The therapeutic efficacy of ascorbic acid 2 phosphate, n-acetylcysteine and metformin against diabetes mellitus associated cellular senescence.

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dc.contributor.advisorVan de Vyver, Marien_ZA
dc.contributor.advisorJohnson, Rabiaen_ZA
dc.contributor.authorGovender, Saiureeen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.en_ZA
dc.date2023-08-30T13:09:12Zen_ZA
dc.date2023-02-13T17:25:13Zen_ZA
dc.date2023-08-30T13:09:12Zen_ZA
dc.date.accessioned2023-08-31T09:18:50Zen_ZA
dc.date.accessioned2023-02-13T17:25:13Zen_ZA
dc.date.available2023-08-31T09:18:50Zen_ZA
dc.date.available2023-02-13T17:25:13Zen_ZA
dc.date.issued2023-03en_ZA
dc.descriptionThesis (MSc)--Stellenbosch University, 2023.en_ZA
dc.description.abstractENGLISH ABSTRACT: Background: The incidence of diabetes mellitus (DM) is widespread. In DM, the systemic pathological microenvironment consists of elevated glucose levels (hyperglycaemia), advanced glycation end products (AGEs), chronic inflammation and oxidative stress. The combined effect of these factors inflicts DNA damage and induce premature cellular senescence. An accumulation of senescent cells within adipose tissue (amongst other) contribute to tissue dysfunction, disease progression and the development of comorbidities. The secretome of premature senescent cells namely, the senescence associated secretory phenotype (SASP) amplifies inflammation and oxidative stress through its paracrine action. There is thus a need for adjuvant treatments aimed at preventing disease progression by either preventing the onset of premature senescence or by restoring the function of senescent cells in DM. The purpose of this study was to develop a physiologically relevant in vitro model of premature cellular senescence associated with DM and investigate the therapeutic efficacy of different preventative and/or restorative therapies. Methods: In this research study, all experiments were conducted using a primary human adipose tissue derived stromal cell line (ADSCs). Serial passaging of ADSCs was performed to determine the point of onset for replicative senescence to rule it out as confounder. All subsequent experiments were conducted at low passages in healthy ADSCs. Cellular growth, morphology viability (crystal violet staining), and senescence (β-galactosidase (SA-β-gal) staining) were assessed in ADSCs following exposure to different concentrations of glucose (low 5mM; high 25mM), AGE-BSA (25, 50,100, 200, 400 µg/mL) and TNF-α (0.001, 0.005, 0.01, 0.02 µg/mL) in isolation or in combination for a period of 3 days. The optimal culture conditions using a combination of these factors were determined by quantifying the ratio of senescent to non-senescent cells. The SA-β-gal staining was corroborated by assessing the SASPs (IFNγ, TNF-α, IL1β, IL6, IL8, IL10) using a multiplex bead array cytokine assay to analyse the conditioned media derived from ADSCs. These results were further corroborated using quantitative polymerase chain reaction (qPCR) to assess the mRNA expression of p53, p16INK4A, p21cip1, TNF-α, IL6, PTX3, IL10, Arg1, NOS2, PPARγ, UCP3. The therapeutic effectiveness of N-acetylcysteine (NAC), Ascorbic acid 2 phosphate (AAP), and Metformin was evaluated using 3 different intervention strategies: a) pre-treatment prior to induction of senescence (preventative); b) combined intervention whilst inducing senescence; c) treatment following induction of senescence (restorative). A dose response experiment was performed to determine the highest non-toxic concentration of NAC (3.75mM) and AAP (0.6mM), whereas the human equivalent dose was used for Metformin (50 µg/mL). Results: Under high glucose (25mM) conditions, the exposure of ADSCs to a combination of AGE-BSA (400ug/mL) and TNF-α (0.02ug/mL) in culture for a period of 3 days, induced a 3- fold increase in the ratio of senescent to non-senescent cells. This observation was accompanied by a significant increase in the expression of SASP factors on both protein (IFNγ, TNF-α, IL1β, IL6, IL10) and mRNA level (TNF-α, IL6). Therapeutic intervention consisting of either pretreatment or restorative treatment with NAC, AAP or Metformin effectively reduced the ratio of senescent to non-senescent cells (SA-β-gal staining) compared to the induced premature senescent ADSCs without intervention. Conclusion: This study developed an in vitro model to induce premature cellular senescence by mimicking the DM microenvironment in culture. It was furthermore demonstrated that therapeutic intervention using either antioxidants such as NAC and AAP or the anti-diabetic drug, Metformin can potentially delay the onset of premature senescence and/or restore the function of senescent cells.