Orexigenic hormone ghrelin attenuates local and remote organ injury after intestinal ischemia-reperfusion

Abstract

Background: Gut ischemia/reperfusion (I/R) injury is a serious condition in intensive care patients. Activation of immune cells adjacent to the huge endothelial cell surface area of the intestinal microvasculature produces initially local and then systemic inflammatory responses. Stimulation of the vagus nerve can rapidly attenuate systemic inflammatory responses through inhibiting the activation of macrophages and endothelial cells. Ghrelin, a novel orexigenic hormone, is produced predominately in the gastrointestinal system. Ghrelin receptors are expressed at a high density in the dorsal vagal complex of the brain stem. In this study, we investigated the regulation of the cholinergic anti-inflammatory pathway by the novel gastrointestinal hormone, ghrelin, after gut I/R.

Methods and findings: Gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery for 90 min in male adult rats. Our results showed that ghrelin levels were significantly reduced after gut I/R and that ghrelin administration inhibited pro-inflammatory cytokine release, reduced neutrophil infiltration, ameliorated intestinal barrier dysfunction, attenuated organ injury, and improved survival after gut I/R. Administration of a specific ghrelin receptor antagonist worsened gut I/R-induced organ injury and mortality. To determine whether ghrelin's beneficial effects after gut I/R require the intact vagus nerve, vagotomy was performed in sham and gut I/R animals immediately prior to the induction of gut ischemia. Our result showed that vagotomy completely eliminated ghrelin's beneficial effect after gut I/R. To further confirm that ghrelin's beneficial effects after gut I/R are mediated through the central nervous system, intracerebroventricular administration of ghrelin was performed at the beginning of reperfusion after 90-min gut ischemia. Our result showed that intracerebroventricular injection of ghrelin also protected the rats from gut I/R injury.

Conclusions: These findings suggest that ghrelin attenuates excessive inflammation and reduces organ injury after gut I/R through activation of the cholinergic anti-inflammatory pathway.

Figure 1. Alterations in plasma levels of ghrelin at the end of 90-min gut ischemia (Ischemia), 2 h reperfusion after ischemia (Ischemia/reperfusion), or sham operation (Sham).

Data are presented as means±SE (n = 5–6) and compared by one-way ANOVA and Student-Newman-Keuls test: * P<0.05 versus Sham group.

Figure 2. Alterations in serum levels of TNF-α (A) and IL-6 (B) in sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or ghrelin (Ghrelin) at 1.5 h after the completion of treatment (i.e., 2 h after reperfusion).

Data are presented as means±SE (n = 6/group) and compared by one-way analysis of variance (ANOVA) and Student-Newman-Keuls test: *P<0.05 versus Sham group; #P<0.05 versus I/R+Vehicle group.

Figure 3. Alterations in intestinal (A) and pulmonary (B) MPO activities in sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or ghrelin (Ghrelin) at 1.5 h after the completion of treatment (i.e., 2 h after reperfusion).

Data are presented as means±SE (n = 5–6) and compared by one-way ANOVA and Student-Newman-Keuls test: * P<0.05 versus Sham group; # P<0.05 versus Vehicle group.

Figure 4. Alterations in intestinal mucosal permeability (A) to fluorescein isothiocyanate dextran with a molecular weight of 4000 Da (FD4) and bacterial translocation to mesenteric lymph nodes (B) in sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or ghrelin (Ghrelin) at 1.5 h after the completion of treatment (i.e., 2 h after reperfusion).

Data are presented as means±SE (n = 6), and compared by one-way ANOVA and Student-Newman-Keuls test: * P<0.05 versus Sham group; # P<0.05 versus Vehicle group.

Figure 5. Alterations in serum levels of lactate (A), gut (B) and lung (C) water content in sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or ghrelin (Ghrelin) at 1.5 h after the completion of treatment (i.e., 2 h after reperfusion).

Data are presented as means±SE (n = 5–6) and compared by one-way ANOVA and Student-Newman-Keuls test: * P<0.05 versus Sham group; # P<0.05 versus Vehicle group.

Figure 6. Alterations in morphologic photomicrography of the small intestine and lungs.

A, a photomicrograph of a small intestinal section from a sham-operated rat; B, a photomicrograph of a small intestinal section from an I/R rat at 2 h after gut ischemia-reperfusion treated with vehicle; C, a photomicrograph of a small intestinal section from an I/R rat at 2 h after gut ischemia-reperfusion treated with ghrelin; D, a photomicrography of a pulmonary section from a sham-operated rat. E, a photomicrography of lung section from an I/R rat at 2 h after gut ischemia-reperfusion treated with vehicle; F, a photomicrography of lung section from an I/R rat at 2 h after gut ischemia-reperfusion treated with ghrelin. Original magnification, ×200.

Figure 7. Alterations in the survival rate at 10 days after gut ischemia/reperfusion with normal saline treatment (Vehicle) and gut ischemia/reperfusion with ghrelin treatment (Ghrelin). Ghrelin (12 nmol/kg BW), or vehicle (1-ml normal saline) was administered intravenously over a period of 30 min to animals that underwent 90 min gut ischemia immediately after removing the microvascular clip.

The animals were then allowed food and water ad libitum and were monitored for 10 days to record survival. There were 12 animals in each group. The survival rate was estimated by the Kaplan-Meier method and compared by using the log-rank test. * P<0.05 vs. Vehicle.

Figure 8. Alterations in intestinal (A) and pulmonary (B) MPO activities, and serum levels of lactate (C) in sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or [D-Arg¹ D-Phe⁵ D-Trp^{7, 9} Leu¹¹]-substance P (GA antagonist) at 1.5 h after the completion of treatment (i.e., 2 h after reperfusion).

Data are presented as means±SE (n = 5–6) and compared by one-way ANOVA and Student-Newman-Keuls test: * P<0.05 versus Sham group; # P<0.05 versus Vehicle group. Alterations in the survival rate (D) at 10 days after gut ischemia/reperfusion with normal saline treatment (Vehicle) and gut ischemia/reperfusion with [D-Arg¹ D-Phe⁵ D-Trp^{7, 9} Leu¹¹]-substance P (GA antagonist). There were 12 animals in each group. Survival rate in the vehicle treated animals is derived from Figure 7. The survival rate was estimated by the Kaplan-Meier method and compared by using the log-rank test. * P<0.05 vs. Vehicle.

Figure 9. Effects of vagotyomy on serum levels of TNF-α (A) and lactate (B), intestinal MPO activities (C), and intestinal mucosal permeability (D) to fluorescein isothiocyanate dextran with a molecular weight of 4000 Da (FD4) in sham-operated animals (Sham) and ischemia/reperfusion animals treated with normal saline (Vehicle) or ghrelin (Ghrelin) at 1.5 h after the completion of treatment (i.e., 2 h after reperfusion).

Data are expressed as means±SE (n = 6–7/group) and compared by one-way analysis of variance (ANOVA) and Student-Newman-Keuls test: *P<0.05 versus sham-operated animals.

Figure 10. Alterations in serum levels of TNF-α (A) and lactate (B), intestinal MPO activities (C), and intestinal mucosal permeability (D) to fluorescein isothiocyanate dextran with a molecular weight of 4000 Da (FD4) in sham-operated animals (Sham) and ischemia/reperfusion animals treated with intracerebroventricular (ICV) injection of normal saline (Vehicle) or ghrelin (Ghrelin) at 1.5 h after the completion of treatment (i.e., 2 h after reperfusion).

Data are presented as means±SE (n = 6) and compared by one-way ANOVA and Student-Newman-Keuls test: * P<0.05 versus Sham group; # P<0.05 versus Vehicle group.