The protective effects of XH‐105 against radiation‐induced intestinal injury

Abstract Radiation‐induced intestinal injury is one of the major side effects in patients receiving radiation therapy. There is no specific treatment for radiation enteritis in the clinic. We designed and synthesized a new compound named XH‐105, which is expected to cleave into polyphenol and aminothiol in vivo to mitigate radiation injury. In the following study, we describe the beneficial effects of XH‐105 against radiation‐induced intestinal injury. C57BL/6J mice were treated by gavage with XH‐105 1 hour before total body irradiation (TBI), and the survival rate was monitored. Histological changes were examined, and survival of Lgr5+ intestinal stem cells Ki67+ cells, villi+ enterocytes and lysozymes was determined by immunohistochemistry. DNA damage and cellular apoptosis in intestinal tissue were also evaluated. Compared to vehicle‐treated mice after TBI, XH‐105 treatment significantly enhanced the survival rate, attenuated structural damage of the small intestine, decreased the apoptotic rate, reduced DNA damage, maintained cell regeneration and promoted crypt proliferation and differentiation. XH‐105 also reduced the expression of Bax and p53 in the small intestine. These data suggest that XH‐105 is beneficial for the protection of radiation‐induced intestinal injury by inhibiting the p53‐dependent apoptosis signalling pathway.

radical or other reactive oxygen species (ROS), chemically repair DNA radicals and deplete oxygen. 8,9 In this study, we examined the natural anti-oxidation agent quercetin, which belongs to flavonoids family derived from plants, as a lead compound and modified the molecular structure. At the same time, we also tried to combine the aminothiol analogue and the natural anti-oxidation agent together with different linkers to retain the efficacy of aminothiol and the safety property of natural anti-oxidation agents respectively. Furthermore, the two functional fragments modulated the pharmacokinetic profile for each other, and they were mutually prodrug to each other. The new compound was expected to cleave into polyphenol and aminothiol in vivo to mitigate radiation injury by their ROS scavenger capabilities. Based on this concept, we designed and synthesized the compound XH-105.
To define the effects of XH-105 on intestinal radiation injury, we used a mouse model with exposure to 9.0 Gy total body irradiation (TBI). We found that XH-105 could improve the survival rate of mice and intestinal epithelium cells (IECs). The crypt-villous structure injuries of small intestinal and the apoptosis of IECs induced by TBI were mitigated by XH-105.

| Synthesis of 2,2-dimethylthiazolidine-3carbonyl chloride
To a solution of bis(trichloromethyl) carbonate (4.05 g, 13.66 mmol) (Energy Chemical, Shanghai, China) in anhydrous tetrahydrofuran (30 mL), 2,2-dimethylthiazolidine (4 g, 44.87 mmol) (Energy Chemical) was added in parts and triethylamine (8.1 mL) was added dropwise; the mixture was stirred under nitrogen atmosphere and in an ice water bath for 1 hour and then at room temperature for 10 hours. The reaction mixture was filtered, and the residue was washed three times with dichloromethane (20 mL). The filtrate phase was combined and evaporated in vacuo. The resultant residue was used directly for the next step.

| Animals
Male C57BL/6 mice (8-10 weeks) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). Animals were bred in the certified animal facility at the Institute of Radiation Medicine (IRM) of the Chinese Academy of Medical Sciences (CAMS).

| Irradiation and treatment
Mice were exposed to ionizing radiation by using a 137 Cs source following an Exposure Instrument Gammacell-40 (Atomic Energy of Canada Lim, Chalk River, ON, Canada) at a dose rate of 1.0 Gy/min. The mice were exposed to 7.5, 9.0 and 11.0 Gy TBI in the survival experiments (n = 10).
In the remaining experiments, animals were divided randomly into three groups (n = 5): (a) control; (b) IR + vehicle; (c) IR + XH-105 and received 9.0 Gy TBI. XH-105 was dissolved in 4% DMSO, 96% of PEG 400 was added after heating for a final concentration of 10 mg/mL. Individual mice in the IR + XH-105 group received a dose of 100 mg/kg XH-105 administered by gavage 1 hour before irradiation. Mice in the Control and IR groups were treated with vehicle similarly to the procedure described for the XH-105 treatment.

