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Curcumin, an antioxidant and anti-inflammatory agent, induces heme oxygenase-1 and protects endothelial cells against oxidative stress

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Abstract

Curcumin, a widely used spice and coloring agent in food, has been shown to possess potent antioxidant, antitumor promoting and anti-inflammatory properties in vitro and in vivo. The mechanism(s) of such pleiotropic action by this yellow pigment is unknown; whether induction of distinct antioxidant genes contributes to the beneficial activities mediated by curcumin remains to be investigated. In the present study we examined the effect of curcumin on endothelial heme oxygenase-1 (HO-1 or HSP32), an inducible stress protein that degrades heme to the vasoactive molecule carbon monoxide and the antioxidant biliverdin. Exposure of bovine aortic endothelial cells to curcumin (5–15 μM) resulted in both a concentration- and time-dependent increase in HO-1 mRNA, protein expression and heme oxygenase activity. Hypoxia (18 h) also caused a significant (P < 0.05) increase in heme oxygenase activity which was markedly potentiated by the presence of low concentrations of curcumin (5 μM). Interestingly, prolonged incubation (18 h) with curcumin in normoxic or hypoxic conditions resulted in enhanced cellular resistance to oxidative damage; this cytoprotective effect was considerably attenuated by tin protoporphyrin IX, an inhibitor of heme oxygenase activity. In contrast, exposure of cells to curcumin for a period of time insufficient to up-regulate HO-1 (1.5 h) did not prevent oxidant-mediated injury. These data indicate that curcumin is a potent inducer of HO-1 in vascular endothelial cells and that increased heme oxygenase activity is an important component in curcumin-mediated cytoprotection against oxidative stress.

Introduction

Curcumin is a major active component of the food flavor turmeric. It is extracted from the powdered dry rhizome of Curcuma longa Linn (Zingiberacee), a perennial herb widely cultivated in tropical regions of Asia. It has been used for centuries in indigenous medicine for the treatment of a variety of inflammatory conditions and other diseases [1]. Several studies in recent years have shown that curcumin is a potent inhibitor of tumor initiation in vivo [2], [3] and possesses antiproliferative activities against tumor cells in vitro [4], [5]. Besides its anticarcinogenic properties, relatively low concentrations of curcumin exhibit remarkable anti-inflammatory and antioxidant effects [1], [6], [7], [8]. Although the exact mechanism(s) by which curcumin promotes these effects remains to be elucidated, the antioxidant properties of this yellow pigment appear to be an essential component underlying its pleiotropic biological activities. In fact, curcumin has been reported to inhibit lipid peroxidation and to effectively scavenge superoxide anion and hydroxyl radicals [7]. In addition to its inherent ability to attenuate the reactivity of oxygen free radical species, curcumin has been shown in vivo to enhance the activities of detoxifying enzymes such as glutathione-S-transferase [9]. Whether induction of distinct antioxidant genes in mammalian tissues contributes to the variety of pharmacological actions mediated by curcumin has yet to be examined.

Among the various genes encoding for proteins that possess antioxidant characteristics, heme oxygenase-1 (HO-1) has attracted particular interest as it is finely up-regulated by stress conditions and generates products that might have important biological activities [10]. Heme oxygenase is a widely distributed enzyme in mammalian tissues and its main function is associated with the degradation of heme to iron, carbon monoxide (CO), and biliverdin [11], the latter being converted to bilirubin by the cytosolic enzyme biliverdin reductase. Both biliverdin and bilirubin possess antioxidant properties [12] and HO-1-derived CO has been implicated in vasoregulation and signal transduction mechanisms [13], [14]. Physiological concentrations of bilirubin have been shown to protect endothelial cells against hydrogen peroxide-mediated injury and prevent protein oxidation in human blood plasma [15], [16]. We have recently reported that increased bilirubin production following HO-1 stimulation is associated with decreased peroxynitrite-mediated cytotoxicity [17] and reduction of postischemic myocardial dysfunction in an isolated rat heart preparation [18]. Otterbein and coworkers have also demonstrated that exposure of rats to low concentration of CO increases tolerance to hyperoxic lung injury [19]. Collectively, the HO-1 pathway appears to: play a key role in the preservation of tissue integrity against oxidative stress [17], [20], [21]; contribute to the modulation of inflammatory responses in vivo [22,23]; and act in synchrony with other crucial enzymatic systems in the maintenance of cellular homeostasis [21].

In the present study we report the effect of curcumin on HO-1 gene regulation, protein expression, and heme oxygenase activity in vascular endothelial cells. In addition, we have examined the interrelation between curcumin and HO-1 in protection against oxidant-mediated injury under normoxic and hypoxic conditions. The biological relevance of these findings will be discussed.

Section snippets

Materials

Tin protoporphyrin IX (SnPPIX), an inhibitor of heme oxygenase activity, was obtained from Porphyrin Products Inc. (Logan, UT, USA). Curcumin and all other chemicals were purchased from Sigma-Aldrich (Poole, UK) unless otherwise specified. Stock solutions of curcumin (5 mM) were prepared freshly on the day of the experiment by dissolving the compound in ethanol. A cDNA probe for rat HO-1 (kindly provided by Prof. Shibahara, Japan) was used to detect HO-1 mRNA expression [24].

Experimental protocol

Bovine aortic

Effect of curcumin on heme oxygenase activity and HO-1 protein expression in vascular endothelial cells

The effect of various concentrations of curcumin (0–30 μM) on endothelial heme oxygenase activity and HO-1 protein expression is shown in Fig. 1. Exposure of endothelial cells to curcumin (1–15 μM) for 18 h resulted in a concentration-dependent increase in heme oxygenase activity (Fig. 1A). The increase was significantly different from control (untreated cells, P < 0.05), with a maximal enzymatic activity (12-fold increase) at 15 μM curcumin. The antioxidant N-acetylcysteine did not

Discussion

In the present study we report that low concentrations of curcumin, the active component of the food flavor turmeric, up-regulate endothelial HO-1 gene, protein expression and heme oxygenase activity in a concentration- and time-dependent manner. Interestingly, this effect was greatly potentiated when endothelial cells were exposed to curcumin under conditions of reduced oxygen tension (hypoxia). Prolonged treatment with curcumin resulted in high resistance to oxidant-mediated cell injury and

Acknowledgements

This work was supported by grants from the National Heart Research Fund, Leeds (R.M.), British Heart Foundation (PG99005, R.M.) and National Kidney Research Fund (R30/1/99, R.M.).

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