Fig 2: Analysis of the radioprotective effect of CeONP60/40 (4 mg/kg) following exposure to levels of radiation able to induce bone loss in vivo. [A] Flow chart of the animal experiment. [B] Representative confocal micrographs of DNA damage (Comet Assay®) in bone marrow cells following exposure of the hind limbs to three fractions of 8 Gy (total 24 Gy) on days 1, 3 and 5 (n = 6). Quantification of tail length and comet length. Scale bar denotes 250 µm (left panel) and 50 µm (right panel). [C] Representative images of RANKL immunohistochemical staining (red arrows) in each of the experimental groups. Scale bar denotes 125 µm. [D] Multinucleated and active osteoclasts were identified via TRAP staining. Red arrows indicate TRAP + cells. The number of osteoclasts per unit of bone surface (cells/mm) were quantified by bone histomorphometric analyses (2 images/rat, n = 6). Scale bar denotes 250 µm. [E] Serum levels of CTX-1 in healthy control animals, X-Ray only, and X-Ray + CeONP60/40. ELISA results showed that CTX-1 concentrations were significantly reduced in rats following 4 mg/kg CeONP60/40 treatment. All values are given as the mean ± SD. ****p < 0.0001. [F] Representative micrographs of SA-ß-gal staining for senescent cells (stained blue). Scale bar denotes 500 µm. [G] Representative images of H&E staining. Note the extensive osteopenia at 14 days post-irradiation in the X-ray group. Scale bar denotes 500 µm. [H] Quantification of trabecular bone area (BA) to total area (TA). *p < 0.05, **p < 0.01. [I] A 3-point bending test was carried out and each tibia loaded to failure at a displacement rate of 0.02 mm/s. Ultimate stress and fracture stress in the control, X-Ray, and X-Ray + CeONP groups are presented (n = 6). The tibial parameters measured to obtain stress values are presented in Supplementary Table S1. All values are given as the mean ± SD. **p < 0.01, ***p < 0.001. [J] Representative 3D reconstruction images of the proximal tibia via nano-Ct scanning.

Fig 3: Analysis of the radioprotective effect of i.p. given P7C3 (20 mg·kg-1) following exposure to irradiation in vivo. a A flow chart of the animal experiment. b Three-dimensional models of the proximal tibia were generated using 3D Slicer™ (v4.11.20210226; Brigham and Women’s Hospital and Massachusetts Institute of Technology). The IR-treated control group showing osteoporosis. Bone architecture and area were maintained in the P7C3 + IR-treated rats. c Representative images of H&E-stained transverse sections through the femoral condyle and osteoblast cells (red arrow). Exposure of rats to harmful levels of irradiation resulted in bone loss and osteoporosis, as indicated by reduced trabecular connectivity, thinner and shorter trabeculae, and increased bone marrow adiposity. P7C3 treatment (20 mg·kg-1) markedly protected against bone loss. d BA/TA% quantification. ***P < 0.001. e Multinucleated osteoclasts were identified by TRAP staining. Red arrows indicate TRAP+ cells (purple?red color). f The number of osteoclasts per unit of bone surface (cells per mm2) was quantified via bone histomorphometric analyses (5 images/rat, n = 6). ****P < 0.000 1. g Representative images of RANKL immunohistochemical staining. Red arrows indicate RANKL+ cells. h Quantification of RANKL+ cells. RANKL was strongly expressed in irradiation-exposed rats, while 20 mg·kg-1 P7C3 treatment reduced RANKL expression. ****P < 0.000 1