| Histological analysis
Three days after IR, mice were killed, and the small intestines were collected, stained with haematoxylin-eosin (H&E) and analysed under a microscope. For morphological analysis, six circular transverse sections were analysed per mouse in a blind manner from coded digital H&E-stained photographs to measure the villi length and crypt number by using ImageJ 1.37 software.

| Immunohistochemistry analysis
The 4-μm-thick sections of paraffin-embedded small intestine sections were dewaxed and rehydrated with citrate buffer. Then, the sections were boiled in 10 mM/L citrate buffer solution (pH 9.0) for antigen retrieval according to standard procedures. After antigen retrieval, the sections were incubated with serum for 1 hour at room temperature to block non-specific antigen-binding sites; then, the sections were incubated with anti-Lgr5 antibody (1:50 dilution; Abcam, Cambridge, MA), anti-Ki67 antibody (1:300 dilution; Novus, Littleton, CO), anti-lysozyme (1:800 dilution; Abcam) or anti-villi (1:800 dilution; Abcam) overnight at 4°C. Sections were then incubated in secondary antibody for 30 minutes at 37°C. Positive cells were detected using a DAB kit (Sigma Aldrich, St. Louis, MO). The images were captured, and positive staining was quantified objectively by the integrated performance primitives (IPP) software as described previously in a blinded fashion.

| TUNEL assay
The 3-μm-thick sections were treated according to the manufacturer's protocols (Roche, Mannheim, Germany). Sections were analysed by light microscopy.

| Isolation of intestinal crypt cells
The method of isolating intestinal crypts was described. 10,11 Briefly, after flushing with ice-cold PBS, the small intestines were chopped into small pieces and then placed into cold crypt chelating buffer for 30 minutes. After rinsing twice with ice-cold PBS, the fragments were resuspended in cold dissociation buffer. The solution was filtered through a 70 μm strainer to remove the villus fraction and to collect the crypt fraction. The crypt fraction was centrifuged to isolate the single cells.

| Western blot analysis
Protein was extracted from small intestinal crypt cells with icecold lysis buffer (Solarbio Science and Technology, Beijing, China).
Then, the membranes were incubated with a suitable horseradish peroxide-conjugated secondary antibody for 1-2 hours at room temperature. Finally, the chemiluminescent substrate is used to detect protein.

| Immunofluorescence analysis
The paraffin-embedded sections of the small intestine were subjected to antigen retrieval as described above and then washed thoroughly with PBS. The sections were blocked with 5% goat serum for 30 minutes at room temperature and incubated with anti- F I G U R E 1 Synthesis Route and Molecular Structure of XH-105. A, The 2,2-dimethylthiazolidine was reacted with bis(trichloromethyl) carbonate with triethylamine as base to prepare thiazolidine-3-carbonyl chloride. B, Thiazolidine-3-carbonyl chloride was coupled with Quercetin in the presence of triethylamine and 4-dimethylaminopyridine to afford the product XH-105

| Statistical analysis
Mice survival curves were analysed by the Kaplan-Meier method using GraphPad Prism 6.0 software for Mac. The data were expressed as the mean ± SD. ANOVA test was used to analyse differences among groups, and Student's t test was used to analyse the difference between two groups.

| Synthesis and characterization of XH-105
Based on the design concept, we designed and prepared XH-105, of which the molecular structure and synthetic routes are shown in Figure 1 Figure S1A and Figure S1B respectively. The results indicated that the new compound XH-105 was successfully prepared with the facile synthetic approach.

| XH-105 improves the survival rate of mice after TBI
To assess the protective effect of XH-105 on TBI-induced lethality in mice, we first observed the survival rates of mice after 7.5 Gy TBI ( Figure 2A). The 7.5 Gy TBI had a 100% mortality in the vehicle-treated group within 20 days compared with the 100 m/kg XH-105-treated group that had a 30% survival rate beyond 30 days. We treated the mice with three doses of XH-105 (50, 100 and 200 mg/kg), and then the mice were exposed to 9.0 Gy TBI ( Figure 2B). All doses improved the mice survival rate compared to that of the vehicle-treated group, and in particular, the 100 mg/kg dose dramatically improved the median survival. There was 80% mortality in vehicle-treated mice at 6 days after 11.0 Gy TBI ( Figure 2C), while 60% of mice survived in the 100 m/kg XH-105-treated group, suggesting that XH-105 may have a protective effect on radiation-induced intestinal injuries in mice. These results indicated that XH-105 effectively mitigates the TBI-induced lethality in mice.

