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. 2022 Sep 26:12:898185.
doi: 10.3389/fonc.2022.898185. eCollection 2022.

Radioprotection of deinococcal exopolysaccharide BRD125 by regenerating hematopoietic stem cells

Hae Ran Park  1 , Ji Hee Lee  1   2 , Hyun Jung Ji  1   3 , Sangyong Lim  1   4 , Ki Bum Ahn  1 , Ho Seong Seo  1   4
Affiliations

Radioprotection of deinococcal exopolysaccharide BRD125 by regenerating hematopoietic stem cells

Hae Ran Park et al. Front Oncol. .

Abstract

There is a substantial need for the development of biomaterials for protecting hematopoietic stem cells and enhancing hematopoiesis after radiation damage. Bacterial exopolysaccharide (EPS) has been shown to be very attractive to researchers as a radioprotectant owing to its high antioxidant, anti-cancer, and limited adverse effects. In the present study, we isolated EPS from a novel strain, Deinococcus radiodurans BRD125, which produces EPS in high abundance, and investigated its applicability as a radioprotective biomaterial. We found that EPS isolated from EPS-rich D. radiodurans BRD125 (DeinoPol-BRD125) had an excellent free-radical scavenging effect and reduced irradiation-induced apoptosis. In addition, bone-marrow and spleen-cell apoptosis in irradiated mice were significantly reduced by DeinoPol-BRD125 administration. DeinoPol-BRD125 enhanced the expression of hematopoiesis-related cytokines such as GM-CSF, G-GSF, M-CSF, and SCF, thereby enhancing hematopoietic stem cells protection and regeneration. Taken together, our findings are the first to report the immunological mechanism of a novel radioprotectant, DeinoPol-BRD125, which might constitute an ideal radioprotective and radiation mitigating agent as a supplement drug during radiotherapy.

