Synthesis and radioprotective effects of novel hybrid compounds containing edaravone analogue and 3‐n‐butylphthalide ring‐opening derivatives

Abstract As the potential risk of radiation exposure is increasing, radioprotectors studies are gaining importance. In this study, novel hybrid compounds containing edaravone analogue and 3‐n‐butylphthalide ring‐opening derivatives were synthesized, and their radioprotective effects were evaluated. Among these, compound 10a displayed the highest radioprotective activity in IEC‐6 and HFL‐1 cells. Its oral administration increased the survival rates of irradiated mice and alleviated total body irradiation (TBI)‐induced hematopoietic damage by mitigating myelosuppression and improving hematopoietic stem/progenitor cell frequencies. Furthermore, 10a treatment prevented abdominal irradiation (ABI)‐induced structural damage to the small intestine. Experiment results demonstrated that 10a increased the number of Lgr5+ intestinal stem cells, lysozyme+ Paneth cells and Ki67+ transient amplifying cells, and reduced apoptosis of the intestinal epithelium cells in irradiated mice. Moreover, in vitro and in vivo studies demonstrated that the radioprotective activity of 10a is associated to the reduction of oxidative stress and the inhibition of DNA damage. Furthermore, compound 10a downregulated the expressions of p53, Bax, caspase‐9 and caspase‐3, and upregulated the expression of Bcl‐2, suggesting that it could prevent irradiation‐induced intestinal damage through the p53‐dependent apoptotic pathway. Collectively, these findings demonstrate that 10a is beneficial for the prevention of radiation damage and has the potential to be a radioprotector.

reactive oxygen species (ROS), which may attack the surrounding macromolecules, this is the indirect effect of IR. 5 As a result, IR can impair the structure of DNA, proteins and lipids, and eventually lead to deleterious consequences, including cell apoptosis and organ system dysfunction. [6][7][8] Research has shown that hematopoietic tissues and the gastrointestinal (GI) tract are sensitive to IR, the former being the most sensitive, 9,10 and there are few effective methods to prevent or mitigate radiation-induced injury of the GI tract. 11,12 Thus, finding remedies to protect these systems from radiation damage is of utmost importance. 13,14 Radioprotectors are compounds used before or at the time of radiation exposure to inhibit radiation damage and protect biomacromolecules. 5 To date, numerous natural products and synthetic compounds have been evaluated for their potential as effective radioprotectors. 3,15 Among these, amifostine is the only radioprotective agent approved by the Food and Drug Administration (FDA) for decreasing adverse reactions in patients undergoing radiation therapy. 4 However, amifostine can only be administrated intravenously and has been shown to have multiple adverse effects, such as nausea, hypotension, cephalalgia, fainting and transient hypocalcemia. 4,5,13 Hence, there is an urgent need to develop efficient, convenient and less toxic radioprotectors to protect normal human tissues from radiation damage.
Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, Figure 1), generally known as an effective free radical scavenger with low toxicity, was approved by the Japanese Ministry of Health in 2001 for the treatment of ischemic stroke. 16 Recent evidence has indicated that edaravone could significantly increase the survival rate of mice exposed to whole-body X-ray irradiation and protect human peripheral blood lymphocytes against γ-ray-induced radiation damage. 17 The bioactivity of edaravone is due to the removal of toxic ROS and the inhibition of DNA damage caused by oxidative stress. 18,19 Unfortunately, its oral use is limited owing to its poor aqueous solubility and low intestinal permeability. 20 The 3-n-butylphthalide (NBP, Figure 1), as a native compound, was approved by the State Food and Drug Administration (SFDA) of China for the therapy of ischemic stroke in 2002. 21,22 Multiple studies have shown that NBP inhibits platelet aggregation and thrombus formation, promotes cerebral microcirculation and decreases oxidative damage. [23][24][25] In addition, toxicology studies have demonstrated safety of NBP as a drug. 26 However, its potency is restricted owing to its poor aqueous solubility and low bioavailability. 23,24 In a previous study, Sheng et al designed and synthesized a range of hybrid compounds containing edaravone analogues and NBP derivative, one of which exhibited potent anti-ischemic stroke activity via the synergistic action of edaravone analogue and NBP, and could be administrated orally to rats. 27,28 They also proved that the mechanisms underlying its bioactivity include inhibiting platelet aggregation, scavenging free radicals and reducing oxidative stress. 27,28 Since the biological activities of the compounds containing edaravone analogue and NBP ring-opening derivative are related to their free radical scavenging effects, our study aimed to investigate whether these compounds exert radioprotective activities. We used the hybrid compound identified by Sheng et al as the lead compound, and synthesized a group of its derivatives ( Figure 1). We hypothesized that these compounds could exhibit potent radioprotective activities via the synergistic action of the edaravone analogue and NBP. To enhance the aqueous solubility and oral bioavailability of the lead compound, we modified its structure by introducing various amines, for example, dimethylamine, pyrrolidine and morpholine, to the side chain of the ring-opening NBP. After obtaining a series of novel hybrid compounds, we examined their in vitro and in vivo radioprotective effects and mechanisms. Our findings provide a new direction for studies on radioprotectors.

