Antioxidants may contribute in the fight against ageing: an in vitro model

Elderly humans have altered cellular redox levels and dysregulated immune r esponses, both of which are key events underlying the progression of chroni c degenerative diseases of ageing, such as atherosclerosis and Alzeimer's d isease. Poorly maintained cellular redox levels lead to elevated activation of nuclear transcription factors such as NFkB and AP-I. These factors are co-ordinately responsible for a huge range of extracellular signalling mole cules responsible for inflammation, tissue remodelling, oncogenesis and apo ptosis, progessess that orchestrate many of the degenerative processess ass ociated with ageing. Tt is now clear that levels of endogenous anti-oxidant s such as GSH decrease with age. This study aimed to investigate the potent ial of exogenous anti-oxidants to influence inflammatory responses and the ageing process itself We investigated the potential of the dietary antioxid ant, quercetin, to reverse the age related influences of GSH depletion and oxidative stress using in vitro human umbilical vein endothelial cells (HUV EC) and human skin fibroblast (HSF) cell models. Oxidative stress-induced i nflammatory responses were investigated in a GSH depletion and a Phorbol 12 -myristate 13-acetate (PMA)-induced stress model. As measured with a sensit ive HPLC fluorescence method, GSH in HUVEC was depleted by the addition of L-buthionine-[S,R]-sulfoxiniine (BSO), a gamma -glutamylcysteine synthetase inhibitor, to the culture medium at a concentration of 0.25 mM. Time cours e studies revealed that the GSH half-life was 4.6 h in HUVEC. GSH depletion by BSO for 24 h led to a slight increase in intracellular adhesion molecul e - 1 (ICAM1) expression and prostaglandin E-2 (PGE(2)) secretion in both t ypes of cells. However, GSH depletion markedly enhanced PMA-induced ICAM an d PGE(2) production in HUVEC. Responses were progressively elevated followi ng prolonged BSO treatment. Inhibition studies showed that 1-(5-Iso-quinoli nylsulfonyl)-2-methylpiperazine (H7), a protein kinase C (PKC) inhibitor, n ot only abolished most of PMA-induced ICAM-1 expression and PGE(2), product ion, but also eliminated GSH depletion-enhanced PMA stimulation. This enhan cement was also inhibited by supplementation with quercetin. The results cl early demonstrate that GSH depletion increased the susceptibility of vascul ar endothelial cells and fibroblasts to oxidative stress associated inflamm atory stimuli. This increased in vitro susceptibility may be extrapolated t o the in vivo situation of ageing, providing a useful model to study the in fluence of micronutrients on the ageing process. In conclusion, these data suggest that dietary antioxidants could play a significant role in the redu ction of inflammatory responses. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.