Compounds specific to Aspalathus linearis protects the diabetic heart against oxidative stress: a mechanistic study

Dludla, Phiwayinkosi Vusi (2016-12)

Thesis (PhD)--Stellenbosch University, 2016

Thesis

ENGLISH ABSTRACT : In diabetics, hyperglycemia, hyperlipidemia and inflammation potentiates the development of cardiovascular diseases (CVDs). These conditions provoke excessive generation of oxidative stress that has been implicated in the pathogenesis of diabetic cardiomyopathy (DCM). In the diabetic state, excessive generation of free radicals can cause oxidative damage to DNA and alter protein and lipids, leading to the activation of various death-induced signaling pathways. Activation of these death pathways result in structural and functional modifications to the myocardium. Current diabetic drug therapies do not protect the diabetic heart at risk from developing cardiovascular complications. Thus, in search for new therapeutics, we aim to unravel the molecular mechanisms associated with the protective effect of two major bioactive compounds from Aspalathus linearis, phenyl pyruvic acid-2-O-β-D-glucoside (PPAG) and aspalathin against hyperglycemia-induced oxidative stress and apoptosis in H9c2 cardiomyocytes. The study showed that PPAG and aspalathin were able to decrease mitochondrial membrane depolarization and prevent hyperglycemia-induced myocardial apoptosis by increasing the Bcl2/Bax ratio. We revealed that while both compounds were able to reduce hyperglycemia-induced apoptosis, only aspalathin could ameliorate lipid toxicity and oxidative stress-associated with insulin resistance. An important feature of the failing heart is the observed shift in mitochondrial substrate preference that precedes the onset of oxidative damage. The current study revealed that aspalathin improved glucose metabolism by decreasing fatty acid uptake and subsequent β-oxidation. This was achieved through decreasing the expression of adenosine monophosphate-activated protein kinase threonine 172 (pAMPK (Thr172)) and carnitine palmitoyltransferase 1 (Cpt1), while increasing that of acetyl-CoA carboxylase (Acc) and glucose transporter 4 (Glut4). Additionally, it is known that cardiomyocytes have a very low antioxidant capacity and a shift in mitochondrial substrate preference can result in accelerated oxidative damage. In this study, we showed that aspalathin ameliorated oxidative stress by increasing the antioxidant capacity of the cells through activation of the antioxidant response pathway, nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) and its downstream target genes. Moreover, we showed that aspalathin was able to reverse lipid toxicity by increasing the expression of Adiponectin, C1Q and collagen domain containing (Adipoq) and concomitantly decreasing cluster of differentiation 36 (Cd36) and Cpt1 mRNA expression. We further observed that Adipoq negatively regulated sterol regulatory element binding transcription factor 1 (Srebf1), stearoyl-Coenzyme A desaturase 1 (Scd1) and solute carrier family 27 (fatty acid transporter), member 3/5 (Slc27a3/5). This led to reduced lipid accumulation in H9c2 cardiomyocytes, with an associated decrease in total cholesterol, triglycerides and low-density lipoprotein in leptin resistant db/db mice. This was accompanied by decreased mRNA expression of inflammation markers in H9c2 cells, including interleukin 3 and 6 (IL3 and IL6), tumor necrosis factor receptor superfamily, member 1b and 13 (Tnfrsf1b and Tnfsf13), Janus kinase 2 (Jak2) and mitogen-activated protein kinase 3 (Mapk3). Together our results infer that aspalathin can slow down the progression of DCM, and thus protect the myocardium against causal factors associated with the development and progression of CVD.