| Synthesis and characterization of the compounds
The synthesis of compound 9 and target compounds 10a-10f are shown in Scheme S1. All intermediates and compound 9 were synthesized as previously described. 21,23,28 The synthesis of compounds 10a-10f are presented in the Supporting Information. All compounds were characterized using 1 H NMR and MS, the target compounds 10a-10f were also characterized using 13 C NMR and HRMS ( Figure S3-S32).

| Cell culture and irradiation
Rat intestinal epithelial cell 6 (IEC-6) and human fetal lung fibroblast 1 (HFL-1) were used for in vitro studies. IEC-6 and HFL-1 cells were cultured in DMEM and Ham's F12K medium, respectively, supplemented with 10% fetal bovine serum and penicillin (100 U/ mL)/streptomycin (100 μg/mL), in a humidified atmosphere with 5% CO 2 at 37°C. A 137 Cs γ-ray source placed in an Exposure Instrument Gammacell-40 (Atomic Energy of Canada Ltd.) was used for in vitro experiments at a dose rate of 0.98 Gy/min.

| Cell viability assay
For the cytotoxicity experiment, IEC-6 and HFL-1 cells were plated in 96-well plates (5 × 10 3 cells per well), incubated overnight, and treated with the tested compounds at concentrations from 0 to 100 μmol/L. After 24 hours, the cells were washed once, and cell viability was measured using the luminescence-based CellTiter Glo TM assay according to the manufacturer's protocols (Promega).
To evaluate the radioprotective effects, IEC-6 and HFL-1 cells were plated in 96-well plates (5 × 10 3 cells per well) and incubated overnight. The cells were pretreated with the tested compounds, with edaravone as the positive control. After 1 hour, the cells were irradiated with 8.0 or 10.0 Gy and were cultured for 24 hours. Cell viability was measured as described above.

| Measurement of intracellular ROS levels in IEC-6 cells
IEC-6 cells were plated in 6-well plates (3 × 10 5 cells per well) and incubated overnight. The cells were pretreated with 10a for 1 hour, irradiated with 4.0 Gy, and harvested after 1 hour. Then, they were incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCFDA; 10 μmol/L) for 20 minutes at 37°C. The levels of intracellular ROS were measured using the mean fluorescence intensity (MFI) of DCFDA through flow cytometry (BD Biosciences).
F I G U R E 1 Structures of edaravone, 3-n-butylphthalide, the lead compound and the target compounds 2.5 | Alkaline single-cell gel electrophoresis (comet assay) IEC-6 cells were plated in 6-well plates at 2 × 10 5 cells per well, incubated overnight, and then pretreated with 10a for 1 hour before 4.0 Gy γ-irradiation. The cells were harvested and suspended in cold PBS. The cell suspension was mixed with 70% low-melting point agarose (7.5 g/L), and 50 μL of the mixture was placed on a comet slide coated with one layer of normal-melting point agarose (7.5 g/L). The slides were submerged in precooled lysis buffer (2.5 mol/L NaCl, 100 mmol/L Na 2 EDTA, 10 mmol/L Tris-HCl, pH 10, 10% DMSO, and 1% Triton X-100) at 4°C for 2.5 hours and in cold electrophoresis buffer (300 mmol/L NaOH, 1 mmol/L Na 2 EDTA, pH >13) at 4°C for 20 minutes. Electrophoresis was performed at 30 V for 20 minutes. The slides were neutralized with Tris-HCl (pH 7.5) for 30 minutes and stained with ethidium bromide (50 μL per slide, 2.5 μg/mL). Finally, the slides were observed using a fluorescence microscope (Olympus). The percentage of DNA in the tail and Olive tail moment were measured using the Comet Assay Software Project.

