Radioprotector WR-2721 and mitigating peptidoglycan synergistically promote mouse survival through the amelioration of intestinal and bone marrow damage

Abstract

The identification of an agent effective for the treatment of intestinal and bone marrow injury following radiation exposure remains a major issue in radiological medicine. In this study, we evaluated the therapeutic impact of single agent or combination treatments with 2-(3-aminopropylamino) ethylsulphanyl phosphonic acid (WR-2721) and peptidoglycan (PGN, a toll-like receptor 2 (TLR-2) agonist) on radiation-induced injury of the intestine and bone marrow in lethally irradiated male C57BL/6 mice. A dose of 3 mg of WR-2721 per mouse (167 mg/kg, intraperitoneally) was given 30 min before irradiation, and 30 μg of PGN per mouse (1.7 mg/kg) was injected intraperitoneally 24 h after 10 Gy irradiation. Bone marrow cluster of differentiation (CD)45(+) and CD34(+) markers of multiple haematopoietic lineages, number of granulocyte-erythroid-macrophage-megakaryocyte (GEMM) progenitor colonies, bone marrow histopathology, leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) expression in the intestines, xylose absorption and intestinal histopathology were all assessed at various time-points after irradiation. Furthermore, nuclear factor kappa B (NF-κB) p65 protein in the ileum was stained by immunofluorescent labelling. PGN-treated irradiated mice showed an increase in CD45(+)CD34(+) cells compared with untreated mice 1.25 days after 10 Gy ionizing radiation (IR) (P < 0.05). Furthermore, combined PGN and WR-2721 treatment had an obviously synergistic radio-protective effect in nucleated cells in the bone marrow, including GEMM progenitors and CD45(+)CD34(+) cells 4 days after 10 Gy IR. Single agent PGN or WR-2721 treatment after 10 Gy IR clearly increased Lgr5-positive pit cells (P < 0.05) and xylose absorption (P < 0.05). However only PGN and WR-2721 combination treatment markedly increased villus height (P < 0.05), number of crypts (P < 0.05) and whole-body weights after 10 Gy whole-body irradiation (WBI). The NF-κB p65 subunit was translocated to the nucleus, and phosphate-IκBα (Ser32/Ser36) was detected after stimulation with either PGN or WR-2721, which indicates that these two agents act synergistically through the activation of the NF-κB pathway. Administration of PGN in combination with WR-2721 was demonstrated to have a synergistic effect on the increase in haematopoietic cells and intestinal reconstitution, as well as improved survival in lethally irradiated mice, but resulted in some degree of an immune disorder.

Keywords: WR-2721; bone marrow; intestine; peptidoglycan; radioprotection.

Fig. 1.

WR-2721 and PGN in combination sustained body weight and improved survival after 10 Gy WBI. (A) All mice except irradiated C57BL/6 mice treated with WR-2721 and PGN in combination lost weight quickly prior to death. (B) Survival of C57BL/6 mice (n = 40) exposed to 10 Gy radiation. The mice were divided into groups of 10, and one group received WR-2721 before IR (3 mg per mouse), another 10 received PGN 24 h post-IR (30 μg per mouse), and the remaining 10 received combination treatment. Survival among the groups was ranked in the following order: W + P group > W group > P group > IR group. Half of the irradiated C57BL/6 mice treated with WR-2721 and PGN survived for more than 30 days. Error bars indicate the SD of the mean for n = 10 mice.

Fig. 2.

