The treatment effect of Rooibos on vascular function and signalling pathways of aortas from diet induced obese rats.

Eldieb, Nada Ahmed (2017-03)

Thesis (MMed)--Stellenbosch University, 2017

Thesis

ENGLISH ABSTRACT : Introduction: Obesity is becoming a worldwide health problem and is a well-documented independent risk factor for the development of endothelial dysfunction (decreased nitric oxide (NO) bioavailability) and cardiovascular disease (CVD). It is also associated with chronic inflammation and decreased insulin sensitivity. As a highly active endocrine organ, perivascular adipose tissue (PVAT) releases a variety of adipokines and inflammatory cytokines that activate several signalling pathways. Recently, observational studies have indicated a role for oral supplementation of Rooibos (RB) in lowering the risk for cardiovascular disease by improving endothelial dysfunction by means of RB’s anti-inflammatory and anti-oxidant properties. The acute effect of RB in an ex vivo aortic model has not been investigated yet. Aims: Therefore, this study aimed to investigate the effect of direct RB administration to aortas from obese and lean Wistar rats, by measuring the aortic function as well as changes in the signalling pathways and the inflammatory markers. . Methods: Aortas were obtained from three groups of euthanized male Wistar rats: young control (aged 16 weeks), aged-matched lean group (aged 22 weeks) on a normal rat chow and diet group (aged 22 weeks) on a 16 week high fat diet, HFD (high in sucrose and cooking fat). Aortic segments, with or without PVAT, were placed into an organ bath containing Krebs buffer at 37⁰C. Aortic ring function was determined by cumulative phenylephrine (PE) induced contraction (PE: 100nM-1μM), followed by cumulative acetylcholine (Ach) induced relaxation (Ach: 30 nM-10μM). RB (0.02%) was administered for 30 min during stabilisation before initiation of contraction and relaxation. Intracellular signalling pathways: eNOS, and the upstream activators PKB and AMPK, MAPKs (ERK1/2, JNK1/2, P38), as well as Nitrotyrosine and CREB were evaluated using Western blot analysis. Inflammatory markers were also analysed using the multiplex kit. Data was analysed using two-way ANOVA with Bonferroni’s post-test. Results: The HFD caused an increase in body weight, intraperitoneal fat, fasting glucose level, as well as HOMA index. Aortas with PVAT of the older lean group showed a significant increase in contractility compared to the young control (p <0.05). However aortas with PVAT of the obese diet group showed a reduced contractile response and enhanced relaxation response compared to the lean group (p <0.05). The addition of RB decreased the contractile response in lean group with PVAT. However, RB enhanced the contractility of the diet group (with and without) PVAT, as well as reduction in relaxation on the diet group without PVAT. The diet group had increased phosphorylation and phospho/total ratio of eNOS and ERK1/2 in aortas with and without PVAT, compared to the lean group. However, RB decreased this effect. Inflammatory markers showed variable results. Conclusion: Sixteen weeks on a HFD was effective to induce insulin resistance. The anti-contractile effect of the HFD on aortic function and the enhancement in the relaxation might be due to the elevation of phosphorylated eNOS that was associated with increased ERK1/2 phosphorylation. This study demonstrated that RB may reverse the contractile inhibition of obese rat aorta with PVAT, with no effect on PVAT relaxation. Furthermore, RB has shown a pro-contractile and anti-relaxant effect in aorta of the diet group without PVAT. This demonstrates that RB in not deleterious when PVAT is present and may be beneficial to endothelial function in obese individuals.

