Mitigation effect of an FGF-2 peptide on acute gastrointestinal syndrome after high-dose ionizing radiation

Abstract

Purpose: Acute gastrointestinal syndrome (AGS) resulting from ionizing radiation causes death within 7 days. Currently, no satisfactory agent exists for mitigation of AGS. A peptide derived from the receptor binding domain of fibroblast growth factor 2 (FGF-P) was synthesized and its mitigation effect on AGS was examined.

Methods and materials: A subtotal body irradiation (sub-TBI) model was created to induce gastrointestinal (GI) death while avoiding bone marrow death. After 10.5 to 16 Gy sub-TBI, mice received an intramuscular injection of FGF-P (10 mg/kg/day) or saline (0.2 ml/day) for 5 days; survival (frequency and duration) was measured. Crypt cells and their proliferation were assessed by hematoxylin, eosin, and BrdU staining. In addition, GI hemoccult score, stool formation, and plasma levels of endotoxin, insulin, amylase, interleukin (IL)-6, keratinocyte-derived chemokine (KC) monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor (TNF)-alpha were evaluated.

Results: Treatment with FGF-P rescued a significant fraction of four strains of mice (33-50%) exposed to a lethal dose of sub-TBI. Use of FGF-P improved crypt survival and repopulation and partially preserved or restored GI function. Furthermore, whereas sub-TBI increased plasma endotoxin levels and several pro-inflammation cytokines (IL-6, KC, MCP-1, and TNF-alpha), FGF-P reduced these adverse responses.

Conclusions: The study data support pursuing FGF-P as a mitigator for AGS.

Figure 1. FGF-P reduces death from AGS

Mice were restrained in a customized jig with right hind-legs extending outside the radiation field while they received sub-TBI (10.5-16 Gy). Ten minutes to four hours post-irradiation, the first of 5 daily i.m. injections of FGF-P was administered (10 mg/kg in 0.2 ml saline). Survival is shown as a function of time after irradiation. A-C) BALB/c mice received 10.5-12 Gy sub-TBI and i.m. injection of FGF-P 10 min post-IR. The survival of rate of the FGF-P-treated group was higher than that of controls (P<0.05). D-F) C57BL/6, C3H/NeN, and NIH Swiss mice received 13.5-16 Gy sub-TBI without/with FGF-P four hours post-IR; the survival rate was 33% to 50% (P<0.05).

Figure 2. FGF-P increases the numbers of proliferating crypts

Mice that had been irradiated (sub-TBI 10.5 Gy, A-F; or 12 Gy, G; or 16 Gy, H) and treated without/with FGF-P (10 mg/kg) daily for 3 days were i.m. injected with 120 mg/kg BrdU and sacrificed 12 hours later. Duodenal, jejunal, and ileal segments (3-4 pieces/segment) in paraffin sections were processed for H&E and BrdU staining. A-C) H&E staining: FGF-P increased the number of chromophilic crypts compared with vehicle controls. D-F) BrdU staining: BrdU incorporated proliferating crypts were greater in the FGF-P groups than in controls (10 ± 5 vs. 34 ± 19, P<0.05). G and H) FGF-P improved crypt count and BrdU staining for all strains and radiation doses tested (*P <0.05).

Figure 3. FGF-P improves stool formation, stool hemoccult, and body mass measures

A and B) On day 3.5 post-IR, BALB/c mice were sacrificed, and the colon was harvested (10 cm). Loose, yellow content in the lumen is indicative of poor stool formation or diarrhea, while solid, dark, granulated content is well-formed stool (indicated by arrows). C) Stool was collected on day 3.5 post-IR and blood content was examined and scored. Mean ± SD of these scores is presented (n=5). IR-induced GI bleeding was reduced by FGF-P administered 10 min post-IR, and Amifostine administered 0.5 hr prior to IR. D) BW of representative surviving BALB/c mice treated with either vehicle alone or FGF-P after 10.5 Gy sub-TBI was measured daily for 2 weeks and on day 32. The daily measurement of two mice per group is presented.

Figure 4. FGF-P reduces toxemia

On day 3.5, sub-TBI mice (A: BALB/c 10.5 Gy; B: C57BL/6 16 Gy) treated without/with FGF-P or Amifostine (indicated as Ami) were sacrificed and levels of endotoxin in plasma was measured. The level of endotoxin increased in control groups but was reduced in FGF-P- and Amifostine-treated groups (i.v. 0.5 hr pre-IR), but not with Amifostine 0.5 hr post-IR (*P<0.05).

Figure 5. FGF-P increases insulin and glucose levels in plasma

Insulin and glucose levels in plasma collected from BALB/c mice on day 3.5 after 10.5 Gy sub-TBI. Insulin and glucose were increased by FGF-P treatment (*P<0.05).

Figure 6. FGF-P reduces inflammatory molecules

Levels of MCP-1, IL6, KC, and TNFα in plasma collected from BALB/c mice on day 3.5 after 10.5 Gy sub-TBI. These IR-induced pro-inflammatory molecules were reduced by FGF-P and Amifostine (0.5 hr pre-IR but not post-IR) (*P <0.05).