| Animal experiments
Male C57BL/6 mice were purchased from Beijing Huafukang Irradiation was performed using a 137 Cs γ-radiation source at a dose rate of 0.98 Gy/min. For the total body irradiation (TBI) survival experiment, mice were randomly assigned to five groups, namely control, TBI + vehicle, TBI + 10a (150 mg/kg), TBI + 10a (300 mg/kg) and TBI + 10a (600 mg/kg) (n = 10 per group). In addition, mice were randomly assigned to 4 groups in the remaining TBI experiments, namely control, 10a (300 mg/kg), TBI + vehicle, and TBI + 10a (300 mg/kg) (n = 5 per group). The mice in the control group and those treated with 10a alone were sham-irradiated. For abdominal irradiation (ABI) studies, a lead shield was used to protect other parts of the body. The mice were randomly divided into three groups in the survival experiment (n = 10 per group) and in the remaining ABI experiments (n = 5), namely control, ABI + vehicle, and ABI + 10a (300 mg/kg). Mice in the control group were sham-irradiated. In all animal experiments, 15% Solutol HS 15 with 20% HPβ-CD in purified water was used to dissolve compound 10a.
The mice of the IR +10a groups were treated with gavage-administered 10a at the indicated dose 1 hour before radiation, whereas those in the 10a groups received 10a at the same dose and through the same route.
Mice in the control and IR +vehicle groups were treated with vehicle using the same method as used for the treatment of 10a.

| Survival study
Mice in the irradiation groups received 8.0 Gy TBI or 15.0 Gy ABI.
The survival status of each mouse was observed for 30 days after irradiation, and the results were expressed as survival rates.

| Peripheral blood cell and bone marrow nucleated cell counts
To assess the hematopoietic injuries, mice in the irradiation groups were exposed to 4.0 Gy of TBI and euthanized after 14 days.
Peripheral blood was obtained from the eyes and placed in EP tubes containing K 3 EDTA. White blood cells (WBCs), platelets (PLTs), the percentage of neutrophils (NE%), and the percentage of lymphocytes (LY%) were analysed by hematology analyzer (Nihon Kohden).
Bone marrow nucleated cells were flushed out from the mouse femurs and were counted similarly.

| Analysis of intracellular ROS levels in HPCs and LSKs
Bone marrow cells (5 × 10 6 ) were stained with HPC and LSK antibodies and incubated with DCFDA at the concentration of 10 μmol/L.
The levels of intracellular ROS were assessed by measuring the MFI of DCFDA using the flow cytometry.

| Histological analysis
To assess the intestinal damage, mice in ABI groups were irradiated with 15.0 Gy, and euthanized on the fifth day after ABI. The small intestinal tissues of the mice were resected, fixed in 10% neutral-buffered formalin solution, dehydrated and embedded in paraffin. The blocks were cut into 4μm-thick sections, stained with haematoxylin-eosin (H&E), and observed under a microscope (Olympus). For the morphological analysis, six circular transverse sections were analysed and quantitated per mouse to determine the villi height and crypts number.

| Immunohistochemistry analysis
Sections (4μm thick) were dewaxed and rehydrated in decreasing concentrations of ethanol. Next, antigen retrieval was then performed and the samples were blocked with serum for 1 hour at 37℃.
Then, the sections were stained overnight at 4℃ with the follow- Subsequently, the cells were stained using DAB kit. The images were captured using a microscope (Olympus), and positive staining was objectively analysed.

| TUNEL assay
Sections (4μm thick) were treated in accordance with the manufacturer's protocols (Roche), observed using a microscope (Olympus), and analysed using a blind method.