WR-2721 + PGN combined treatment decreased bone marrow damage and increased numbers of CFU-GEMM and CD45⁺CD34⁺ cells. (A) Representative histological sections of the femur from mice in each of the 5 treatment groups at 1.25 and 4 days after 10 Gy WBI. N = unirradiated mice, IR = 10 Gy WBI mice, P = 10 Gy WBI mice treated with PGN, W = WR-2721-treated 10 Gy WBI mice, P + W = PGN and WR-2721 combination treated 10 Gy WBI mice (original magnification ×200). After 10 Gy irradiation, C57BL/6 mice could be seen to have lost most white cells in their bone marrow, and cavities in the bone marrow appeared. After single agent treatment with PGN or WR-2721, the numbers of nucleated cells increased, but haemorrhages appeared in the W group at 1.25 days and cavities still remained. On Day 4 post-IR, marrow microvessels appeared in femora of the P + W group and haematopoiesis commenced. (B) Number of CD45⁺CD34⁺ cells per 10 000 total cells from femoral marrow detected after red blood cells were removed using Zapoglobin, as analysed by fluorescence-activated cell sorting (FACS). On Day 1.25, only PGN-treated 10 Gy irradiated mice showed a marked increase in the proportion of CD34⁺CD45⁺ cells. On Day 4, the number of CD45⁺CD34⁺ cells was higher in the WR-2721 + PGN group than in other 10 Gy irradiated mice. (C) Only WR-2721 + PGN combined treatment induced growth of mouse CFU-GEMM haematopoietic progenitors following 10 Gy irradiation, and that growth was considerably subnormal on Days 4, 9, 16 and 25 days post IR. However, on Day 40, no marked difference between the W + P group and the N group was observed. Error bars indicate the SD for n = 10 mice (*P < 0.05).

Fig. 3.

Treatment with PGN and WR-2721 in combination normalized ileum villus height and number of crypts after 10 Gy WBI. N = unirradiated mice, IR = 10 Gy WBI mice, P = 10 Gy WBI mice treated with PGN, W = WR-2721-treated 10 Gy WBI mice, P + W = PGN and WR-2721 combination-treated 10 Gy WBI mice (original magnification ×200). After 10 Gy WBI the small intestine became vacuolated, villus height was clearly shortened, and half or fewer of the crypts remained. On Day 1.25 post IR, villus height and number of crypts in the P and W groups group had not changed obviously compared with the IR group, and marked differences could be observed in comparison with the N group. However, on Day 4 post-IR, the P + W group had longer villi and more crypts compared with the IR group (*P < 0.05), while the P and W groups were unchanged. Error bars indicate the SD for n = 10 mice.

Fig. 4.

WR-2721 and PGN promoted recovery of intestinal absorption in irradiated mice. Xylose uptake assay of 10 mice per group was performed 1 day before mice were euthanized. Samples of urine were collected, and xylose levels were measured using a modified micro-method and normalized to data from unirradiated mice at the same time-points. On Day 3 after 10 Gy IR, the absorption rate of mice treated with WR-2721 + PGN demonstrated a clear difference from that of mice exposed to 10 Gy irradiation (P < 0.01). On Day 7, the absorption rate of mice treated with WR-2721 or PGN alone, or with WR-2721 + PGN, was obviously higher than that of the 10 Gy irradiated mice. Error bars indicate the SD for n = 10 mice (*P < 0.05).

Fig. 5.

Both single treatment with WR-2721 or PGN and combination treatment augmented intestinal Lgr5 expression and activated NF-κB following irradiation. (A) The percentage of Lgr5⁺ cells in the ilea of mice treated with WR-2721 and PGN was higher than that in the ilea of mice irradiated with 10 Gy, but lower than the percentage in the ilea of normal mice (*P < 0.05). (B) The NF-κB p65 subunit is located in the cytoplasm 1.25 days post 10 Gy irradiation. Both WR-2721 and PGN treatment may activate NF-κB and induce it to move into the nuclei of such cells as enterocytes and smooth muscle cells. (C) Western blot data of phospho-IκBα in the ileum 1.25 days post 10 Gy irradiation.

Fig. 6.

GM-CSF, IL-2, IL-5, IL-6 and IL-17 showed significant changes over the course of the experiments. WR-2721 reversed the radiation-induced changes in IL-2 and IL-6. In the PGN group, IL-5 and IL-6 levels were similar to those in the IR group. The combination of WR-2721 and PGN was unable to reverse the increase in IL-6 observed following IR (A). IL-6, IL-17 and GM-CSF in the serum of mice treated with WR-2721 and PGN were all significantly higher than in the unirradiated mice on Day 9 (B) (*P < 0.05).