| XH-105 reduces the damages of intestinal morphology after TBI
To determine the effect of XH-105 on radiation-induced intestinal injuries, the morphological changes of the small intestine in mice were evaluated. At 3.5 days after 9.0 Gy TBI, irradiated mice treated with vehicle showed significantly shorter villous length (P < 0.005) and fewer crypts (P < 0.005) than the control group ( Figure 3A,B). In comparison to vehicle-treated mice, XH-105-treated mice showed more survival crypts (P < 0.01) and an increased villous height (P < 0.01). The expression of villi + enterocytes was also affected by radiation ( Figure 3C

| XH-105 enhances Lgr5 + ISC survival and maintains the regeneration of intestinal cells after TBI
To evaluate the effect of XH-105 on the proliferation and differentiation ability of crypt cells, Lgr5 and Ki67 were identified by immunohistochemistry staining. Lgr5 + intestinal stem cells are indispensable for F I G U R E 2 XH-105 improves the survival of mice after 9.0 Gy total body irradiation (TBI). Kaplan-Meier survival analysis of mice exposed to 7.5, 9.0 or 11.0 Gy TBI. A, 100 mg/kg XH-105 treated mice have 30% survival beyond 30 d post 7.5 Gy TBI, compared with IR mice with 100% mortality within 20 d of radiation exposure (P < 0.05, n = 10 per group). B, three doses (50, 100, 200 mg/kg) of XH-105 treated mice show reduced mortality following lethal doses of TBI (9.0 Gy) within 13 d, compare with IR group 100% mortality within 5 d (P < 0.05, n = 10 per group). C, The Kaplan-Meier survival curve of vehicle and 100 mg/kg XH-105 treated mice (P < 0.05, n = 10 per group) after 11.0 Gy TBI. The data were expressed as the percent of surviving mice intestinal regeneration following radiation. 12 At 3.5 days after 9.0 Gy TBI, the number of Lgr5 + intestinal stem cells (ISCs) was significantly increased in the XH-105-treated mice than in the vehicle-treated group ( Figure 4A,B). Similarly, the number of Ki67 + positive cells in XH-105treated mice was also markedly greater than those in vehicle-treated mice after TBI ( Figure 4C,D). Paneth cells located at the bottom of intestinal crypts produce lysozymes. We also investigated the changes in Paneth cells 3.5 days after 9.0 Gy TBI ( Figure 4F). Similarly, we found that the number of lysozyme + positive cells ( Figure 4E) of XH-105treated group were greater than that of the vehicle-treated group. In summary, these data clearly indicated that XH-105 enhances the regenerative response of radiation-induced intestinal injuries by promoting the differentiation and proliferation of ISCs in the small intestine.

| XH-105 decreases apoptosis of the small intestine after TBI
To investigate the effect of XH-105 on apoptosis of the small intestines after IR, we evaluated apoptosis in the small intestine by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The results indicated that XH-105 had a protective role in preventing radiation-induced intestinal damage by suppressing apoptosis ( Figure 5A,B). To further validate our observations, we also analysed the rate of apoptosis by caspase-8 antibody and caspase-9 antibody immunofluorescence staining of small intestinal sections from mice after 9.0 Gy TBI. The immunofluorescence staining images showed expression of apoptosis-related proteins ( Figure 5C,E). The mice exposed to radiation increase the num-

| XH-105 attenuates DNA damage of the small intestine after TBI
To determine whether XH-105 treatment could reduce TBI-induced DNA damage, histone H2AX phosphorylation was analysed. As demonstrated in Figure 6, there was an increase in γH2AX in intestinal sections from the IR group compared with the control group.
XH-105 treatment decreased H2AX phosphorylation in intestinal sections compared with vehicle-treated mice after 9.0 Gy irradiation. The results indicated that XH-105 could reduce IR-induced DNA damage to the small intestine.

