Synergistic actions of FGF2 and bone marrow transplantation mitigate radiation-induced intestinal injury

Abstract

Unwanted radiological or nuclear exposure remains a public health risk for which effective therapeutic countermeasures are lacking. Here, we evaluated the efficacy of fibroblast growth factor-2 (FGF2) in treating radiation-induced gastrointestinal syndrome (RIGS) incurred by lethal whole-body irradiation (WBI) when administered in conjunction with bone marrow transplantation (BMT). In vitro experiments indicated FGF2 treatment increased proliferation, reduced apoptosis, and upregulated AKT-GSK3β/β-catenin signaling in irradiated IEC-6 cells. We next established and analyzed mice cohorts consisting of sham irradiation (Group Sh); 12 Gy WBI (Group A); WBI with BMT (Group B); WBI with FGF2 treatment (Group F); and WBI with BMT and FGF2 treatment (Group BF). At 2 weeks post-irradiation, Group BF showed a dramatic increase in survival over all other groups. Intestinal epithelium of Group BF, but not Group B or F, showed augmented proliferation, decreased apoptosis, and preserved crypt numbers and morphology. Furthermore, Group BF maintained intestinal barrier function with minimal inflammatory disturbances in a manner comparable to Group Sh. In accordance, transcriptomic analyses showed significant upregulation of intestinal barrier and stem cell markers in Group BF relative to Groups A and B. Taken together, parenteral FGF2 synergizes with BMT to confer potent mitigation against RIGS.

Fig. 1. Evaluation of the radiomitigative effects of various FGF family proteins on irradiated IEC-6 cells.

In all experiments, FGFs were added 30 min after irradiation. Asterisks indicate statistical significance between FGF2 and vehicle. a Clonogenic survival curve for irradiated IEC-6 cells. IEC-6 cells were treated with vehicle (PBS, Gibco), FGF1, FGF2, or FGF19 (each 10 ng/ml) for 24 h after the indicated doses of irradiation. n = 3 in each point. b Cell viability measured by CCK-8 assay 48 h after 2 Gy and 4 Gy irradiation, which were normalized to vehicle-treated group. n = 3 for each group. c Analyses of signaling pathways by Western blot with cell extracts from IEC-6 cells irradiated (4 Gy) and treated with FGFs for 4 h. Data are representative results from at least two independent experiments. Values are mean ± SEM; *p < 0.05, **p < 0.01

Fig. 2. Effects of FGF2 injection with or without BMT on clinical parameters of C57BL/6 J mice after 12 Gy WBI.

a Schematics of irradiation, FGF2 injection, BMT, and analyses at various time points. b Kaplan–Meier survival analyses of treatment groups; the number of mice per group and p-value compared with Group BF are shown in parentheses. c Survival time to death of mice that died within 2 weeks post-irradiation. d Body weight trends for mice that died within day 7 post-irradiation; n = 15 in each group. Asterisks indicate statistical significance between Group BF and Group A. e Body weight trends for mice in Group BF and Group B. Body weights of all surviving mice at the time of measurement are reflected in each data point; n = 13 per group on Day 0. Values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3. Effects of FGF2 injection with or without BMT on histologic parameters and intestinal inflammation.

a Representative hematoxylin and eosin-stained sections of mouse small intestine at day 5 post-irradiation. Magnification ×40 (first row, scale bar 500 μm), ×200 (second row, scale bar 100 μm), and ×400 (third row, scale bar 50 μm). b Quantification of crypt counts per circumference, villi height, and basal lamina length. n = 5 in each group. c Intestinal IL-1α and IL-6 levels were measured in tissue lysates obtained at day 3 post-irradiation. Values are means ± SEM; n = 4 in each group. d In vivo intestinal permeability was determined by FITC–dextran serum concentration at 4 h after oral gavage; day 3 post-irradiation, n = 3 in each group. e Cldn15, Epcam, and Vil1 expression levels at day 3 post-irradiation; n = 4 in each group. Values are mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4. Effects of FGF2 treatment combined with BMT on intestinal crypt proliferation, apoptosis, and LGR5 expression.

a BrdU incorporation measured by immunohistochemistry (magnification ×200, scale bar 100 μm) at day 5 post-irradiation. b Quantification of BrdU-positive crypts per circumference. At least five circumferences per mouse were counted; n = 3 in each group. c Ki-67 and TUNEL immunofluorescence staining (Ki-67: magnification ×400, scale bar 50 μm; TUNEL: magnification ×1000, scale bar 20 μm). d, e Quantification of Ki67- and TUNEL-positive cells per crypt at day 5 post-irradiation. At least 10 well-oriented crypts per mouse were counted; n = 3 in each group. f Intestinal tissue lysates were examined by Western blot at day 3 post-irradiation. α-tubulin was used as the loading control and LC3B was used as the positive control for radiation damage (autophagy). Data are representative results from at least two independent experiments. g Lgr5 and Axin2 expression levels at day 3 post-irradiation; n = 3 in each group. Values are mean ± SEM; *p < 0.05, **p < 0.01

Fig. 5. Effects of FGF2 treatment combined with BMT on radiation-induced alterations of serum cytokines.

Serum cytokine profiles from mice on day 3 and day 5 post-irradiation. a IL-10. b TNFα. c IL-4. d IL-6. n = 3–6 in each group. Values are mean ± SEM; *p < 0.05, **p < 0.01

Fig. 6. FGF2 treatment reveals transcriptomic signatures of reduced inflammation and restoration of intestinal tissue homeostasis in radiation-damaged intestines.

a Heatmap of differentially expressed genes in intestinal tissue from indicated treatment groups (day 5 post-irradiation). Intestinal tissue RNA samples from three mice in each treatment group were pooled together for RNA sequencing. Red indicates higher normalized expression levels and green indicates lower expression levels. b Number of upregulated and downregulated genes (≥twofold change) between indicated treatment groups. c Heatmap of genes involved in intestinal tissue homeostasis and tissue inflammation. d qPCR validation of select genes from (c). n = 3 in each group. Values are mean ± SEM; *p < 0.05, **p < 0.01