Ghrelin, a novel therapy, corrects cytokine and NF-κB-AKT-MAPK network and mitigates intestinal injury induced by combined radiation and skin-wound trauma

Abstract

Background: Compared to radiation injury alone (RI), radiation injury combined wound (CI) further enhances acute radiation syndrome and subsequently mortality. We previously reported that therapy with Ghrelin, the 28-amino-acid-peptide secreted from the stomach, significantly increased 30-day survival and mitigated hematopoietic death by enhancing and sustaining granulocyte-colony stimulating factor (G-CSF) and keratinocyte chemoattractant (KC) in the blood and bone marrow; increasing circulating white blood cell depletion; inhibiting splenocytopenia; and accelerating skin-wound healing on day 30 after CI. Herein, we aimed to study the efficacy of Ghrelin on intestinal injury at early time points after CI.

Methods: B6D2F1/J female mice were exposed to ⁶⁰Co-γ-photon radiation (9.5 Gy, 0.4 Gy/min, bilateral), followed by 15% total-body-surface-area skin wounds. Several endpoints were measured: at 4-5 h and on days 1, 3, 7, and 15.

Results: Ghrelin therapy mitigated CI-induced increases in IL-1β, IL-6, IL-17A, IL-18, KC, and TNF-α in serum but sustained G-CSF, KC and MIP-1α increases in ileum. Histological analysis of ileum on day 15 showed that Ghrelin treatment mitigated ileum injury by increasing villus height, crypt depth and counts, as well as decreasing villus width and mucosal injury score. Ghrelin therapy increased AKT activation and ERK activation; suppressed JNK activation and caspase-3 activation in ileum; and reduced NF-κB, iNOS, BAX and Bcl-2 in ileum. This therapy recovered the tight junction protein and mitigated bacterial translocation and lipopolysaccharides levels. The results suggest that the capacity of Ghrelin therapy to reduce CI-induced ileum injury is mediated by a balanced NF-κB-AKT-MAPK network that leads to homeostasis of pro-inflammatory and anti-inflammatory cytokines.

Conclusions: Our novel results are the first to suggest that Ghrelin therapy effectively decreases intestinal injury after CI.

Keywords: AKT; Apoptosis; BAX; Bacteria; Bcl-2; Caspase; ERK; GI; Ghrelin; Ionizing radiation; JNK; MAPK; NF-κB; Skin wound; Tight junction; iNOS.

Fig. 1

Effect of Ghrelin therapy on IL-1β, IL-6, IL-12p70 and IL-17A after RI and CI. Animals were irradiated alone or followed by wounding. Blood samples at different time points were collected for measuring IL-1β, IL-6, IL-12p70 and IL-17A concentrations in serum (N = 6 per group). The data presented are mean ± sem. *p < 0.05 vs. Sham vehicle group; ^p < 0.05 vs. respective vehicle group; #p < 0.05 vs. RI + Veh group. Veh or V: vehicle; Ghr or GHR: Ghrelin; W wounding; RI: 9.5 Gy; CI: 9.5 Gy + wounding

Fig. 2

Effect of Ghrelin therapy on IL-15, IL-18, MIP-2 and Eotaxin after RI and CI. Animals were irradiated alone or followed by wounding. Blood samples at different time points were collected for measuring IL-15, IL-18, MIP-2 and Eotaxin A concentrations in serum (N = 6 per group). The data presented are mean ± sem. *p < 0.05 vs. Sham vehicle group; ^p < 0.05 vs. respective vehicle group; #p < 0.05 vs. RI + V group. V vehicle, GHR Ghrelin, W wounding; RI: 9.5 Gy; CI: 9.5 Gy + wounding

Fig. 3

Effect of Ghrelin therapy on G-CSF, GM-CSF, KC and TNF-α after RI and CI. Animals were irradiated alone or followed by wounding. Blood samples at different time points were collected for measuring G-CSF, GM-CSF, KC and TNF-α concentrations in serum (N = 6 per group). The data presented are mean ± sem. *p < 0.05 vs. Sham vehicle group; ^p < 0.05 vs. respective vehicle group; #p < 0.05 vs. RI + V group. V vehicle, GHR Ghrelin, W wounding; RI: 9.5 Gy; CI: 9.5 Gy + wounding

Fig. 4

Effect of Ghrelin therapy on IL-18, G-CSF, KC and MIP-1α after RI and CI in ileum. Animals were irradiated alone or followed by wounding. Ileum samples at different time points were collected for measuring IL-18, G-CSF, KC and MIP-1α concentrations in ileum (N = 6 per group). The data presented are mean ± sem. *p < 0.05 vs. Sham vehicle group; ^p < 0.05 vs. respective vehicle group; #p < 0.05 vs. RI + V group. V vehicle, GHR Ghrelin, W wounding; RI: 9.5 Gy; CI: 9.5 Gy + wounding