| XH-105 protects the small intestine against radiation-induced injury at least in part via the p53 signalling pathway
To investigate the mechanisms by which XH-105 protects against radiation-induced intestinal injuries, the expression of p53 was

| D ISCUSS I ON
The gastrointestinal (GI) system is one of the most sensitive organs to radiation, and radiation-induced intestinal injuries seriously affect the efficacy of tumour radiotherapy. 13 Therefore, development of an effective method and drug to mitigate the radiation-induced intestinal injuries needs to be explored. Many studies have reported that Chinese herbal medicines or extracts and other drugs may be able to reduce TBI-induced injuries in the brain, oesophagus and haematopoietic system of irradiated animals, [14][15][16][17][18] but the study of protective drugs in IR-induced intestinal injuries still needs to be improved. [19][20][21] In the present study, we observed that the new compound XH-105 had protective effects on radiation-induced intestinal injuries.
After receiving radiation, various degrees of villi blunting and fusion may occur; villous epithelial cell attenuation and hypertrophy and severe loss of crypts lead to the destruction of epithelial cell homeostasis and epithelial integrity. 22 Intestinal epithelial cells cannot easily maintain intestinal absorption and defense functions. 23 Our results demonstrated that the intestinal crypt-villus structure in XH-105-treated mice was well preserved after 9.0 Gy Many studies have shown that IR-induced tissue damage increases the amount of apoptotic cells. [31][32][33] Caspases are a family of genes important for maintaining homeostasis by regulating apoptosis and inflammation. 34 Caspases involved in apoptosis have been subclassified by their mechanism of action into initiator caspases (caspase-8 and -9) and executioner caspases (caspase-3, -6 and -7).
We found that XH-105 decreased the number of apoptotic cells in the small intestines by inhibiting the expression of caspase-8 and caspase-9.
Phosphorylated H2AX is a variant form of histone H2A, which has been widely used as a marker for DNA double-strand breaks. 35 In this study, we observed that the expression of γH2AX decreased in XH-105-treated mice after TBI compared with the vehicle-treated mice. Radiation induces DNA damage directly through ROS 36 and destroys the expression of proteins in cells, 37 activating p53. 32,33,36,[38][39][40] Radiation activates p53 in the GI epithelium, and p53-mediated apoptosis has been implicated in regulating the intestinal injuries. 22,41 It is well known that p53 activates genes that regulate cell cycle checkpoints, DNA damage and repair and apoptosis. 42 In addition, p53 can promote apoptosis through interactions with Bcl-2 family proteins, such as Bax, in the cytoplasm. 41,43 Studies reported that Bax −/− and Bak1 −/− mice reduced the apoptosis of epithelial cells after exposure to irradiation. 44,45 Treatment with XH-105 decreased the expression levels of Bax and p53. These data suggested that XH-105 mitigated DNA damage induced by IR and radiation-induced intestinal injuries via the p53-dependent apoptosis signalling pathway.
Our studies show protective effects of XH-105 against radiation-induced intestinal injury, which may attenuate radiation-induced intestinal damage via the p53 signalling pathway. XH-105 is a promising novel compound to be used as a radioprotector, and the mechanism of radiation enteritis and therapy methods need to be further investigated.
F I G U R E 7 XH-105 decreases the expression of p53 and Bax of the small intestine after 9.0 Gy total body irradiation (TBI). The small intestinal sections of control, IR + vehicle and IR + 105 mice were gained at 3.5 d after 9.0 Gy TBI. A, Representative immunofluorescence images for the expression of p53 of the small intestines (red, p53; blue, DAPI). B, Histogram showing Quantitative analysis of p53-positive cells per field of view. C, Western blot for Bax and tubulin in the intestinal crypts from non-IR mice, vehicle-treated mice and XH-105 treated mice at 3.5 d after 9.0 Gy TBI. The results are represented as mean ± SEM, n = 5 mice per group. ***P < 0.005. Scale bar: 10um