| Statistical analysis
All data are expressed as the mean ± SEM, unless indicated otherwise. The Kaplan-Meier method and log-rank test were used to analyse the survival rates, whereas unpaired t-tests (two-tailed) were used for mean comparisons. Statistical analysis was performed using the GraphPad Prism version 5 software. Differences were considered statistically significant at P < 0.05.

| Synthesis of compound 9 and target compounds 10a-10f
The compounds were synthesized as described in the Supporting Information. Compounds 9 and 10a-10f with purities > 95% were used for all the experiments (see Supporting Information) and their oil-water partition coefficient (LogP) values are listed in Table S1.
We found that the LogP values of 10a, 10e, and 10f were less than 5, indicating good aqueous solubilities.

| Cytotoxicity and radioprotective activity of the tested compounds
Considering that intestines and lungs are sensitive to radiation, 31,32 we used IEC-6 and HFL-1 cells to investigate the in vitro radioprotective activities of the tested compounds. Initially, we evaluated the toxicity of compound 9 and target compounds 10a-10f on IEC-6 and HFL-1 cells. As illustrated in Figure 2A and B, the tested compounds had no toxic effect on IEC-6 and HFL-1 cells at lower concentrations and exhibited more pronounced inhibitory effect on the cells at concentrations higher than 10 μmol/L. Thus, a concentration of 1 μmol/L was used for subsequent in vitro experiments.
The radioprotective effects of the tested compounds were displayed in Figure 2C

| Compound 10a inhibited IR-induced oxidative stress in IEC-6 cells
Excessive ROS levels triggered by irradiation may lead to oxidative stress, contributing to cell and tissue damage. As demonstrated in Figure 2E, the ROS level increased significantly in the IR group compared with that in the control group (P < 0.01), and that in the 10a treatment group was only 60% of the IR group (P < 0.05). These results suggested that 10a can scavenge ROS and reduce oxidative stress in irradiated IEC-6 cells.

| Compound 10a protected IEC-6 cells from IRinduced DNA damage
Nuclear DNA is an intracellular target of radiation injury. Both direct deposition of radiant energy and excessive oxidative stress in cells induced by rays would aggravate the accumulation of DNA damage. 29 Figure 2F Figure 2G, H). Compared with the IR group, the percentage of DNA in the tail and Olive tail moment were dramatically reduced upon 10a pretreatment (P < 0.01). These data demonstrated that 10a played a crucial role in protecting DNA from radiation damage.

| Compound 10a upregulated the expression of Nrf2 and downstream antioxidant proteins in irradiated IEC-6 cells
Nrf2 is a transcription factor which can regulate redox homeostasis in cells. 33 When activated under stress, it translocates from the cytoplasm to the nucleus and induces the transcription of downstream cytoprotective proteins. 33,34 Therefore, we analysed the distribution of Nrf2 in IEC-6 cells by immunofluorescence assay. As displayed in Figure 3A, exposure to IR caused Nrf2 foci formation in the nucleus of the cells, which was enhanced by 10a treatment.
Furthermore, we investigated the expression of Nrf2 in the nucleus of IEC-6 cells by Western blotting. As shown in Figure 3B, the expression of Nrf2 was upregulated by irradiation, and compound 10a further increased its expression level. Together, these results suggested that 10a could induce the nuclear translocation of Nrf2 in irradiated cells.
NQO1 and HO-1 are enzymes which can be upregulated by Nrf2, and they are known to protect against oxidative injury. 34, 35 We evaluated the levels of antioxidant enzymes NQO1 and HO-1 in IEC-6 cells using Western blotting. As illustrated in Figure 3C, the levels of these compounds in IEC-6 cells significantly increased after irradiation (P < 0.01), and 10a treatment further enhanced their expressions compared to the IR group (P < 0.01). These results suggested that 10a may reduce oxidative stress injury in irradiated cells via the activation of the Nrf2 pathway.

| Compound 10a protected IEC-6 cells against irradiation partially through the modulation of the apoptotic pathway
To further investigate the radioprotective mechanisms of 10a, we analysed the levels of the pro-apoptotic protein Bax and antiapoptotic protein Bcl-2 in IEC-6 cells using Western blotting ( Figure 3D). After irradiation, Bax levels in IEC-6 cells remarkably increased (P < 0.01), whereas Bcl-2 levels significantly decreased (P < 0.05). However, 10a treatment dramatically alleviated the increased abundance of Bax (P < 0.05) and enhanced that of Bcl-2 (P < 0.05) compared with the IR group ( Figure 3D). These results indicated that 10a can suppress IR-induced apoptosis in IEC-6 cells by regulating the apoptotic pathway.

