Gastrointestinal
Ergothioneine Modulates Proinflammatory Cytokines and Heat Shock Protein 70 in Mesenteric Ischemia and Reperfusion Injury

https://doi.org/10.1016/j.jss.2007.04.020Get rights and content

Background and aim

Ergothioneine (EGT) is a natural compound that is synthesized by soil bacteria in fungal substrates and exhibits antioxidant functions in many cell models. The purpose of this study was to investigate the effect of EGT on mesenteric ischemia and reperfusion injury.

Materials and methods

Rats were supplemented with or without l-ergothioneine (10 mg/kg/d) for 15 days prior to intestinal ischemia. Animals were subjected to ischemia induced by clamping the superior mesenteric artery for 60 min followed by reperfusion. Serum tumor necrosis factor (TNF)-α and interleukin-1β (IL-1β) levels, tissue malondialdehide (MDA), myleoperoxidase (MPO), and heat shock protein (HSP) 70 levels, as well as histological findings, were evaluated after 1, 2, and 4 h of reperfusion.

Results

Serum TNF-α and IL-1β levels, and tissue MDA and MPO activities at 1, 2 and 4 h after reperfusion in the EGT group, were significantly lower than the control group (P < 0.05). Tissue HSP-70 levels of the study group were significantly greater than the control group at any time point of reperfusion. No significant differences in tissue damage including morphological changes ranging from villous denudation to focal necrosis, ulceration, hemorrhage, and architectural disintegration at 1 and 2 h after reperfusion exist between the two groups; however, after 4 h of reperfusion, the tissue damage based on histopathologic scores by Chiu was considerably lower in the study group (P < 0.05). After 4 h of reperfusion, focal epithelial lifting and occasional areas of denuded villi could be seen in the samples of the treated animals, thus preserving villous height and mucosal architecture.

Conclusion

EGT attenuates mesenteric ischemia reperfusion injury in rat intestine by increasing tissue HSP-70 and decreasing TNF-α, IL-1β, MDA, and MPO levels. EGT also improves morphological alterations, which occurred after IR injury after prolonged periods of reperfusion.

Introduction

Ischemic injury in the intestine occurs when the intestinal tissue is deprived of oxygen and other nutrients necessary to maintain cellular functions. Interruption of blood supply to the intestine may result from several situations including strangulated hernias, abdominal aortic aneurysm, cardiopulmonary bypass, and poor systemic perfusion due to shock. Whatever the cause, ischemic injury leads to damage of metabolically active tissues. Re-establishing blood flow to the ischemic tissues initiates a sequence of events that, paradoxically, injure the same tissues known as reperfusion injury [1]. During reperfusion, reintroduction of molecular oxygen into the ischemic tissue results in production of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, peroxinitrite, and hydroxyl/radicals [2]. Particularly, hydroxyl radicals typically cause biological damage by stimulating the free chain reaction known as lipid peroxidation [3, 4]. On the other hand, reports have shown that some intracellular proteins called heat shock proteins (HSPs) accumulate after a variety of stress stimuli such as heat shock, hypoxia, starvation, inflammation, and ischemia. HSPs function as molecular chaperones and appear as a defense mechanism that cells use when confronted with oxidative stress [5, 6]. In the rat intestine, tolerance to ischemic injury has been associated with the production of various inducible HSPs (e.g., HSP-70, HSP-72, HSP-73) [5, 7, 8]. Although the exact mechanism through which HSP-70 protects the intestine against ischemia/reperfusion (IR) is unknown, Stojadinovic et al. [9] suggested that a possible mechanism involves the inhibition of leukotriene B4 production and subsequent prevention of neutrophil activation and migration. Furthermore, many studies in rats have revealed that tolerance to IR injury could be conferred by creating hyperthermic preconditioning [7, 10]. However, such manipulations cannot be suitable for clinical management. Therefore, pharmacological agents that induce expression of HSPs in the ischemic gut without harmful side effects should be focused on a therapeutic approach. Ergothioneine (EGT) is a fungal metabolite with antioxidant actions. In humans, EGT is only absorbed through the consumption of plant diet, primarily by the intake of edible mushrooms, and has been shown to have some protective effects in cells [8, 11]. We also previously demonstrated that EGT pretreatment in rats could protect the liver from IR injury through the induction of HSP-70 [12]. Likewise, in this study, we investigated whether EGT pretreatment in rats could protect against intestinal IR injury and induce expression of HSP-70 in intestine.

Section snippets

Experimental Protocol

Eighty-four adult male Wistar rats weighing between 250 and 280 g were cared for under laboratory conditions at 22 ± 2°C with a 12 h day-night cycle in stainless-steel cages. Animals were allowed to acclimatize, with free access to standard pellet chow and tap water ad libitum. All procedures in this study were maintained after approval of the local ethics committees of Gazi University, School of Medicine (Ankara, Turkey) and in accordance with the National Institutes of Health Guide for the

Results

During the experiment, all animals survived. Some animal tests were conducted to investigate whether EGT pretreatment could attenuate intestinal IR injury and its effects on expression of HSP-70.

As shown in Fig. 1, compared with the control groups, EGT pretreatment significantly decreased serum TNF-α levels at 1, 2, and 4 h of reperfusion following 60 min ischemia, respectively (P < 0.001). Similarly, blood IL-1β levels were also found to be significantly lower in each group of EGT pretreatment

Discussion

After exposure to stress stimuli, specifically oxidative stress, the living cells of the organism acquire enhanced ability to tolerate subsequent stress. This stress response is known as hyperthermic preconditioning and has been associated with the production of HSPs. In response to stressful stimuli, expression of HSPs is induced to trigger stabilizing denaturated proteins within the damaged cells, thereby helping improve cell function [17]. Tsuruma et al. [5] reported that induction of HSPs

Acknowledgments

This study was presented at the 41st Congress of European Surgical Research, Rostock, Germany, 17-21 May, 2006. This work was supported by a grant from Gazi University, Science and Research Center, Turkey.

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