AFRIKAANSE OPSOMMING : In diabete, hiperglukemie, hiperlipidemie en inflammasie versnel die ontwikkeling van kardiovaskulêre siektes (CVDs). Hierdie toestande lei tot oormatige generasie van oksidatiewe stres wat betrokke is by die patogenese van diabetiese kardiomiopatie (DCM). In die diabetiese toestand, kan oormatige generasie van vrye radikale oksidatiewe skade aan DNA asook proteïne en vette verander, wat tot die aktivering van verskeie seldood seinpaaie kan lei. Aktivering van hierdie seldood seinpaaie veroorsaak strukturele en funksionele wysigings aan die miokardium. Huidige medikasie beskerm nie die diabetiese hart teen die ontwikkeling van kardiovaskulêre komplikasies nie. In die studie ondersoek ons die beskermende effek van twee hoof bioaktiewe verbindings van Aspalathus linearis, fenielpirodruiwesuur-2-O-β-D-glukosied (PPAG) en aspalatien, as nuwe terapeutiese middels, teen oksidatiewe stres en hiperglukemie-geïnduseerde apoptose in H9c2 kardiomiosiete en ontrafel ons die molekulêre meganismes wat betrokke is. Die studie het getoon dat PPAG en aspalatien mitokondriale membraan depolarisasie verminder en hiperglukemie-geïnduseerde miokardiale apoptose voorkom deur die Bcl2/Bax verhouding te verhoog. Die studie toon verder dat alhoewel beide verbindings in staat was om hiperglukemie-geïnduseerde apoptose te verminder, net aspalatien lipiedtoksisiteit en oksidatiewe stres, wat verband hou met insulien weerstand, verbeter. 'n Belangrike kenmerk van hartversaking is die verskuiwing in mitokondrialesubstraat voorkeur wat die aanvang van oksidatiewe skade voorafgaan. Die huidige studie het getoon dat aspalatien glukose metabolisme verbeter deur vetsuuropname en daaropvolgende β-oksidasie te verminder. Dit word bereik deur die verlaagde uitdrukking van adenosienmonofosfaat geaktiveerde proteïenkinase threonien 172 (pAMPK (Thr172)) en karnitien palmitoyltransferase 1 (Cpt1), terwyl die ekspresie van asetiel-KoA karboksilase (Rek) en glukose transporter 4 (Glut4) verhoog word. Verder is dit bekend dat kardiomiosiete baie lae antioksidant kapasiteit het en gevolglik dat 'n verskuiwing in mitokondrialesubstraat oksidatiewe skade kan veroorsaak. Die studie wys dat aspalatien die antioksidantkapasiteit van die selle beskerm en oksidatiewe stres verlig deur aktivering van kernfaktor erythroïd-faktor (NF-E2)-verwante faktor 2 (Nrf2) antioksidant response element seintransduksie-pad. Aspalatien kon ook lipiedtoksisiteit verhoed deur adiponektien, C1Q en kollageen domein-verwante (Adipoq) ekspressie te verhoog met gepaardgaande afname van groepering van differensiasie 36 (Cd36) and Cpt1 mRNA uitdrukking. Ons het verder opgemerk dat Adipoq sterol regulatoriese element bindende transkripsie faktor 1 (Srebf1), stearoyl-koënsiem A desaturase 1 (Scd1) en opgeloste stof-draer familie 27 vetsuur transporter lid 3/5 (Slc27a3/5) ekspresie onderdruk, wat lipied akkumulasie in H9c2 kardiomiosiete teenwerk. In leptien weerstandige db/db muise het aspalatien totale cholesterol, trigliseriede en lae-digtheid lipoproteïen verlaag. Dit het gepaard gegaan met 'n afname in mRNA uitdrukking van inflammasie merkers, wat interleukin 3 and 6 (IL3 and IL6), tumor nekrose faktor reseptor superfamilie, lede 1b and 13 (Tnfrsf1b and Tnfsf13), Janus kinase 2 (Jak2) en mitogen-activated protein kinase 3 (Mapk3) insluit. In samevatting toon ons resultate dat aspalatien die ontwikkeling van DCM kan vertraag en daardeur die miokardium teen oorsaaklike faktore wat met die ontwikkeling en vordering van CVD verbind word, beskerm.

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