The effect of green rooibos extract on rat hearts in a pre-diabetic model : an evaluation of the function and mechanisms involved

Smit, Sybrand Engelbrecht (2019-03)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Background— Cardiovascular diseases (CVD) remains the leading cause of death globally, with a rising prevalence of individual risk factors such as obesity and insulin resistance. Furthermore, diabetic patients are particularly at risk for developing ischemic heart disease and strokes. In patient suffering an ischemic event, most of the myocardial damage incurred happens once blood flow is restored – a phenomena known as reperfusion injury. Mitochondrial ROS production is implicated as one of the major contributors to cell death in the reperfused heart and the importance of mitophagic processes (mitochondrial housekeeping) could be a potential therapeutic target to prevent ischemia/reperfusion injury (I/R-I). Aspalathus linearis (commonly known as rooibos) is an indigenous South African plant grown exclusively in the Western Cape fynbos region. Rooibos is rich in bioactive phenolic compounds, including aspalathin, a C-linked dihydrochalcone glucoside unique to rooibos, known for its hypoglycemic and strong antioxidant potential. Methods— The present study made use of 300 Wistar rats randomly allocated into controls and rats receiving a 16-week high-fat, high-caloric diet (HCD). After 10 weeks on the respective diets, half of each group received 60 mg/kg/day Afriplex green rooibos extract (GRT), containing 12% aspalathin, for 6 weeks. The primary aim was to investigate its therapeutic potential in treating 20 min global ischemia/reperfusion injury (I/R-I) in rats with increased susceptibility for CVD, as well as determine the mitochondrial oxidative phosphorylation (OxPhos) during four stages of I/R-I. The secondary aim was to elucidate GRT’s effect on cardiac signaling and mitophagy with regards to I/R-I. Results— HCD over 16 weeks resulted in daily increased food intake (22.30±1.27 g vs 18.00±0.54 g = ↑24%; p<0.001), decreased daily water intake (19.55±0.58 mL vs 29.74±0.74 mL = ↓34%; p<0.001), leading to increased body weight (403.9±5.1 vs373.6±4.8 g = ↑8%; p<0.001) and increased intraperitoneal fat (23.69±0.89 vs 13.98±0.43 g = ↑69%; p<0.001) compared to age-matched controls. Furthermore, HCD increased fasting blood glucose (5.75±0.16 vs 5.21±0.16 mM = ↑10%; p<0.05), fasting blood insulin (6.20±0.57 vs 4.31±0.54 ng/mL = ↑44%; p<0.05) and insulin resistance through raised HOMA-IR (3.52±0.35 vs 2.30±0.28 = ↑53%; p<0.05) compared to age-matched controls. GRT supplementation for 6 weeks had no significant effect on biometric parameters in either controls or HCD. Pre-ischemia, HCD rats presented with worse functional heart parameters, such as diastolic and systolic pressure, aortic output (AO), cardiac output (CO) and total work (WT) (10.69±0.14 vs 12.6±0.19 mW = ↓15%; p<0.001) compared to controls, while GRT supplementation was able to significantly improve these parameters in both controls (13.91±0.23 mW = ↑10%; p<0.001) and HCD (11.98±0.20 mW = ↑12%; p<0.001). GRT administration also lowered the heart rate during stabilization in both control and HCD by an average 10 bpms (p<0.05). Post-ischemia, HCD hearts were weaker than controls and had a lower WT recovery (55.51±2.28 vs 64.8±2.04% = ↓14%; p<0.01), while GRT restored WT recovery in HCD (63.35±2.49% = ↑14%; p<0.05). HCD rats also had a greater infarct size compared to controls (34.99±11.56 vs 16.42±8.71% = ↑113%; p<0.01) following a 35 min regional ischemia protocol. GRT supplementation led to a remarkable reduction in infarct size (13.22±6.66% = ↓62%; p<0.001), while conferring no added protection to controls (14.58±5.43%). Regarding cardiac and mitochondrial signaling, prior to ischemia HCD heart had a decreased dependence on insulin-dependent AMPK and increased inflammation via upregulated p38, whereas GRT treatment presented with decreased insulindependent PKB and AS160 signaling (together with increased FA OxPhos compared to carbohydrates, however showed more mitochondrial uncoupling, decreasing basal metabolic rate and thereby potentially less ROS production), inhibited GSK3β and conferred an anti-inflammatory effect by significantly reducing p38 activation. HCD also had higher mitophagy rates through Parkin and LC3 signaling. After 20 min ischemia, in HCD, ATM and AMPK were upregulated and insulin-dependent PKB downregulated with GSK3β. Mitophagy signals, such as PINK1 and p62 were elevated, but autophagic flux remained low during ischemia in all groups. GRT supplementation resulted in an opposite profile with AMPK downregulated and showing inhibition of ERK1/2. In early reperfusion, all protective signaling were downregulated in HCD including AMPK, PKB, AS160, GSK3b and ATM. Mitophagy was also activated through Parkin, p62 and LC3 while having increased OxPhos potential in FA compared to carbohydrates. GRT supplementation reduced oxidative stress and inflammation through downregulation of JNK1/2 and p38, and initiated mitophagy through an AMPKdependent mechanism and Parkin. GRT supplementation also inhibited mitochondrial OxPhos after reperfusion culminating in a potentially decreased ROS production. Conclusion— This study showed the cardioprotective effect of Afriplex GRT supplementation by improving functional heart recovery and reducing infarct size post-ischemia in rats with elevated risk for CVD. Another novel find is the reduction in heart rate induced by GRT treatment through inhibition of pacemaking cells. This could have potential therapeutic application in patients suffering from ischemic heart disease. GRT ilicits a cardiotonic effect in I/R-I through anti-inflammatory mechanisms pre- and post-ischemia. In early reperfusion, GRT treatment resulted in decreased oxidative stress, inhibition of mitochondrial OxPhos and enabled AMPKdependent mitophagy. GRT shows promise as a strong antioxidant and anti-inflammatory agent in managing adverse outcomes in patients at risk for CVD.

