Fluacrypyrim Protects Hematopoietic Stem and Progenitor Cells against Irradiation via Apoptosis Prevention

Abstract

Ionizing radiation (IR)-induced hematopoietic injury has become a global concern in the past decade. The underlying cause of this condition is a compromised hematopoietic reserve, and this kind of hematopoietic injury could result in infection or bleeding, in addition to lethal mishaps. Therefore, developing an effective treatment for this condition is imperative. Fluacrypyrim (FAPM) is a recognized effective inhibitor of STAT3, which exhibits anti-inflammation and anti-tumor effects in hematopoietic disorders. In this context, the present study aimed to determine whether FAPM could serve as a curative agent in hematopoietic-acute radiation syndrome (H-ARS) after total body irradiation (TBI). The results revealed that the peritoneally injection of FAPM could effectively promote mice survival after lethal dose irradiation. In addition, promising recovery of peripheral blood, bone marrow (BM) cell counts, hematopoietic stem cell (HSC) cellularity, BM colony-forming ability, and HSC reconstituting ability upon FAPM treatment after sublethal dose irradiation was noted. Furthermore, FAPM could reduce IR-induced apoptosis in hematopoietic stem and progenitor cells (HSPCs) both in vitro and in vivo. Specifically, FAPM could downregulate the expressions of p53-PUMA pathway target genes, such as Puma, Bax, and Noxa. These results suggested that FAPM played a protective role in IR-induced hematopoietic damage and that the possible underlying mechanism was the modulation of apoptotic activities in HSCs.

Keywords: Fluacrypyrim; apoptosis; hematopoietic stem cells; ionizing radiation; p53-PUMA signaling.

Figure 1

FAPM ameliorated pancytopenia and improved survival in the mice subjected to irradiation. (A–C) The mice were administered with vehicle or FAPM at different doses and then exposed to 6.5 Gy TBI. The (A) platelets, (B) white blood cells, and (C) red blood cells were analyzed in peripheral blood (n = 6). (D–F) Survival rates of the mice administrated with vehicle or FAPM followed by exposure to 8.0, 8.5, or 9.0 Gy TBI (n = 10). Data represent the mean ± standard deviation. * p < 0.05 and ** p < 0.01 versus the vehicle group by two-tailed Student’s t-test. # p < 0.05 and ### p < 0.001 versus the vehicle group by log-rank test.

Figure 2

FAPM alleviated the irradiation-induced injury in BM and enhanced colony-forming ability in the mice subjected to irradiation. The vehicle and FAPM group mice were administered vehicle and FAPM, respectively, followed by exposure to 6.5 Gy TBI (n = 7). (A) Bone marrow mononuclear cell (BMNC) counts and (B) colony-forming units were determined for the mice treated with the vehicle or FAPM at 10 days after exposure to 6.5 Gy TBI. Data represent the mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the vehicle group by two-tailed Student’s t-test.

Figure 3

FAPM accelerated hematopoietic stem and progenitor cell recovery in the mice subjected to irradiation. The vehicle and FAPM group mice were administered vehicle and FAPM, respectively, followed by exposure to 6.5 Gy TBI (n = 7). (A) Representative gating strategy of LT-HSC, ST-HSC, and MPP was analyzed by flow cytometry. Bone marrow cells were harvested 10 days after the irradiation and evaluated using flow cytometry. (B,C) Absolute numbers and (D,E) and percentages of LK (Lin–Sca1–c-Kit+) cells, LSK (Lin–Sca1+c-Kit+) cells, long-term hematopoietic stem cells (LT-HSC) (LSK Flt3– CD34–), short-term HSCs (ST-HSCs) (LSK Flt3–CD34+), and multipotent progenitor (MPP) (LSK Flt3+ CD34+) cells in the bone marrow nuclear cells (BMNCs). Data represent the mean ± standard deviation. ** p < 0.01 and *** p < 0.001 versus the vehicle group by two-tailed Student’s t-test.

Figure 4

FAPM enhanced the repopulating ability of murine hematopoietic stem cells (HSCs), as revealed in the competitive repopulation experiment. (A) Schematic of the experimental procedure. (B) Representative FACS plots. (C) The percentage of the donor (CD45.2) chimeras in the peripheral blood of the recipient mice (CD45.1) in the vehicle and FAPM groups at 4, 8, 12, and 16 weeks after transplantation (n = 7). (D) Frequency of vehicle-derived and FAPM donor (CD45.2)-derived T (CD45.2+CD3+) lymphocytes, B(CD45.2+B220+) lymphocytes, and myeloid cells (CD45.2+CD11b+ Gr-1+) in the peripheral blood of the recipient mice (CD45.1) at 12 weeks after the transplantation. (E) Percentages of LSK cells among the donor (CD45.2)-derived lymphocytes when the sacrifice was performed 16 weeks after the transplantation. Data represent the mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the vehicle group by two-tailed Student’s t-test.

Figure 5

FAPM alleviated the irradiation-induced cell apoptosis both in vitro and in vivo. (A) The ratio of apoptosis in BMNCs at 3, 6, 9, and 12 h after the 6.5 Gy irradiation (n = 3). (B) The ratio of apoptosis in the Lin– cells and Lin–c-kit+ cells at 9 h after the 6.5 Gy irradiation. (C) Representative FACS plots. (D) Percentage of Annexin V ⁺ cells in BMNC, LK, and LSK cells at 6 h after 6.5 Gy TBI (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the IR group by two-tailed Student’s t-test; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus the control group by two-tailed Student’s t-test.

Figure 6

FAPM inhibited the irradiation-induced expressions of the genes related to the p53-PUMA pathway, both in vitro and in vivo. (A) Mice were sacrificed to collect and prepare the BMNCs, which were then pre-incubated with FAPM (5 µM) for 1 h followed by exposure to 6.5 Gy γ-irradiation and culture for another 9 h to obtain the total RNA. (B) The IR and FAPM groups were administered with the vehicle and FAPM, respectively, followed by exposure to 6.5 Gy TBI (n = 3). Control mice were administered with the vehicle and shielded from irradiation. The mice were sacrificed at 6 h after the irradiation to harvest BMNCs, from which the total RNA was obtained. (C) HSPCs in these BMNCs were enriched using magnetic-activated cell sorting (MACS) and then incubated with FAPM (5 µM) for 1 h, followed by exposure to 6.5 Gy γ-irradiation and culture for another 9 h to obtain the total RNA. RT-qPCR was performed to determine the mRNA expressions of Puma, Bax, and Noxa. (D,E) After incubation with FAPM (5 μM) for 1 h, BMNC cells were exposed to γ-irradiation at a dose of 6.5 Gy and cultured for another 3 h, and the BMNCs were harvested. The cell lysates were subjected to SDS-PAGE followed by Western blotting with p-p53, p53, Puma, Bax, Noxa, cleaved caspase-3, phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), STAT3, and GAPDH antibodies. Data represent the mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus IR group by two-tailed Student’s t-test; ## p < 0.01 and ### p < 0.001 versus control group by two-tailed Student’s t-test.