Antioxidant supplementation as protective strategy against stem cell impairment in Type 2 Diabetes

Anti-oksidant aanvulling as beskermende strategie teen stamsel disfunksie in Tipe 2 Diabetes

Maartens, Michelle (2021-03)

Thesis (MSc)--Stellenbosch University, 2021.

Thesis

ENGLISH ABSTRACT: Type 2 Diabetes Mellitus (T2DM) is a global epidemic. It is a complex disorder that leads to cellular dysfunction and the development of co-morbidities. The underlying pathologic microenvironment in T2DM, associated with hyperglycaemia, include the accumulation of advanced glycation end products, excessive oxidative stress, and chronic inflammation. Bone marrow mesenchymal stem cells (MSC) are especially susceptible to this damaging microenvironment and as consequence the endogenous repair mechanisms within the body fail giving rise to secondary complications such as non-healing wounds, retinopathy, and neuropathy. There is thus a need for a holistic approach when it comes to disease management in which anti-diabetic drugs focussed on glucose control is complemented by supplementary treatment aimed at restoring homeostasis. Natural and synthetic antioxidants such as Ascorbic acid-2-phosphate (AAP) and N-acetyl-l-cysteine (NAC) are known to protect cells against oxidative stress-induced damage in vitro and have been shown to reduce inflammation in various models. The efficacy of these antioxidants to restore homeostasis and prevent cellular dysfunction in T2DM is however still unknown. The aim of this study was to investigate the protective effects of combined NAC and AAP supplementation against MSCs impairment using an animal model of obese diabetic (B6.C-Lepob/J) (ob/ob) (n=14) and wild type control (C57BL6/J) (n=20) mice. All mice received jelly cubes containing either antioxidants (NAC7.5mM+AAP0.6mM) or placebo (vehicle control) for a period of 6-weeks. Metabolic parameters (weight and blood glucose) were assessed on a weekly basis and the overall antioxidant status of animals assessed at the end of the 6-week period by analysing the total antioxidant capacity (TAC) and Malondialdehyde (MDA) levels in serum. Bone marrow MSCs were isolated from mice in each of the respective treatment groups and their ex vivo growth rate, viability, multi-lineage differentiation capacity and paracrine responsiveness upon stimulation with wound fluid assessed. Compared to the wild type (C) mice, excessive weight gain (weight: C 28.7±1.6 g; DM 44.3±3.5 g) (p<0.05) and hyperglycaemia (blood glucose: C 10.1±1.3 mmol/L; DM 23.6±5.7 mmol/L) (p<0.05) was evident in the DM animals validating the animal model as representative of T2DM. The antioxidant supplementation did not affect metabolic parameters indicating that differences observed between supplement and placebo treated mice were not due to weight loss or changes in glucose metabolism. NAC/AAP significantly (p<0.05) reduced lipid peroxidation in DM animals (DM:P 39.0±14.7 nmol/L; DM:S 21.5±11.6 nmol/L) to a level comparable to that of controls (C:P 22.9±11.4 nmol/L; C:S 30.5±3.3 nmol/L) and increased the overall TAC (C:P 3.7±1.3 U/mL; C:S 4.0±0.7 U/mL; DM:P 5.2±0.9 U/mL; DM:S 5.8±1.5 U/mL). The ex vivo growth rate of cells derived from DM animals were impaired (Time to confluence: C 8 days; DM 12 days). NAC/AAP supplementation did however improve the growth rate and viability of MSCs derived from both animal models. NAC/AAP furthermore reduced the adipogenic differentiation capacity of MSCs but could not restore osteogenesis in DM MSCs. Upon stimulation with wound fluid, slightly increased IL6 (DM:S+WF 1583.3±481.9 pg/mL) and IL10 (DM:S+WF 27.1±9.8 pg/mL) release was evident in MSCs derived from NAC/AAP supplemented animals. In conclusion, NAC/AAP supplementation was able to reduce oxidative stress in animals and improved MSCs viability.