| Compound 10a improved the survival of TBI-and ABI-exposed mice
As illustrated in Figure 4A, all mice in the TBI +vehicle group died within 15 days, but no death was observed in the control group. All doses of 10a treatment increased the survival rate of mice compared with the vehicle treatment. The survival rate in the group pretreated with 300 mg/kg 10a was 50% (P = 0.0003 vs. TBI + vehicle group), whereas that in the 600 mg/kg 10a group was only 30% (P = 0.0006 vs. TBI + vehicle group), indicating that a higher dose of 10a may have toxic effects on mice. Consequently, a dose of 300 mg/kg was used for subsequent in vivo experiments.
In Figure 4B, all mice in the ABI +vehicle group died within 16 days. Particularly, the survival rate of mice administered with 300 mg/kg 10a before radiation exposure was 40% (P = 0.0047 vs. ABI + vehicle group). These findings suggested that 10a could effectively protect against irradiation-induced intestinal damage and improve survival after a fatal dose of ABI.

| Compound 10a ameliorated myelosuppression and myeloid skewing of TBIexposed mice
TBI can cause myelosuppression, and thus result in significant reduction in peripheral blood cell counts. 30 IR may also affect the differentiation of HSCs, leading to myeloid skewing in peripheral blood. 36,37 As illustrated in Figure 4C-F, compared with the control   Figure 5A). As demonstrated in Figure 5B and C, compared with the control group, the TBI + vehicle group showed a significant reduction in the frequencies of LSKs and HPCs (P < 0.01), whereas 10a treatment dramatically increased the LSK and HPC frequencies (P < 0.01). These findings suggested that 10a could prevent against radiation-induced HSPC injury.

| Compound 10a decreased TBI-induced oxidative stress in the hematopoietic system
As we already demonstrated that 10a could scavenge ROS at the cellular level, we proceeded to assess its effect on IR-induced ROS levels in vivo. In Figure 5D and E, the ROS levels in LSKs and HPCs of irradiated mice increased dramatically compared with those of the control (P < 0.05), whereas 10a treatment markedly reduced these ROS levels (P < 0.05). These results suggested that 10a could scavenge ROS and consequently attenuate oxidative stress in irradiated HSPCs.

F I G U R E 4
Compound 10a increased the survival rates of mice and ameliorated TBI-induced myelosuppression and myeloid skewing after irradiation. A, Survival analysis of mice exposed to 8.0 Gy TBI. All doses of 10a treatment reduced mortality of mice following TBI compared with vehicle treatment (P < 0.05, n = 10). B, Survival analysis of mice exposed to 15.0 Gy ABI (P = 0.0047 10a vs vehicle, n = 10). C, WBC counts, D, PLT counts, E, NE% and F, LY% in peripheral blood were analysed 14 days after 4.0 Gy TBI, data are presented as the mean ± SEM (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001

| Compound 10a reduced intestinal damage of ABI-exposed mice
IR can induce necrosis and apoptosis in small intestinal epithelial cells, block the renewal of the villus epithelium, and lead to the loss of intestinal mucosal barrier function. 12,38 The morphological changes in the mouse small intestine are shown in Figure 6A. Five days after exposure to 15.0 Gy ABI, the mice in the vehicle group had significantly decreased villi height and number of crypts than the control (P < 0.01).
Compared with the irradiated mice, those treated with 10a exhibited increased number of crypts and villi height ( Figure 6A, B). The expression of villin + enterocytes was also decreased by ABI but increased by 10a pretreatment ( Figure 6B). These findings suggested that 10a could prevent intestinal villus-crypt structural damage in irradiated mice.

