Medical Physiology

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Browsing Medical Physiology by Subject "Advanced glycation end products"

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    The therapeutic efficacy of ascorbic acid 2 phosphate, n-acetylcysteine and metformin against diabetes mellitus associated cellular senescence.
    (Stellenbosch : Stellenbosch University, 2023-03) Govender, Saiuree; Van de Vyver, Mari; Johnson, Rabia; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    ENGLISH 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.