Action of reactive oxygen species on colonic mucus secretions

Abstract

Reactive oxygen species (ROS) have been implicated in the pathogenesis of many colonic diseases. Mucus is the colon's first line of defence against luminal agents. This study has therefore characterised ROS action on colonic mucus secretions. ROS were produced using peroxide-based systems of different concentrations. The effects of these systems were tested on native colonic mucus gels, isolated colonic mucins, and in vivo models. Colonic mucus gels were resistant to ROS breakdown. Mucins were susceptible to ROS attack, causing loss of terminal sugars and protein and mucin fragmentation. The in vivo thickness of the mucus barrier was reduced by up to 50% by ROS (above 5 mM peroxide). A 5 mM peroxide caused a significant increase in resting mucus thickness of ca. 15%. All ROS-generating systems caused mucosal damage once the loosely adherent mucus had been removed. As native mucus gel is more resistant to ROS damage than purified mucin, nonmucin components of mucus may have extensive ROS-scavenging properties. Low levels of luminal colonic ROS increase the protection afforded by the mucus barrier in vivo. Higher levels of ROS significantly reduce this protection. In vitro modeling of mucus degradation by ROS does not necessarily correlate with the dynamic, in vivo situation.

Introduction

A continuous mucus layer lines the mucosal surface of the lower bowel, protecting the mucosa, while at the same time lubricating the passage of luminal contents, thus lowering shear stress-induced damage [1]. These actions help to maintain mucosal integrity. The barrier-like functions of the mucus layer are elicited by its functional component, mucin, which makes up about 4% of the weight of the mucus gel [2]. In effect, the colonic mucus barrier acts as the colon's first line of defence against the wide range of potentially damaging agents that occur within the lumen.

One type of damaging agent that has been hypothesised to occur within the colonic lumen is reactive oxygen species (ROS) [3]. Increased ROS activity has been linked to a number of colonic disorders, most notably ulcerative colitis (UC) [4], [5]. Increased levels of oxidant stress and decreased levels of antioxidant defence are witnessed in the colonic tissue of active UC patients [5], [6], [7], [8], [9], [10].

In active UC, the thickness and continuity of the colonic mucus barrier have been shown to be reduced [11], [12], [13]. Mice deficient of Muc-2, the gene that encodes for the major intestinal secreted mucins, have been reported to spontaneously develop colitis after 5 weeks [14], demonstrating the importance of the colonic mucus barrier in preventing ulcerative colitis. A number of in vitro studies have shown that the gastric mucins of various species are degraded by the presence of ROS [15], [16], [17], [18], [19]. The colonic mucus gel may behave differently to gastric mucus as it contains mucins with a higher negative charge and larger quantities of bacterial cells, both of which could have a role in ROS quenching [20]. Colonic mucus may be subjected to elevated ROS produced within the intestinal lumen, either as a result of dietary components (e.g., high iron [21] or low fibre/high fat [3]), release of endogenous, ROS-producing enzymes from sloughed cells, such as xanthine and aldehyde oxidases [22], [23], or via infiltration of neutrophils and macrophages toward the colonic epithelium [24].

Increased levels of metabolites of anaerobic colonic bacteria [25] may also bring about raised ROS levels. Further from this, higher counts of aerobic bacteria have also been suggested to occur in the mucosa-associated microflora of pediatric patients with inflammatory bowel disease [26] and feces of ulcerative colitis patients [27]. The colonic mucus layer could protect from luminal ROS by increasing the diffusion distance from the lumen to the mucosa, as well as by acting to directly quench ROS before they cause damage to the underlying mucosa.

Whether the increase in oxidative stress to the colon and the thinning of the colonic mucus barrier seen in UC are the cause, or an effect, of the disease process remains unclear. For luminal ROS to cause damage to the colonic mucosa, we hypothesise that it must first reduce the protective potential of colonic mucus. To test whether this occurs, this paper aimed to determine the effects of ROS on polymeric colonic mucins, the native gel layer in vitro, and also in an in vivo colonic mucus barrier, thereby aiming to demonstrate the molecular and macroscopic effects that increased colonic ROS could have on mucus barrier integrity in vivo.