| Compound 10a enhanced the regenerative ability of Lgr5 + intestinal stem cells (ISCs) and maintained homeostasis in intestinal cells of ABIexposed mice
The intestinal epithelium constantly self-renews and can quickly regenerate after injury. [39][40][41] Lgr5 is a marker for intestinal stem cells, and Lgr5 + ISCs have been shown to be essential for intestinal regeneration after radiation injury. 12,42,43 Paneth cells are intestinal epithelium cells that secrete abundant antibacterial proteins including lysozyme, which are important for maintaining intestinal homeostasis. 39 Ki67 is a marker for the regenerating epithelium, and can be used to determine the fraction of proliferating crypts. 12 Therefore, we evaluated the effects of 10a on ABI-induced intestinal damage F I G U R E 5 Compound 10a attenuated TBI-induced hematopoietic system injury. A, Representative fluorescence-activated cell sorting plots of HPCs and LSKs. B, HPC and C, LSK frequencies in Lineagebone marrow cells, and the intracellular ROS levels of D, HPCs and E, LSKs were analysed 14 d after TBI. Data are presented as the mean ± SEM (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001  Figure 6C-E). Five days after ABI, the numbers of Lgr5 + ISCs, lysozyme + cells, and Ki67 + TACs were notably reduced in the ABI + vehicle group compared with the control (P < 0.001). However, these changes were markedly reversed by 10a treatment (P < 0.05), indicating that 10a could enhance the regenerative ability of ISCs and maintain intestinal homeostasis by stimulating the proliferation and differentiation of intestinal crypt cells.

| Compound 10a attenuated DNA damage in the small intestine of ABI-exposed mice
Phosphorylation of H2AX at Serine 139 (γ-H2AX) is a marker for DNA double-strand breaks (DSBs). 12,44 To investigate whether 10a could mitigate ABI-induced DSBs, we analysed the levels of γ-H2AX in the small intestine using an immunofluorescence method. As illustrated in Figure 7B, immunofluorescence revealed a significant increase in the expression of γ-H2AX in the irradiated mice compared with the control (P < 0.001), and, 10a treatment significantly inhibited the phosphorylation of H2AX in the mouse small intestine (P < 0.01). These results suggested that 10a can reduce DSBs in the small intestines of irradiated mice.

| Compound 10a reduced the apoptotic rate in the small intestine of ABI-exposed mice
We used the TUNEL assay to evaluate the effect of 10a on the apoptosis of small intestinal tissues. Figure 7A show an increase number of apoptotic cells in the irradiated mice than in the control (P < 0.001), whereas 10a treatment dramatically decreased the number of apoptotic cells (P < 0.01). These results suggested that 10a could play a positive role in the inhibition of apoptosis. We further studied the effects of 10a on ABI-induced apoptosis using immunofluorescence assay. In the ABI + vehicle group, the levels of p53 ( Figure 7C), caspase-3 ( Figure 7D) and caspase-9 ( Figure 7E) in the intestinal cells were significantly higher than those in the control (P < 0.01). In the 10a treatment group, the levels of these proteins decreased significantly compared with those in the ABI + vehicle group (P < 0.05). Taken together, these data demonstrated that 10a treatment could protect mice from ABI-induced intestinal injuries by suppressing radiation-induced apoptosis.

