerular corpuscle, glomeruli and PCT size remained constant compared to controls. No overt histopathology was present indicative of ART induced pancreatic toxicity, hepatotoxicity or nephrotoxicity. The results indicated no major changes between the various tea solutions indicating that the rats received similar concentrations of rooibos polyphenols throughout the week. The study maintains that ART, when given to rats, does not cause significant changes to organ histomorphometry. While rooibos tea administration did not worsen, or improve organ histomorphometry.

AFRIKAANSE OPSOMMING : Die bekendstelling van antiretrovirale terapie (ART), het die gesondheidslas van die menslike immuniteitsgebreksvirus (MIV) verbeter. Om optimale voordeel uit die terapie te verkry, moet ART nakoming optimaal gehandhaaf word. Nakoming word egter belemmer deur newe-effekte en ongunstige geneesmiddelreaksies, wat gedeeltlik aan geneesmiddel geïnduseerde oksidatiewe stress toegeskryf kan word. Rooibos (Aspalathus linearis) is ‘n struikagtige gewas wat inheems aan Suid Afrika is en word hoofsaaklik as ‘n kruietee gekweek. Rooibos bevat verskeie polifenole wat tot die algehele antioksidatiewe effek van die tee bydra. Aspalathin, die hoof polifenol wat in rooibos teenwoordig is, het sterk antioksidantiewe eienskappe wat in vitro waargeneem is, en wat dit dus ‘n aanloklike model vir terapie maak. Die studie poog om te bepaal of die waarneembare histologiese effekte wat deur ART veroorsaak word, met die toediening van rooibostee in vivo verminder kan word. Die doel van hierdie studie was om die effek van ‘n kombinasie van ART en rooibostee op die histologie van die pankreas, lewer, en nier in ‘n MIV-negatiewe rotmodel waar te neem. Rotte (n=40) was in vier groepe verdeel, naamlik ‘n kontrole groep (K), kontrole ART groep (K-ART), kontrole rooibos groep (K-R) en eksperimentele (ART+R) groep. Die K-ART en ART+R groepe het ‘n kombinasie ART middel ontvang, wat bestaan het uit 200 mg Emtricitabine (EMT), 600 mg Efavirenz (FTC) en 300 mg Tenofovir (TDF) ingesluit het. ‘n Twee persent (w/v) rooibostee oplossing is ad libitum aan die K+R en ART+R groepe toegedien. Monsters van die rooibos tee was met behulp van ‘n vloeibare chromatografie-massaspektrometrie tegniek geanaliseer om te bepaal of die rotte dieselfde polifenol konsentrasie gedurende die eksperiment ontvang het. Monsters van die pankreas, lewer, en nier het standaard histologiese prosesering ondergaan, waartydens roetine Hematoksilien en Eosin-kleuring in kombinasie met toepaslike immunohistochemiese -en spesiale kleuring gedoen is. Gedetailleerde morfometriese afmetings is van die eilandgroottes van die pankreas gedoen, en die aantal en persentasie van α-en β-selle, is bepaal. Om lewerfunksie te bepaal, is serum alanien aminotransferase (ALT) en aspartaat aminotransferase (AST) vlakke geëvalueer. Veranderinge in die grootte van die glomerulêreliggaampie, glomeruli en proksimale kronkelbuise (PKB) in die nier is gemeet. ‘n In-diepte analise van die orgaan argitektuur, sellulêre struktuur, en gepaardgaande patologie is in elke orgaan gedoen. Die analise is deur ‘n histopatoloog bevestig. Die grootte en aantal pankreaseilande en die persentasie van α en β-selle in die eksperimentele groep het nie aansienlik van die kontrole groep verskil nie. Serum AST en ALT vlakke wat aanduidend van normale lewerfunksie is, het nie beduidend verskil van die kontrole groep nie, wat aanduidend van normale lewerfunksie is. In vergelyking met die kontroles, het die grootte van die glomerulêre liggaampie, glomeruli en PKB konstant gebly. Geen duidelike histopatologie was teenwoordig, wat ART geïnduseerde pankreas-toksisiteit, hepatotoksisiteit of nefrotoksisiteit kon aandui nie. Die resultate het geen groot veranderinge tussen die verskeie tee oplossings getoon nie, wat bevestig dat die rotte soortgelyke polifenol konsentrasies gedurende die week ontvang het. Die studie bevind dat wanneer ART aan rotte toegedien word, dit nie betekenisvolle veranderinge aan die orgaan histomorfometrie veroorsaak nie. Verder, toediening van rooibostee versleg of verbeter nie die orgaanhistomorfometrie nie.

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