AFRIKAANSE OPSOMMING: Agtergrond- Kardiovaskulêre siektes (CVD) is steeds die wêreld se nommer een oorsaak van sterftes. Die teenwoordigheid hiervan kan grotendeels toegeskryf word aan ‘n toenemende voorkoms van individuele risikofaktore, insluitende vetsug en insulienweerstandigheid. Verder, diabetiese pasiënte in besonder dra hoë risiko vir die ontwikkeling van iskemiese hartsiektes en beroertes. In pasiënte wat 'n iskemiese gebeurtenis ly, word die meeste van die myokardiale skade eers opgedoen wanneer bloedvloei herstel word - 'n verskynsel bekend as reperfusiebesering. Mitochondriale reaktiewe oksidant spesie (ROS)-produksie word geïmpliseer as een van die belangrikste bydraers tot seldood tydens reperfusie en die belangrikheid van mitofagiese prosesse (mitochondriale huishouding) kan 'n potensiële terapeutiese doelwit dien in voorkoming van iskemie/reperfusiebesering (I/R-I). Aspalathus linearis (algemeen bekend as rooibos) is 'n inheemse Suid-Afrikaanse plant wat uitsluitlik in die Wes-Kaapse fynbosstreek gegroei word. Rooibos is ryk aan bioaktiewe fenoliese verbindings, insluitende aspalatien, 'n C-gekoppelde dihidrochalcoon-glukosied, uniek aan rooibos, en bekend vir sy sterk anti-inflammatoriese antioksidant en vermoë om bloedsuiker te verlaag. Metodes- In die teenwoordigende studie het ons gebruik gemaak van 300 Wistar-rotte wat lukraak verdeel is in ‘n 16-week hoë-vet, hoë-kalorie-dieet (HCD) groep, en ‘n ouderdom-gekontroleerde beheergroep wat slegs standard rotkos ontvang het. Na 10 weke op die onderskeie diëte het die helfte van elke groep vir 6 weke 60 mg / kg / dag Afriplex Green Rooibos Extract (GRT), wat 12% aspalatien bevat, ontvang. Die primêre doel was om die terapeutiese potensiaal as behandeling van 20 min globale ischemie / reperfusiebesering (I /R-I) te ondersoek in rotte met verhoogde vatbaarheid vir KVS, asook om die mitochondriale oksidatiewe fosforilering (OxPhos) tydens vier stadiums van I/R-I te bepaal. Die sekondêre doelwit was om GRT se effek op hart seintransduksie en mitofagie met betrekking tot I/R-I te verduidelik. Resultate-HCD administrasie, teenoor kontrole dieet, vir 16 weke het gelei tot verhoogde daaglikse voedselinname (22,30±1,27 g teenoor 18,00±0,54 g = ↑ 24%; p<0.001) en verhoogde daaglikse waterinname (19.55±0.58 mL vs 29.74±0.74 mL = ↓ 34%; p<0.001) wat gelei het tot verhoogde liggaamsgewig (403.9±5.1 vs 373.6±4.8g = ↑ 8%; p<0.001) en verhoogde intraperitoneale vet (23.69±0.89 teenoor 13.98±0.43g = ↑ 69%; p<0.001). Daarbenewens het HCD ook verhoogde vastende bloedglukose (5.75±0.16 vs 5.21±0.16 mM = ↑10%; p<0.05), vastende bloedinsulien (6.20±0.57 vs 4.31±0.54 ng / mL = ↑44%; p<0.05) en insulienweerstandigheid deur verhoogde HOMA-IR (3.52±0.35 vs 2.30± 0.28 = ↑53%; p<0.05) in vergelyking met ouderdom-gekontroleerde kontrole groepe. GRT-aanvulling vir 6 weke het geen beduidende effek gehad op biometriese parameters in beide die kontroles en HCD nie. Tydens stabilisering van die geïsoleerde harte, het dié van HCD-rotte verswakkende funksionele hartparameters getoon, soos diastoliese en sistoliese druk, aorta-uitset (AO), hartuitset (CO) en totale werk (WT) (10.69±0.14 teenoor 12.6±0.19 mW = ↓15%; p<0.001) in vergelyking met kontroles, terwyl GRT-aanvulling hierdie parameters aansienlik kon verbeter in beide kontroles (13.91±0.23 mW = ↑10%; p<0.001) en HCD (11.98±0.20 mW = ↑12%; p<0.001). GRT-administrasie