Transplantation of Bone Marrow Mesenchymal Stem Cells Prevents Radiation-Induced Artery Injury by Suppressing Oxidative Stress and Inflammation

Abstract

The present study aims to explore the protective effect of human bone marrow mesenchymal stem cells (hBMSCs) on radiation-induced aortic injury (RIAI). hBMSCs were isolated and cultured from human bone marrow. Male C57/BL mice were irradiated with a dose of 18-Gy 6MV X-ray and randomly treated with either vehicle or hBMSCs through tail vein injection with a dose of 10³ or 10⁴ cells/g of body weight (low or high dose of hBMSCs) within 24 h. Aortic inflammation, oxidative stress, and vascular remodeling were assessed by immunohistochemical staining at 3, 7, 14, 28, and 84 days after irradiation. The results revealed irradiation caused aortic cell apoptosis and fibrotic remodeling indicated by aortic thickening, collagen accumulation, and increased expression of profibrotic cytokines (CTGF and TGF-β). Further investigation showed that irradiation resulted in elevated expression of inflammation-related molecules (TNF-α and ICAM-1) and oxidative stress indicators (4-HNE and 3-NT). Both of the low and high doses of hBMSCs alleviated the above irradiation-induced pathological changes and elevated the antioxidant enzyme expression of HO-1 and catalase in the aorta. The high dose even showed a better protective effect. In conclusion, hBMSCs provide significant protection against RIAI possibly through inhibition of aortic oxidative stress and inflammation. Therefore, hBMSCs can be used as a potential therapy to treat RIAI.

Figure 1

Morphology and features of human bone marrow mesenchymal stem cells (hBMSCs). (a) The morphological features of cultured hBMSCs at the 5th day; the 3rd and 6th passages (P3 and P6) were evaluated by the light microscope or HE staining. (b) Cell cycle analysis by FACS showed that (86.65% ± 2.8%) of P5 hBMSCs was in the G₀/G₁ phase and (14.35% ± 2.8%) was in the S + G₂/M phase. (c) Flow cytometry analysis disclosed that more than 90% of P5 hBMSCs were positive for CD44, CD105, CD166, and CD73; however, they were negative for CD34, CD31, and CD45. (d) Immunofluorescence staining revealed that P5 hBMSCs expressed the antigens of CD73, CD44, CD105, and CD166. (e) hBMSCs differentiated into adipose cells that formed lipid droplets in the cytoplasm, indicated by positive Oil Red staining (upper). The differentiation of hBMSCs to bone was demonstrated by positive von Kossa staining (bottom left). The differentiation to cartilage was reflected by positive Alcian blue staining (bottom right). Scale bar, 50 μm.

Figure 2

hBMSCs alleviated radiation-induced aortic pathological changes. Male C57BL/6 mice were irradiated by 6MV X-ray of 18Gy once with their lungs were shielded to establish the RIAI model. hBMSCs were injected by tail vein in a dose of 10³ or 10⁴ cells/g of body weight within 24 h after radiation. Therefore, the mice were evenly divided into four groups: the control group (control), the radiation group (IR), the radiation with low or high dose of the hBMSC group (IR + LD hBMSCs and IR + HD hBMSCs). At 3, 7, 14, 28, and 84 days after radiation, the aortas were isolated for histological studies. The pathological changes of aortas were examined by HE staining (a) and the accumulation of collagen was detected by Sirius red staining (b), followed by semiquantitative analysis. Data were presented as means ± SD (n = 7). ^∗P < 0.05 versus control group; ^&P < 0.05 versus IR group; ^#P < 0.05 versus IR + LD hBMSC group. Scale bar, 50 μm.

Figure 3

hBMSCs alleviated radiation-induced aortic fibrosis. Aortic fibrosis was examined by immunohistochemical staining for the expression of CTGF (a) and TGF-β (b), followed by semiquantitative analysis. Data were presented as means ± SD (n = 7). ^∗P < 0.05 versus control group; ^&P < 0.05 versus IR group; ^#P < 0.05 versus IR + LD hBMSC group. Scale bar, 50 μm.

Figure 4

hBMSCs reduced radiation-induced aortic inflammation. Aortic inflammation was examined by immunohistochemical staining for the expression of TNF-α (a) and ICAM-1 (b), followed by semiquantitative analysis. Data were presented as means ± SD (n = 7). ^∗P < 0.05 versus control group; ^&P < 0.05 versus IR group; ^#P < 0.05 versus IR + LD hBMSC group. Scale bar, 50 μm.

Figure 5

hBMSCs attenuated radiation-induced aortic oxidative damage. Aortic oxidative damage was examined by immunohistochemical staining for the expressions of 4-HNE (a) and 3-NT (b), followed by semiquantitative analysis. Data were presented as means ± SD (n = 7). ^∗P < 0.05 versus control group; ^&P < 0.05 versus IR group; ^#P < 0.05 versus IR + LD hBMSC group. Scale bar, 50 μm.

Figure 6

hBMSCs reduced radiation-induced aortic apoptosis. The apoptotic cell was examined by TUNEL staining followed with semiquantitative analysis. Data were presented as means ± SD (n = 7). ^∗P < 0.05 versus control group; ^&P < 0.05 versus IR group; ^#P < 0.05 versus IR + LD hBMSC group. Scale bar, 50 μm.

Figure 7

hBMSC upregulated antioxidant enzymes expression of HO-1 and catalase in aortas. The antioxidant enzyme expression of HO-1 (a) and catalase (b) was examined by immunohistochemical staining followed with semiquantitative analysis. Data were presented as means ± SD (n = 7). ^∗P < 0.05 versus control group; ^&P < 0.05 versus IR group; ^#P < 0.05 versus IR + LD hBMSC group. Scale bar, 50 μm.