Fig. 5

Ghrelin therapy mitigates ileum injury. Animals were irradiated alone or followed by wounding. Ileum samples on day 15 were collected for histology with H&E staining (N = 4 per group). Villus height, villus width, crypt depth and crypt counts were measured. Mucosal injury scores were assigned according to the criteria described in the Materials and Methods section above. The data presented are mean ± sem. *p < 0.05 vs. Sham vehicle group; ^p < 0.05 vs. respective VEH group; #p < 0.05 vs. RI + VEH group. VEH vehicle, GHR Ghrelin, W wounding; RI: 9.5 Gy; CI: 9.5 Gy + wounding

Fig. 6

Ghrelin therapy decreases cell apoptosis and caspase-3 activation in ileum. Animals were irradiated alone or followed by wounding. A Slides from ileum samples from day 15 were stained for apoptosis assessment. The representative slides for each group are presented. B The apoptotic cells per crypt were counted. C-E Ileum samples at different time points were collected for measuring caspase-3 activation (N = 6 per group). The data presented are mean ± sem. *p < 0.05 vs. Sham VEH group; ^p < 0.05 vs. respective VEH group. VEH vehicle, GHR Ghrelin, W wounding; RI: 9.5 Gy; CI: 9.5 Gy + wounding

Fig. 7

Ghrelin therapy increases AKT activation in ileum. Animals were irradiated alone or followed by wounding. Ileum samples collected on day 3 were analyzed for AKT activation (N = 4 per group). The data presented are mean ± sem. ^p < 0.05 vs. respective VEH group. VEH vehicle, GHR Ghrelin, W wounding; RI: 9.5 Gy; CI: 9.5 Gy + wounding

Fig. 8

Ghrelin therapy increases ERK activation and decreases JNK activation in ileum. Animals were irradiated alone or followed by wounding. Ileum samples collected on day 3 were analyzed for AKT activation (N = 4 per group). The data presented are mean ± sem. ^p < 0.05 vs. respective VEH group. VEH vehicle, GHR Ghrelin, W wounding; RI: 9.5 Gy; CI: 9.5 Gy + wounding

Fig. 9

Ghrelin therapy decreases NF-κB, iNOS, BAX and Bcl-2 in ileum. Animals were irradiated alone or followed by wounding. Ileum samples collected on day 3 were for analyzed for NF-κB, iNOS, BAX and Bcl-2 (N = 4 per group). The data presented are mean ± sem. *p < 0.05 vs. sham vehicle group; **p < 0.05 vs. sham vehicle and wound vehicle groups; ***p < 0.05 vs. sham vehicle, wound vehicle and RI vehicle groups; ^p < 0.05 vs. respective VEH group. VEH vehicle, GHR Ghrelin, W wounding; RI: 9.5 Gy; CI: 9.5 Gy + wounding

Fig. 10

Ghrelin therapy increases tight junction protein in ileum and mitigates bacterial translocation and lipopolysaccharides (LPS) levels. a Animals were irradiated alone or irradiated followed by wounding. Ileum lysate samples collected on day 7 were used to detect claudin 2, a biomarker for the tight junction (N = 4 per group). b The liver lysate samples (100 mg per sample) from each animal (N = 6) were used for measuring bacterial DNA, presented as ng/g liver. c The liver lysate samples from each animal (N = 6) were used for measuring LPS levels using the LPS ELISA Kit, presented as O.D./µg protein. The data presented are mean ± sem. *p < 0.05 vs. RI + VEH. W wounding; RI: 9.5 Gy; CI: 9.5 Gy + wounding; VEH vehicle, GHR Ghrelin

Fig. 11

Possible mechanisms of Ghrelin therapy in mitigating ileum injury. RI and CI increase miR-34a and miR-696, as well as NF-κB. Increases in NF-κB expression trigger cytokines and chemokines in ileum. Increases in IL-1β/18, IL-6, and TNF-α stimulate Myd88, which reduces AKT activation and elevates MAPK activation, thereby increasing apoptosis. On the other hand, increases in NF-κB transcribe iNOS that triggers caspase-3 activation, also resulting in apoptosis. Ghrelin therapy inhibits NF-κB, reinforces RI/CI-induced increases in G-CSF, MIP-2 and KC and promotes tissue repair. This therapy also increases AKT activation which promotes cell survival