AFRIKAANS OPSOMMING: Tipe 2 Diabetes Mellitus (T2DM) is ‘n wêreldwye epidemie. Dit is ‘n ingewikkelde siekte wat tot selwanfunksionering en die ontwikkeling van komorbiditeite lei. Die onderliggende patologie van T2DM word met hiperglukemie geassosieer en sluit die vermeerdering van gevorderde glykasie-eindprodukte, buitengewone oksidatiewe-spanning en chroniese inflammasie in. Beenmurg-mesenchimale-stamselle (MSS) is veral vatbaar tot hierdie skadelike mikro-omgewing. Gevolglik word die liggaam se inwendige herstelmeganismes beskadig wat tot die ontstaan van sekondêre komplikasies soos ongeneesbare wonde, retinopatie en neuropatie lei. Dus word daar ‘n holistiese-aanvlug ten opsigte van behandeling benodig. Diabeetmedisyne, gefokus op die beheer van glukosevlakke, word met aanvullende behandeling wat beoog om homeostase reg te stel, gepaar. Natuurlike en sintetiese anti-oksidante soos askorbiensuur-2-fosfaat (AAP) en N-asetiel-I-sisteïen (NAC) is daartoe in staat om selle in vitro teen oksidatiewe-spanning-geïnduseerde skade te beskerm. Dit is met behulp van verskeie modelle bewys dat dié anti-oksidante ook inflammasie verminder. Daar is egter nog onsekerheid met betrekking tot hierdie anti-oksidante se vermoë om homeostase te herstel en selwanfunksionering te voorkom. Die doel van hierdie studie is om aanvullings NAC en AAP se beskermende effek op diabeet-aangetaste MSS deur middel van ‘n diermodel wat uit vetsugtige diabeet-B6.C-Lepob/J-muise (ob/ob) (n=14) en wilde-tipe-C57BL6/J-muise (n=20) bestaan, te ondersoek. Al die muise was vir ‘n tydperk van 6 weke jellieblokkies wat óf die aanvulling (NAC7.5 mM+AAP0.6 mM) óf die plasebo (kontrole) bevat het, gevoer. Die metaboliese parameters (gewig en bloedglukosevlakke) was weekliks geëvalueer. Die diere se universele anti-oksidant-status was teen die einde van die 6-week-periode in terme van totale anti-oksidant-kapasiteit (TAC) en Malondialdehiedvlak (MDA) in serum geëvalueer. Die muise se geïsoleerde beenmurg-MSS se ex vivo groeitempo, lewensvatbaarheid, multi-kapasiteit differensiasievermoë en parakrien-reaksie op wondvog-stimulasie was geassesseer. In vergelyking met die wilde-tipe-muise (WT), het die ob/ob-muise buitensporig gewig opgetel (gewig: WT 28.7±1.6 g; ob/ob 44.3±3.5 g) (p<0.05) en hiperglukemie (bloedglukose: WT 10.1±1.3 mmol/L; ob/ob 23.6±5.7 mmol/L) (p<0.05) ontwikkel, wat bekragtig dat die diermodel ‘n geldige verteenwoordiging van T2DM is. Die anti-oksidantaanvulling het nie die metaboliese parameters geaffekteer nie wat aandui dat die verskille tussen die aanvulling- en plasebo-behandelde muise nie weens gewigsverlies of veranderinge in hul glukosemetabolisme is nie. Die NAC/AAP-aanvulling het lipiedperoksidasie in ob/ob-diere (plasebo: 39.0±14.7 nmol/L; behandeling: 21.5±11.6 nmol/L) aansienlik (p<0.05) verlaag en is vergelykbaar met dié van WT-diere (plasebo: 22.9±11.4 nmol/L; behandeling: 30.5±3.3 nmol/L). Dit het die totale TAC (WT: plasebo: 3.7±1.3 U/mL; behandeling: 4.0±0.7 U/mL; ob/ob: plasebo: 5.2±0.9 U/mL; behandeling: 5.8±1.5 U/mL) verhoog. Die ex vivo groeitempo van die ob/ob-dierselle was vertraag (tyd tot samevloeiing: WT 8 dae; ob/ob 12 dae). Die NAC/AAP-aanvulling het egter die groeivermoë en lewensvatbaarheid van MSS van beide die ob/ob- en WT-diermodel verhoog. Bowendien het die NAC/AAP-aanvulling die adipogenese-differensiasiekapasiteit van MSS verlaag. Ongelukkig kon dit nie die osteogenese-differensiasie in ob/ob-MSS herstel nie. Daar was ‘n effense toename in die vrystelling van IL6 (ob/ob-MSS: 1583.3±481.9 pg/mL) en IL10 (ob/ob-MSS: 27.1±9.8 pg/mL) in MSS van NAC/AAP-aangevulde diere na wondvog-stimulasie. Die gevolgtrekking is dat die NAC/AAP-aanvulling in staat is om oksidatiewe-spanning in diere te verminder, asook MSS se lewensvatbaarheid te verbeter.

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