Keywords: DeinoPol; Deinococcus; exopolysaccharide; hematopoietic stem cell (HSC); mitigator; radiation; radioprotectant; radiotherapy.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
High exopolysaccharide (EPS)-producing Deinococcus radiodurans BRD125 isolated from the crater of Mt. Halla. (A) Quantification of EPS production in Deinococcus sp. using the anthrone assay. EPS in the culture supernatants was precipitated using 80% ethanol, and then quantified using an anthrone reaction. The bacteria culture. (B) Comparison of the R1 and BRD125 EPS biosynthesis gene clusters. ExoP: regulatory protein, Gly: Glycosyltransferase, WbaP: undecaprenyl-phosphate galactose phosphotransferase, RfbX: polysaccharide transporter, RfbA: glucose-1-phosphate thymidyltransferase. Blue arrow: hypothetical protein.
Figure 2
Figure 2
Inhibition of radical scavenging and radiation-induced cell death by DeinoPol-BRD125 in vitro. (A, B) Radiation induced radical scavenging effect by DeinoPol-BRD125. CHO-K1 cells were pre-treated with DeinoPol-R1, EPS isolated from Lactococcus plantarum, or DeinoPol-BRD125 (0.5 or 1.0 μg) followed by irradiation (4 Gy). DPPH scavenging activity was calculated by comparing the proportion of the amount of DPPH generated in irradiated cells with and without DeinoPol-BRD125 treatment (A). *p<0.05 compared to DeinPol-R1 and EPS-LP. Intracellular ROS production in CHO-K1 cells after irradiation (4 Gy) were quantified by labeling with DCHF-DA using flow cytometry (B). (C, D) Protection of radiation-induced cell death by DeinoPol-BRD125. CHO-K1 cells were pretreated with DeinoPol-BRD125 (50 μg/ml) for 2 h and cell survival was calculated at 7 days after irradiation using clonogenic assay (C). Mouse bone marrow cells or splenocytes were pretreated with DeinoPol-BRD125 (50 μg/ml) for 2 h and the ratio of apoptosis was calculated at 1 day after irradiation (2 Gy) by staining with propidium iodide (PI) (D). *p<0.05 and **p<0.001 compared to PBS-treated irradiated cells.
Figure 3
Figure 3
Enhancement of the proliferation of immune cells by DeinoPol-BRD125. (A, B) Splenic lymphocytes (3×105/well) were pretreated with indicated concentration of DeinoPol-BRD125 for 2 h followed by irradiation (1 Gy). Cell proliferation was measured with bromodeoxyuridine (BrdU) proliferation assay kit at day 1(A) and day 3 (B) after irradiation. *p<0.05 compared to cells without DeinoPol-BRD125.
Figure 4
Figure 4
Protection of radiation-induced cell death by DeinoPol-BRD125 in vivo. (A–C) Mice (n=6) were injected intraperitoneally with DeinoPol-BRD125 (50 mg/kgBW) twice before and once after irradiation at a dose of 4 Gy. Bone marrow cells (BMCs) and spleen cells were collected at 4 h after irradiation. Schematic schedule of radiation-induced immune cell death experiment (A). The ratio of apoptosis was calculated by staining BMCs (left) or spleen cells (right) with propidium iodide (PI) (B). Protection of chromosomal DNA damage after irradiation by DeinoPol-BRD125 was measured by DNA fragmentation analysis. Chromosomal DNA from individual mice spleen and BMC were harvested at 4 h after irradiation and the pattern of DNA damage was visualized on 2% agarose gel. M: DNA ladder, NC: No DNA, PBS: PBS treated mice, BRD125: DeinoPol-BRD125 (00 μg/mice) treated mice (C). *p<0.01 and **p<0.001 compared to PBS-injected irradiated mice.
Figure 5
Figure 5
Rapid regeneration of hematopoietic stem cells by DeinoPol-BRD125. (A) Mice (n=12) were injected intraperitoneally twice before radiation and an additional third time after radiation with DeinoPol-BRD125 (50 mg/kgBW). Schematic schedule of endogenous spleen colony assay (top). At 9 days after 6.5 Gy radiation exposure, the colonies on the surface of the spleens were visualized (middle) and counted (bottom). (B, C) Mice (n=6) were injected intraperitoneally with DeinoPol-BRD125 (50 μg/BW). Schematic schedule of spleen and blood cell analysis (top). At 7, 14, and 24 days after 4 Gy radiation exposure, the spleens were visualized (middle) and total splenocytes were counted (bottom) (B). At 7, 14, and 24 days after 4 Gy radiation exposure, the blood were collected in K3EDTA tubes. WBC, lymphocytes, neutrophiles, monocytes, red blood cells, and platelets were analyzed using automatic blood analyzer (C). *p<0.05, **p<0.01 and ***p<0.001 compared to PBS-injected irradiated mice.
Figure 6
Figure 6
DeinPol-BRD125 enhances multilineage engraftment of irradiated hematopoietic stem cells after BM transplantation. (A–C) Mice were injected intraperitoneally with DeinoPol-BRD125 (50 mg/kgBW) two times before and three time after irradiation at a dose of 4 Gy. At 7 days after irradiation, bone marrow (BM) cells were injected into the tail veins of recipient mice previously administered lethal irradiation. Schematic schedule of BM transplantation experiment (A). Cells in peripheral blood at 2 and 4 weeks after BM transplantation were counted by automatic blood analyzer (B). Cell populations in the spleen at 2 weeks after BM transplantation were was analyzed by flow cytometry (C). *p<0.05, **p<0.01, and **p<0.001 compared to recipient mice transplanted with PBS-injected normal mice. †p<0.05 and ††p<0.01 compared to recipient mice transplanted with BM cells from PBS-injected irradiated mice.
Figure 7
Figure 7
Modulation of hematopoiesis-related cytokine expression by DeinoPol-BRD125 in irradiated mice. (A–C) Mice (n=3) were injected intraperitoneally twice before irradiation and an additional third time after irradiation with DeinoPol-BRD125 (50 mg/kgBW). Bone marrow cells (BMC) and spleen cells were collected at 3 days after irradiation. Schematic schedule of hematopoietic stem cell cytokine expression experiment (A). mRNA expressions of hematopoietic-related cytokine (GM-CSF, G-CSF, M-CSF, SCF) in BMC (B) and spleen (C) were quantified using real time PCR. †p<0.05 and ††p<0.01, compared to normal control; *p<0.05 and **p<0.01 compared to PBS-injected irradiated mice.
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