Modulation of oxidative stress and inflammation using a grape seed derived polyphenol

Petersen, Kelly Shirley (2016-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Oxidative stress and inflammation are intricately interlinked, especially in the context of ageing/accelerated ageing, as well as several chronic disease states, where they are implicated as aetiological factors. In the past decade polyphenols have been investigated for their potent anti-oxidant as well as anti-inflammatory properties. Previous research by our group has highlighted the potential of a grape seed-derived polyphenol as both anti-oxidant and anti-inflammatory modality. However, no data has been generated in human models and the mechanism(s) of action is still largely unknown. Thus the aim of this study was to more comprehensively investigate potential anti-oxidant or anti-inflammatory mechanisms of a grape seed-derived polyphenol (PCO) on acute (as found with exercise) and chronic (as in ageing) models of oxidative stress. After obtaining ethical clearance, blood samples were obtained from healthy human subjects from two different groups (aged (n=7) and young (n=14). In the young group a further distinction was made between sedentary (n=8) and fit individuals (n=6) whom we exposed to an HIIT exercise intervention to induce acute oxidative stress. Oxidative status was assessed in plasma using validated photometric and colorimetric assays and plasma myeloperoxidase (MPO) assessed as baseline inflammatory indicator. Neutrophils were isolated at rest (and after exercise from young fit subjects) and treated with the polyphenol in vitro for 1 hour. The chemokinetic capacity of treated and control neutrophils in response to a chemotactic signal was then determined using a Dunn chamber and live cell imaging. In addition, neutrophils were analysed for the expression of functional capacity markers (intracellular MPO, Fcγ Receptor IIIb (CD16) and CEACAM 8 (CD66b)) via flow cytometry. Oxidative status of neutrophils were also performed on isolates. The results indicate that the aged population had significantly worse oxidative and inflammatory profiles than young sedentary controls - this was evident in their conjugated dienes content as well as decreased ferric reducing ability of plasma (FRAP). In neutrophils, aged and young subjects had increased chemokinetic accuracy and capacity after 1 hour of in vitro polyphenol treatment. The treatment was also associated with a shedding of CD16 and increased expression of CD66b - both linked to improved neutrophil motility. The intracellular MPO content of neutrophils indicated that PCO had an anti-inflammatory effect across groups. The exercise intervention did not induce measurable changes in oxidative stress or inflammation. We therefore concluded that grape seed-derived polyphenols modulate inflammation and oxidative responses by facilitating more efficient neutrophil motility. This decreases the number of neutrophils required per response, effectively resulting in less secondary tissue damage, less oxidative stress and faster resolution of inflammation.

AFRIKAANS OPSOMMING: Oksidatiewe stres en inflammasie is fyn verweef, veral in die konteks van veroudering en versnelde veroudering. Verder word beide prosesse geïmpliseer in die etiologie van veroudering en verskeie kroniese siektes. In die verlede is polifenole al ondersoek vir hul aansienlike anti-inflammatoriese en anti-oksidant effekte. Vorige navorsing deur ons groep het die potensiaal van ‘n druif-afgeleide polifenool al bewys in hierdie konteks. Daar is tans nog geen data uit mensmodelle gegenereer nie en die meganisme waardeur hierdie effekte uitgevoer word, is nog grootliks onbekend. Die doel van hierdie studie was dus om op ‘n meer omvattende manier, die moontlike anti-inflammatoriese en anti-oksidant megansismes van ‘n druif-afgeleide polifenool te ondersoek, deur modelle van akute (soos na harde oefening) en kroniese (soos met veroudering) oksidatiewe stres. Nadat etiese klaring verkry is, is bloedmonsters verkry van gesonde vrywilligers in twee groepe (oud (n=7) en jonk (n=14)). In die jong groep is onderskeid ook getrek tussen fikse (n=6) en onfikse (n=8) individue. Die fikse groep is addisioneel onderwerp aan hoë intensiteit oefening om akute oksidatiewe stres te veroorsaak. Oksidatiewe status in plasma is bepaal duer erkende fotometriese en kolorimetriese toetse te gebruik, terwyl plasma MPO as indikasie van inflammasie getoets is. Neutrofiele is geïsoleer uit bloed wat in ‘n rustende toestand getrek is, asook na oefening in die fiske jong groep. Chemokinetiese kapasiteit van neutrofiele is voor en na 1 uur in vitro polifenool behandeling bepaal deur gebruik te maak van ‘n Dunn kamer en lewende sel mikroskopie. Neutrofiel uitdrukking van merkers vir funksionele kapasiteit (intrasellulêre MPO, Fcγ Receptor IIIb (CD16) and CEACAM 8 (CD66b)) is ook met vloeisitometrie bepaal. Oksidatiewe status merkers is ook op geïsoleerde neutrofiele bepaal. Resultate dui aan dat die ouer populasie beduidend slegter oksidatiewe en inflammatoriese profiele gehad het as hulle jonger eweknieë – dit was bv. Die geval vir CD en FRAP in die plasma. In neutrofiele het beide ouer en jong vrywilligers verbeterde chemokinetiese kapasiteit en akkuraatheid getoon na polifenool blootstelling. Die polifenool blootstelling het ook verhoogde CD66b en verlaagde CD16 uitdrukking tot gevolg gehad, wat altwee met verbeterde neutrofiel beweeglikheid in verband gebring word. Verder het intrasellulêre MPO ook aangedui dat die polifenool ‘n anti-inflammatoriese effek in al die groepe gehad het deurdat selle minder MPO vrygestel het. Die oefening ingreep het nie beduidende verskille teweeggebring in die gekose merkers vir oksidatiewe stres of inflammasie nie. Ons gevolgtrekking is dus dat druif-afgeleide polifenole die oksidatiewe en inflammatoriese response moduleer, ten minste gedeeltelik deurdat dit neutrofiele meer effektief maak. Dit het tot gevolg dat minder neutrofiele per respons nodig is, dat minder sekondêre skade aan weefsel aangerig word en dat inflammasie vinniger opklaar.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100193
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