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Agtergrond: Die voorkoms van diabetes mellitus (DM) is wydverspreid. In DM, bestaan die patologiese mikro-omgewing uit verhoogde glukose vlakke (hiperglukemie), gevorderde glykasie eindprodukte (AGEs), kroniese inflammasie en oksidatiewe spanning. Die gekombineerde effek van hierdie faktore veroorsaak DNS-skade en voortydige sellulêre veroudering word geïnduseer. ‘n Opbouing van verouderdeselle in vetweefsel (onder andere) dra by tot weefsel disfunksie, siekte vordering en komorbiditeit ontwikkeling. Die secretome van verouderdeselle ook genoem senescence associated secretory phenotype (SASP) versterk inflammasie en oksidatiewe spanning deur middel van die selle se parakriene funksie. Dus is daar ‘n behoefte aan bykomende behandelinge in DM wat mik om siekte vordering te verhoed deur óf die aanvang van verouderdeselle te verhoed, óf die funskie van verouderdeselle te herstel. Hierdie studie het beoog om ‘n fisiologiese relevante in vitro model van voortydige sellulêre veroudering geassosieer met DM te ontwikkel, asook die terapeutiese effektiwiteit van verskillende voorkomende en/of herstellende terapieë te ondersoek. Metodes: In hierdie navorsingstudie was ‘n primêre stromale sellyn afkomstig van menslike vetweefsel (ADSCs) gebruik om alle eksperimente uit te voer. Die sellyn het reeks subkulturing ondergaan om die begin van repliserende veroudering te bevestig en dit as ‘n verwarrings faktor uit te skakel. Alle daaropvolgende eksperimente was by lae subkulturing in gesonde ADSCs uitgevoer. Sellulêre groei, morfologie, lewensvatbaarheid (Crystal Violet kleuring) en veroudering (β-galactosidase (SA-β-gal) kleuring) was in ADSCs geassesseer nadat die selle aan verskillende konsentrasies glukose (laag 5mM; hoog 25mM), AGE-BSA (25, 50,100, 200, 400 µg/mL) en TNF-α (0.001, 0.005, 0.01, 0.02 µg/mL) afsonderlik of in kombinasie vir ‘n tydperk van 3 dae blootgestel was. Die SA-β-gal kleurstof resultate was bevestig deur die SASPs (IFNγ, TNF-α, IL1β, IL6, IL8, IL10) in gekonditioneerde medium afgelei van ADSCs te analiseer deur van ‘n multipleks krale-skikking sitokien-toets gebruik te maak. Verdere bevestiging van die resultate was gedoen deur die mRNS uitdrukking van p53, p16INK4A , p21cip1, TNF-α, IL6, PTX3, IL10, Arg1, NOS2, PPARγ, UCP3 deur middel van kwantitatiewe polimerase kettingreaksie (qPCR) te kwantifiseer. Die terapeutiese effektiwiteit van Nasetielsistien (NAC), Askorbiensuur 2 fosfaat (AAP), en Metformin was deur drie veskillende intervensie strategieë naamlik, a) voorbehandeling voor die induksie van veroudering (voorkomend); b) gekombineerde intervensie gedurende geïnduseerde veroudering; c) behandeling wat na geïnduseerde veroudering volg (herstellend), geëvalueer. Om die hoogste nie-toksiese konsentrasie van NAC (3.75mM) en AAP (0.6mM) te bepaal was ‘n dosisreaksie eksperiment uitgevoer, maar in die geval van Metformin (50 µg/mL) was die menslike ekwivalente dosis gebruik. Resultate: Onder hoë glukose kondisies (25mM) in selkultuur het die kombinasie AGE-BSA (400ug/mL) en TNF-α (0.02ug/mL) vir ‘n periode van 3 dae die verhouding van verouderde tot nie-verouderdeselle met 3-voudig verhoog. Hierdie observasie was ondersteun deur 'n beduidende verhoging in die uitdrukking van SASP faktore op beide protein (IFNγ, TNF-α, IL1β, IL6, IL10) en mRNS vlak (TNF-α, IL6). Terapeutiese intervensie bestaande uit óf voorbehandeling, óf herstellende behandeling met NAC, AAP of Metformin het die verhouding van verouderde tot nie-verouderdeselle (SA-β-gal) verlaag, in vergelyking met geïnduseerde voortydige veroudering in ADSCs sonder intervensie. Gevolgtrekking: Hierdie navorsingstudie het ‘n in vitro geïnduseerde voortydige sellulêre verouderings model ontwikkel deur die DM mikro-omgewing in selkultuur na te boots. Verder demonstreer die studie dat ‘n terapeutiese intervensie deur óf antioksidante soos NAC en AAP, óf die anti-diabetiese middel Metformin te gebruik, die aanvang van voortydige veroudering kan vertraag en/of die funksie van verouderdeselle herstel.af_ZA
dc.description.versionMastersen_ZA
dc.embargo.terms2023-08-16en_ZA
dc.formatapplication/pdfen_ZA
dc.format.extent89 pages : illustrationsen_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/128479en_ZA
dc.languageen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshMetformin -- Therapeutic use -- Effectivenessen_ZA
dc.subject.lcshDiabetes -- Treatmenten_ZA
dc.subject.lcshCellular senescenceen_ZA
dc.subject.lcshAdipose tissuesen_ZA
dc.subject.lcshAdvanced glycation end productsen_ZA
dc.subject.lcshIn vitro modelen_ZA
dc.subject.lcshUCTDen_ZA
dc.titleThe therapeutic efficacy of ascorbic acid 2 phosphate, n-acetylcysteine and metformin against diabetes mellitus associated cellular senescence.en_ZA
dc.typeThesisen_ZA
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