AFRIKAANSE OPSOMMING : Inleiding: Vetsug is besig om 'n wêreldwye gesondheidsprobleem te word en is 'n goed-gedokumenteerde onafhanklike risikofaktor vir die ontwikkeling van endoteel disfunksie (afgeneem in stikstofoksied, NO, biobeskikbaarheid) en kardiovaskulêre siekte (KVS). Dit word ook geassosieer met chroniese inflammasie en verminderde insulien sensitiwiteit. As 'n hoogs aktiewe endokriene orgaan, stel perivaskulêre vetweefsel (PVAT) 'n verskeidenheid van adipokines en inflammatoriese sitokiene vry wat verskeie seintransduksiebane aktiveer. Onlangs het waarnemingstudies 'n rol vir orale aanvulling van Rooibos (RB) ‘n verlaging van die risiko vir KVS aangedui deur die verbetering van endoteel disfunksie deur middel van RB se anti-inflammatoriese en anti-oksidant eienskappe. Die akute effek van RB in 'n ex vivo aorta model is nog nie ondersoek nie. Doelwitte: Hierdie studie is daarop gemik om die effek van direkte RB administrasie op aortas van oorgewig en maer Wistar rotte te ondersoek, deur die meting van die aorta funksie asook veranderinge in die sein paaie en die inflammatoriese merkers. Metodes: Aortas is verkry van drie groepe van kant gemaakte manlike Wistar rotte: 'n jong kontrole (16 weke oud), ‘n maer groep (22 weke oud) op 'n normale rotdieet, en 'n dieet groep (22 weke oud) op 'n hoë vet dieet, HVD (hoog in sukrose en kookvet) vir 16 weke. Aorta segmente, met of sonder PVAT, is in 'n orgaan bad met Krebs buffer teen 37⁰C geplaas. Aorta ring funksie is bepaal deur kumulatiewe fenielefrien, PE, geïnduseerde kontraksie (PE: 100nM-1μM), gevolg deur kumulatiewe asetielcholien, Ach, geïnduseerde verslapping (Ach: 30 nM-10μM). Die RB (0,02%) is toegedien vir 30 min tydens stabilisering voor aanvang van kontraksie en verslapping. Intrasellulêre sein paaie: eNOS, en die aktiveerders PKB en AMPK, asook MAPKs (ERK1/2, JNK1/2, p38), Nitrotyrosine en CREB is geëvalueer met behulp van Western-klad analise. Inflammatoriese merkers is ook geanaliseer met behulp van die multiplex kit. Data is geanaliseer deur van twee-rigting ANOVA met Bonferroni se post-toets gebruik te maak. Resultate: Die HVD veroorsaak 'n toename in die liggaamsgewig, intraperitoniale vet, vastende glukose vlak, asook HOMA indeks. Aortas met PVAT van die ouer maer groep het ‘n beduidende toename in kontraktiliteit vergeleke met die jong kontroles getoon (p <0.05). Die aortas met PVAT van die vetsugtige dieet groep het 'n afname kontraktiele respons getoon en verbeterde verslapping in vergelyking met die maer groep (p <0.05). Die toediening van RB het die kontraktiele respons in die maer groep met PVAT verlaag. RB het die kontraktiliteit van die dieet groep (met en sonder PVAT) verhoog, asook die verslappings effek in die dieet groep sonder PVAT verlaag. Die dieet groep het toename in fosforilering en fosfo / totale verhouding van eNOS en ERK1/2 in aortas met en sonder PVAT getoon, in vergelyking met die maer groep. Maar RB hierdie effek laat afneem. Inflammatoriese merkers toon veranderlike resultate. Gevolgtrekking: Sestien weke op 'n hoë vet dieet was effektief om insulienweerstandigheid te veroorsaak. Die anti-kontraktiele effek van die HVD op aorta funksie en die pro-verslappings effek kan verband hou met die toename van gefosforileerde eNOS en 'n verhoogde ERK1/2 fosforilering. Hierdie studie bewys dat RB die kontraktiele inhibisie van oorgewig rot aortas met PVAT kan omkeer, met geen effek op PVAT ontspanning nie. Verder het RB 'n pro-kontraktiele en anti-verslappings effek in aorta van die dieet groep sonder PVAT getoon. Dit dui aan dat RB nie nadelige is wanneer PVAT teenwoordig is nie en dat dit tot voordeel van endoteel funksie in vetsugtig individue kan wees.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100932
This item appears in the following collections: