The Anti-apoptotic Effect of Ghrelin on Restraint Stress-Induced Thymus Atrophy in Mice

Abstract

Thymic atrophy is a complication that results from exposure to many environmental stressors, disease treatments, and microbial challenges. Such acute stress-associated thymic loss can have a dramatic impact on the host's ability to replenish the necessary naïve T cell output to reconstitute the peripheral T cell numbers and repertoire to respond to new antigenic challenges. We have previously reported that treatment with the orexigenic hormone ghrelin results in an increase in the number and proliferation of thymocytes after dexamethasone challenge, suggesting a role for ghrelin in restraint stress-induced thymic involution and cell apoptosis and its potential use as a thymostimulatory agent. In an effort to understand how ghrelin suppresses thymic T cell apoptosis, we have examined the various signaling pathways induced by receptor-specific ghrelin stimulation using a restraint stress mouse model. In this model, stress-induced apoptosis in thymocytes was effectively blocked by ghrelin. Western blot analysis demonstrated that ghrelin prevents the cleavage of pro-apoptotic proteins such as Bim, Caspase-3, and PARP. In addition, ghrelin stimulation activates the Akt and Mitogen-activated protein kinases (MAPK) signaling pathways in a time/dose-dependent manner. Moreover, we also revealed the involvement of the FoxO3a pathway in the phosphorylation of Akt and ERK1/2. Together, these findings suggest that ghrelin inhibits apoptosis by modulating the stress-induced apoptotic signal pathway in the restraint-induced thymic apoptosis.

Keywords: Apoptosis; Ghrelin; Restraint stress; Thymic atrophy.

Figure 1. Experimental design and the result of behavioral assessments. (A) A diagrammatic representation of the experimental schedule. Mice were treated with ghrelin and mifepristone from D-1 to D+6 30 min before restraint stress. Mice were immobilized for 1.5 hr daily over a period of 7 days. (B, C) The weight of thymus (mg) and thymus cell numbers were measured at D+7 of restraint stress. (D, E) Percentage of thymic lymphocyte populations in the thymus after restraint stress. Flow cytometry plot (C) showing the gating strategy for thymocyte subsets. DN, double-negative; DP, double-positive; CD4SP, single-positive CD4; and CD8SP, single-positive CD8 thymocytes, respectively. Each bar represents the mean±SEM from 10 animals per group. ^*p<0.05, ^**p<0.01 for control (–STR) versus stressed (+STR) groups; ^#p<0.05, ^##p<0.01 for stressed (+STR) groups.

Figure 2. The thymic protective effects of ghrelin upon restraint stress. (A, B) Flow cytometry scatter plots for thymic cells from one control (left) and one stressed (right) mouse. (A) Total cells, (B) CD4/CD8-double positive cells.

Figure 3. Effects of ghrelin treatment on the levels of apoptosis-related 14-3-3-sequestered proteins in restraint stress-induced thymic apoptosis. (A, B) Cell lysates were obtained from control (–STR) versus stressed (+STR) thymus tissues, and were subjected to western blot analysis with indicated antibodies. Actin was used as a loading control.

Figure 4. Effect of ghrelin on the phosphorylation and activity of Akt and Akt-mediated downstream molecules in thymocytes. Cells were stimulated with 100 nM ghrelin for the indicated times. Lysates were assayed by immunoblotting with the indicated antibodies. β-actin was used as the loading control. (A) Tyrosine phosphorylation of Akt, Erk1/2, and p38 upon ghrelin stimulation. (B) Inhibitory effects of Akt inhibitor IV. Cells were pretreated with Akt inhibitor IV for 30 min as indicated, followed by stimulation with 100 nM ghrelin for 15 min. (C) Effects of ghrelin on the phosphorylation of Akt and FoxO3a. (D) Effects of Ghrelin receptor inhibitor ([D-Lys-3]-GHRP-6) on ghrelin-mediated Akt and MAPK activation. Cells were stimulated with 20 µM [D-Lys-3]-GHRP-6 and/or 100 nM ghrelin for 1 hr.

Figure 5. Effect of ghrelin on the binding of FoxO3a to 14-3-3 upon restraint stress-induced thymic apoptosis. (A, B) Cell lysates were obtained from control (–STR) versus stressed (+STR) thymus tissues, then immunoprecipitated with FoxO3a (A) or 14-3-3 (B). The immune complexes were subjected to western blot analysis using the indicated antibodies.