| D ISCUSS I ON
In this study, we designed and synthesized a series of hybrid compounds containing edaravone analogue and NBP ring-opened derivatives and evaluated their radioprotective effects in vitro and in vivo. The anionic form of edaravone can eliminate free radicals through a single-electron transfer mechanism. 28 Owing to the electron-withdrawing ability of the fluorine substituent on the benzene ring, the edaravone analogue moiety of these compounds can exhibit higher free-radical scavenging activities. 28 In addition, analysis of structure-activity relationships (SAR) showed that different substituted amines in the side chain of the ring-opening NBP could have different effects on the solubility and permeability of the parent compound, which might affect the release of the edaravone analogue and NBP, and thus lead to varying bioactivities of these compounds. We observed that compound 10a, which bears a dimethylamino moiety in side chain of ring-opened NBP, displayed the highest radioprotective activities in IEC-6 and HFL-1 cells among all tested compounds. We speculate that the enhanced solubility of compound 10a (LogP = 4.97) contributed to its good cell permeability. Besides, its higher biological activity than those of 10e and 10f may be due to the relatively low molecular weight and high absorbability of 10a.
Since the hematopoietic system is the most radiosensitive system in the body, we investigated whether 10a could prevent TBI-induced hematopoietic tissue damage. We found that the oral administration of 10a not only enhanced the survival of mice exposed to a fatal dose of TBI, but also mitigated TBI-induced hematopoietic tissue damage.
Furthermore, 10a provided protection against radiation-induced myelosuppression and myeloid skewing in mice by increasing WBCs, PLTs and LY%, and decreasing NE%. We also observed that 10a consumption significantly increased the proportions of HSCs and HPCs in TBI mice. Overall, these results suggested that 10a could ameliorate hematopoietic injury by promoting the recovery of HSC density and function.
Radiotherapy for abdominal and pelvic malignant tumours may injure the GI system, and the small intestine is particularly sensitive to the damaging effects of IR. 45,46 To evaluate whether 10a could function as a radioprotector to treat radiation-induced intestinal injury, we established an ABI mouse model and found that 10a improved the survival of mice and preserved the intestinal crypt-villus structure following 15.0 Gy ABI. The intestinal epithelium is one of the most rapidly self-renewing tissues in mammals. 40 ISCs, specifically marked by Lgr5, are indispensable for maintaining epithelial homeostasis and promoting structural regeneration. Lgr5 + ISCs can differentiate into Paneth cells, which can produce lysozymes. Our findings revealed that 10a treatment increased the number of Lgr5 + cells and lysozymes + Paneth cells after irradiation. Additionally, the abundance of Ki67, a proliferative marker of epithelial layer, was promoted in the mice pretreated with 10a. Therefore, we concluded that 10a might exert protective effects on ABI-induced intestinal damage and could maintain the regenerative capacity of ISCs by stimulating the proliferation and differentiation of intestinal epithelial cells.
Oxidative stress due to ROS accumulation after radiation exposure can contribute to tissue damage. We found that the ROS levels in irradiated cells were decreased by 10a, indicating that 10a can scavenge ROS generated by ionizing radiation. Besides, results showed that 10a increased the expression levels of Nrf2, NQO1 and HO-1 in IEC-6 cells after irradiation, suggesting that 10a may target used as a biomarker. 12,29 We observed that γ-H2AX levels in the small intestines of irradiated mice were reduced by 10a, suggesting that 10a could alleviate DNA damage by reducing oxidative stress in irradiated cells. Furthermore, the pro-apoptotic molecule p53 is mainly activated by DNA strand breaks and can activate genes that regulate radiation-induced DNA damage and repair, and cell apoptosis. 47 Studies have shown that p53 promotes apoptosis by regulating the expression of Bcl-2 protein family members, such as Bax, Bak, and Bcl-2, in the cytoplasm. 2,42 We observed decreased p53 levels and Bax levels, and increased Bcl-2 levels in irradiated cells after 10a treatment. Moreover, previous studies have indicated that apoptosis is induced by mitochondrial outer membrane permeabilisation, which is affected by the Bcl-2 family members. 48,49 After the release of cytochrome C and the formation of apoptosome, apoptosis is executed by a series of sequentially activated caspases, which can be categorized into initiator caspases (caspase-8 and −9) and effector caspases (caspase-3, −6 and −7). 42, 50 We found that 10a treatment downregulated the expression of caspase-3 and caspase-9 after irradiation. Together, these results suggested that 10a could mitigate DNA damage induced by oxidative stress, thereby reduce apoptosis through the p53 pathway ( Figure 8).
In summary, hybrid compounds with edaravone analogue and NBP ring-opened derivatives were synthesized in the present study.
Among these, compound 10a displayed the most potent radioprotective activity in vitro. We showed that 10a effectively increased the survival rates of irradiated mice, prevented TBI-induced HSC injury, improved the structure and function of the small intestine, and decreased apoptosis of intestinal epithelium cells in ABI-exposed mice. Our results proved that 10a could reduce IR-induced oxidative stress and DNA damage both in vitro and in vivo. Moreover, 10a might mitigate IR-induced intestinal damage via the p53-dependent apoptotic pathway. We demonstrate that compound 10a is a promising radioprotector, and the detailed molecular mechanisms underlying its radioprotective effects should be further studied.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest. Hongqqi Tian: Project administration (lead); Writing-review and editing (supporting).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.