Elsevier

Experimental Gerontology

Volume 41, Issue 8, August 2006, Pages 800-804
Experimental Gerontology

Short Report
Long-term treatment with N-acetylcysteine, but not caloric restriction, protects mesenchymal stem cells of aged rats against tumor necrosis factor-induced death

https://doi.org/10.1016/j.exger.2006.05.003Get rights and content

Abstract

The survival of mesenchymal stem cells (MSCs) to tumor necrosis factor α (TNFα) stimulation was evaluated after a long-term antioxidant treatment, or caloric restriction, in aged rats. MSCs were isolated from bone marrow of 30-month-old rats which orally received N-acetylcysteine in the last 18 months. The necrotic cell death-induced in vitro by TNFα, determined by trypan blue exclusion, was markedly attenuated in MSCs obtained from treated vs. control aged rats (percent mean ± SEM: 10.9 ± 2.17 vs. 17.8 ± 0.53; p < 0.05). Also, the proliferation rate of MSCs from control, but not N-acetylcysteine-treated, aged rats evaluated up to 2 weeks was significantly higher than that of MSCs from younger (4-month-old) rats. No significant effect was observed relative to the parameters investigated when the aged rats were previously subjected to a hypocaloric diet for 18 months. In conclusion, a prolonged supplementation with N-acetylcysteine in rats can increase resistance to necrotic death of MSCs and may also counteract an excessive rate of MSC proliferation.

Introduction

The levels of circulating inflammatory mediators, such as TNFα and interleukin-6, are 4–6-fold increased in elderly population (Krabbe et al., 2004). Several cytokines and acute phase proteins have been invoked as potential factors inducing and/or increasing the risk of age-associated pathologies. Indeed, low-grade inflammatory activity is strongly associated with atherosclerosis, type-2 diabetes, osteoporosis, and demenzia (Grimble, 2003). Moreover, inflammation represents a key factor in the progressive loss of lean tissue (i.e., sarcopenia) and in the immune system dysfunction observed during ageing (Krabbe et al., 2004). Age-related increase in pro-inflammatory cytokine levels have been also described in rats, as well as the biological relevance of this condition with respect to ageing has been underlined (Foster et al., 1992, Hotamisligil et al., 1993, Siren et al., 1993, Belmin et al., 1995, Csiszar et al., 2003).

Although the effects of inflammatory mediators on a variety of mammalian cells have been widely investigated, there is only few knowledge about the impact of these factors on the biology of adult and aged stem cells. Somatic stem cells, which are present in several human tissues, show a number of age-related abnormalities (Van Zant and Liang, 2003, Park and Gerson, 2005). Since the raised level of oxidative stress (Finkel and Holbrook, 2000) might both result from, and contribute to, a stimulation of inflammation in elderly subjects, an open question is whether an antioxidant therapy can be protective against the damages induced by the increased circulating levels of inflammatory cytokines. Through this mechanism, antioxidant treatments could also favorably affect the biological function of aged stem cells, i.e., their proliferation potential and ability to survive in the presence of damaging conditions.

The aim of this research was to evaluate the effects of either long-term oral administration of N-acetylcysteine, a well known antioxidant agent, or hypocaloric diet, on the main biological functions of MSCs isolated from aged rats and in response to a cytotoxic treatment with TNFα.

Section snippets

Materials

TNFα was purchased from Peprothec Inc. (NJ, USA). N-acetylcysteine was from Chemicon International (Temecula, CA, USA). MG132 and all other biochemical reagents were Sigma (St. Louis, MO, USA) products.

Animal treatment

Four groups of male Wistar rats were scheduled: 4-month-old rats (Young), 30-month-old rats (Aged), 30-month-old rats fed in the last 18 months with standard diet implemented with 0.1% N-acetylcysteine (NAC), and 30-month-old rats subjected to hypocaloric alimentary regimen in the last 18 months

Results

Administration of TNFα plus MG132 to young and aged MSCs for 24 h triplicated the number of both necrotic and apoptotic dead cells (Table 1). Eighteen months of oral treatment in rats with N-acetylcysteine significantly increased resistance against TNFα/MG132-induced necrotic, but not apoptotic, cell death. No protective effect was attributable to the hypocaloric diet.

After one-week expansion, the MSCs of the Aged group proliferated more quickly than those of Young and NAC groups (Fig. 1a),

Discussion

The MSCs isolated from aged rats showed to be sensible to the cytotoxic stress induced by the treatment with TNFα and MG132. Although N-acetylcysteine was effective in protecting MSCs of aged rats against TNFα/MG132-induced necrosis, apoptosis was not affected. The mechanism responsible for the selective effect of TNFα inducing cell necrosis, but not apoptosis, has been recently described (Lin et al., 2004). In particular, inhibitors of the nuclear factor NF-κB, such as MG132, increase

Acknowledgments

We thank Prof. Antonio Contestabile, Department of Evolutionistic and Experimental Biology, University of Bologna, Italy, for providing rats. We thank also Mr. Massimo Sgarbi for his technical assistance. This research was carried out with funds from MIUR, Rome (FIRB 2001, PRIN 2005) and Compagnia di San Paolo, Turin, Italy.

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