Resveratrol decreases TNFα-induced ICAM-1 expression and release by Sirt-1-independent mechanism in intestinal myofibroblasts

Highlights

• Resveratrol (RE) reduces TNFα-induced up-regulation of ICAM-1 expression and release.

• The mechanism involved in this RE effect is redox-regulated and Sirt-1-independent.

• RE down-regulates ICAM-1 levels by reducing ROS and IκB-α degradation.

• NF-κB is not the only factor involved in TNFα-induced ICAM-1 up-regulation.

Abstract

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) and its soluble form are involved in the chronic inflammation. For the first time, we demonstrated that resveratrol (RE), a natural polyphenol with antioxidant and anti-inflammatory properties, reduces the increase of expression and release of ICAM-1, due to TNFα-induced oxidative stress, in a myofibroblast cell line derived from human colonic (18Co cells). RE is scavenger of radical oxygen species (ROS) and modulates signaling pathways in which Sirt-1 and NF-κB are involved. Effectively, in TNFα-stimulated 18Co cells RE decreases ROS production and increases Sirt-1 expression and activity, but it reduces TNFα-induced ICAM-1 up-regulation by a Sirt-1-independent mechanism, as demonstrated by EX527 and Sirt-1 siRNA treatments. RE inhibits TNFα-induced activation of NF-κB by reducing both ROS and the degradation of IκB-α, an endogenous inhibitor of NF-κB, with consequent decrease of NF-κB nuclear translocation. This study also shows that NF-κB is not the only factor involved in the TNFα-induced ICAM-1 up-regulation and confirms our previous evidence according to which TNFα increases ICAM-1 levels by redox- and non-redox-regulated mechanisms. RE can represent good and useful support in therapies for intestinal inflammatory diseases in which TNFα plays a crucial role in the increase of adhesion molecule expression.

Introduction

Intercellular adhesion molecule-1 (ICAM-1)¹, a glycoprotein expressed on the surface of various cell types, represents an important regulator of inflammation contributing to the leukocyte infiltration into inflammatory site by inducing adhesion and activation [1]. ICAM-1 expression is up-regulated by pro-inflammatory factors and/or oxidative stress [[2], [3], [4], [5]] in various inflammatory and immunological disorders [[6], [7], [8], [9]]. Moreover, ICAM-1 up-regulation is related to intestinal pathological conditions such as inflammatory bowel disease (IBD) [10,11]. In particular, IBD patients present an up-regulation of ICAM-1 expression on membranes of intestinal epithelial and fibroblast cells, and an enhancement of soluble ICAM-1 (sICAM-1) form, probably deriving from ICAM-1 shedding on the surface of intestinal cells, occurs in serum of Crohn’s disease (CD) patients [[11], [12], [13], [14]]. The increase of ICAM-1 and sICAM-1 can be crucial in chronic inflammation and this contributes to excessive infiltration of leukocytes to the inflammatory site [15] and stimulates cytokine production [16,17]. We have previously reported that tumor necrosis factor-alpha (TNFα), a crucial pro-inflammatory mediator involved in IBD inflammatory processes [18], up-regulates ICAM-1 expression and its release in a myofibroblast cell line derived from human colonic mucosa, CCD-18Co (18Co) cells [5]. In these cells, TNFα increases the production of radical oxygen species (ROS) that are in part responsible for TNFα-induced ICAM-1 expression and release. In fact, N-acetylcysteine (NAC), an antioxidant precursor of GSH synthesis, prevents the increase of expression and release of ICAM-1 due to TNFα-induced oxidative stress [5]. 18Co cells show many properties of intestinal subepithelial myofibroblasts (ISEMFs) that are involved in different functions, including the mucosal repair and inflammatory processes, both in healthy and ill intestine [19]. These cells play an important role in the physiopathology of IBD by secreting pro-inflammatory mediators such as cytokines, chemokines, metalloproteinases and adhesion molecules [12,[20], [21], [22]]. In particular, ISEMFs isolated from colonic mucosa of CD patients are characterized by oxidative stress that induces the up-regulation of pro-inflammatory mediators that in turn can cause an overproduction of ROS [[23], [24], [25], [26], [27]]. For this, the effect of natural phytochemicals with antioxidant properties, such as flavonoids and other polyphenols, in the modulation of intestinal inflammation has been investigated [[28], [29], [30]]. Resveratrol (RE) (trans-3,5,4’-trihydroxy-stilbene), a polyphenol present in red wine, some berries and peanuts, shows antioxidative and anti-inflammatory functions in IBD [29]. In experimental models for chronic inflammatory intestinal disease RE reduces neutrophil recruitment, TNFα production and the activity of adhesion molecules [31,32]. Studies performed in intestinal cells treated with lipopolysaccaride show that RE exerts its role by decreasing cyclooxygenase-2 expression and inhibiting nuclear factor-kappa B (NF-κB) activation [33,34]. Considering this and that a therapeutic role for RE has been suggested in IBD [29], in this study we investigated the effect of RE on TNFα-induced oxidative stress and on consequent up-regulation of ICAM-1 expression and release in 18Co cells. RE effect on NF-κB was also studied in TNFα-up-regulated expression of ICAM-1 by evaluating the involvement of sirtuin-1 (Sirt-1), a deacetylase that regulates various biological anti-inflammatory processes [35,36]. In fact, RE up-regulates activity and expression of Sirt-1 that is related also to down-regulation of NF-κB activity by deacetylating RelA/p65 subunit at Lysine 310 [37]. In fact, numerous properties of RE are due to Sirt-1-dependent mechanisms [38,39].