A Combined Therapy of Pegylated G-CSF with Ciprofloxacin Mitigates Damage Induced by Lethal Ionizing Radiation to the Bone Marrow, Spleen, and Ileum by Increasing AKT Activation but Decreasing IL-18, C3, and miR-34a.

Abstract

Ciprofloxacin (CIP) was found to enhance pegylated G-CSF therapy (PEG, Neulasta®)-induced survival from 30% to 85% after ionizing radiation exposure. This combined therapy significantly mitigated radiation-induced brain hemorrhage through its capability to improve platelet recovery. This study tested whether this combined treatment also mitigated gastrointestinal damage from radiation. B6D2F1 female mice were exposed to 60Co γ radiation. CIP was fed daily to mice for up to 14 days. PEG was injected on day 1, and then weekly up to day 14. For the early time point study, blood, femurs, spleen, and ileum were collected on days 2, 4, 9, and 15 postirradiation. Bone marrow cells were counted; spleen weights and splenocyte counts were measured; and ileum histopathology was examined and analyzed. AKT, ERK, JNK, p38, claudin 2, NF-kB, Bax, Bcl-2, and gasdermin D were measured in ileum lysates using Western blotting while miR-34a was measured by reverse transcription followed by real-time-PCR, and citrulline was measured by colorimetric assay. In serum, interleukin-18 (IL-18) was measured by Luminex assay and complement protein 3 (C3) was detected by ELISA. The bacterial DNA load in livers was measured by real-time PCR. Radiation depleted bone marrow cells in femurs beginning day 2 through day 15 postirradiation, which was mitigated by PEG or CIP+PEG on day 9 through day 15 and by CIP on day 15, respectively. Radiation exposure led to decreased spleen weight on day 2 through day 15, while PEG or CIP+PEG significantly mitigated the reduction on day 9 through day 15. Radiation exposure reduced splenocyte counts on day 2 through day 15 postirradiation, but that was mitigated by PEG or CIP+PEG on day 15. Ileum histology showed that radiation decreased villus height on day 2 through day 15; CIP mitigated the reduction on day 15, whereas PEG+CIP mitigated it on day 2 through 15. Villus widths were increased on day 2 through day 15, while PEG+CIP effectively decreased them on day 4 through day 15. Crypt depth was reduced by radiation on day 2, but returned to the baseline on day 4 through 15. CIP or CIP+PEG transiently increased the depth only on day 4. Crypt counts were reduced by radiation on days 2 and 4, but returned to the baseline on days 9 and 15, regardless of individual drugs or combinations. Citrulline data confirmed the villus height recovery. Radiation significantly increased pro-inflammatory cytokine IL-18 on days 4 and 9, which was mitigated by PEG alone or PEG+CIP, but not by CIP alone. Radiation increased C3 on day 9 in ileum and serum. The serum C3 was positively associated with the serum IL-18 levels and negatively correlated with the crypt depth. Radiation-induced decreases in claudin 2 (a tight junction marker) in ileum and increases in bacterial DNA in livers were mitigated by PEG+CIP. Radiation did not reduce NF-kB and its activation but reduced Bcl-2 expression, which was not significantly recovered by any individual drug or combination. However, the PEG and CIP combination significantly decreased NF- kB and BAX. In contrast, radiation increased miR-34a and cleaved gasdermin D, which CIP+PEG effectively mitigated. This was confirmed by immunohistochemistry. The results taken together suggest that PEG+CIP combined treatment was effective in mitigating the radiation-induced bone marrow, spleen, and ileum injury. The mitigative effect of this combined treatment was mediated by increases in G-CSF levels that suppress miR-34a, thereby probably leading to decreased gasdermin D-mediated pyroptosis.