het ook die hartklop verlaag tydens stabilisering met 'n gemiddelde 10 hartkloppens per minuut in beide beheer en HCD groepe (p<0.05). Tydens reperfusie het HCD harte swakker gevaar as kontroles met 'n verlaagde WT herstelling (55.51±2.28 teenoor 64.8±2.04% = ↓14%; p<0.01), terwyl GRT WT kon herstel in HCD (63.35±2.49% = ↑14%; p<0.05). HCD-rotte het ook groter infarkt-groottes gehad in vergelyking met beheer groepe (34,99±11,56 teenoor 16,42±8,71% = ↑113%; p<0.01) na aanleiding van 'n 35 min streeks-ischemie protokol. GRT-aanvulling het gelei tot 'n merkwaardige vermindering in infarkt-grootte (13.22±6.66% = ↓62%; p<0.001), terwyl dit geen ekstra beskerming aan kontroles verleen het nie (14.58±5,43%). Met betrekking tot die hart- en mitochondriale seintransduksie het die HCD-hart 'n afname in afhanklikheid van insulien-afhanklike AMPK en toename van inflammasie via opgereguleerde p38 getoon. GRT-behandeling het ‘n beduidende verlaging van insulien-afhanklike PKB- en AS160-sein geïnduseer (saam met verhoogde vetsuur teenoor koolhidraat oksidatiewe fosforilasie (OxPhos) vertoning, maar terselfdertyd verhoogde mitochondriale ontkoppeling wat gevolglik ‘n stadiger basale metaboliese tempo tot volg het en moontlik minder reaktiewe oksidante spesie (ROS)-produseer). GRTbehandeling het ook GSK3β geïnhibeer en 'n anti-inflammatoriese effek toegedien deur die p38-aktivering aansienlik te verminder. HCD het ook hoër mitofagiese prosesse getoon deur Parkin- en LC3-seine te opreguleer. Na 20 min van iskemie in HCD harte is ATM en AMPK opgereguleer en insulien-afhanklike PKB is afgereguleer saam met GSK3β. Mitofagiese seine, soos PINK1 en P62, was verhef, maar autofagie as ‘n geheel was laag gedurende ischemie in alle groepe. GRT aanvulling het gelei tot 'n teenoorgestelde profiel met ‘n afname in AMPK aktivering asook inhibisie van ERK1/2. Tydens vroeë reperfusie is alle beskermende seinwerk in HCD afgestel, insluitende AMPK, PKB, AS160, GSK3β en ATM. Mitofagie is ook geaktiveer deur Parkin, P62 en LC3, terwyl OxPhos potensiaal toegeneem het in vetsure in vergelyking met koolhidrate. GRT-aanvulling het verder oksidatiewe stres en inflammasie verminder deur JNK1/2 en p38 af te reguleer, en verhoging in mitofagie teweeg gebring deur middel van 'n AMPK-afhanklike meganisme en Parkin. GRTaanvulling het ook mitochondriale OxPhos geïnhibeer na reperfusie wat 'n potensiële verlaagde ROSproduksie tot volg gehad het. Gevolgtrekking - Hierdie studie het die hartbeskermende effek van Afriplex GRT-aanvulling getoon deur die verbetering van funksionele hartherstel en die vermindering van infarkt-grootte na ‘n ischemiese episode uit te wys in pre-diabetiese rotte met verhoogde risiko vir CVD. Nog 'n nuwe bevinding is die verlaging in hartklop geïnduseer deur GRT behandeling deur inhibisie van pasaangee selle en kan potensiële terapeutiese toediening hê by pasiënte wat aan iskemiese hartsiektes ly. GRT gee 'n kardiotoniese effek in I/R-I deur middel van anti-inflammatoriese meganismes voor- en na-ischemie. In vroeë reperfusie het GRTbehandeling gelei tot verminderde oksidatiewe stres, inhibisie van mitochondriese OxPhos en AMPKafhanklike mitofagie. GRT toon belowende effekte as 'n sterk antioksidant en anti-inflammatoriese middel in die hantering van nadelige uitkomste vir pasiënte met 'n risiko vir CVD.

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