Currently, radiation therapy is widely used in a variety of cancer treatments. The small intestine is one of the most sensitive organs to ionizing radiation (IR) in the human body. High doses of ionizing radiation induce acute damage to epithelial cells of the intestines and produce death within 10 days reflecting toxicity to the gastrointestinal (GI) tract [
Natural antioxidant agents and aminothiol compounds have been intensively investigated for the radiation protection application [
In this study, we take the natural antioxidation agent quercetin which belongs to flavonoid family derived from plants as a lead compound and modify the molecular structure. At the same time, we also try to combine the aminothiol analogue and the natural antioxidation agent together with different linkers in order to retain the efficacy of aminothiol and the safety property of natural antioxidation agent, respectively. The quercetin group and aminothiol group can modulate pharmacokinetic (PK) profile mutually. Finally, we designed and synthesized the compound TZ (XH-103) (Scheme
In this study, to define the effect of XH-103 on intestinal repair and regeneration following radiation injury, we used a mouse model of radiation-induced intestinal damage by exposure to 9.0 Gy total body irradiation (TBI). We found that the XH-103 could improve the survival rate of mice and intestinal epithelium cells (IECs). We also found that the crypt-villous structure injuries of the small intestines and the apoptosis of IECs induced by TBI were mitigated by XH-103. And XH-103 could protect the proliferation and differentiation of intestinal stem cells (ISCs). Here, we evaluated the possibility and mechanisms of the radiation protective effects of XH-103 on radiation-induced intestinal injury.
To a solution of bis(trichloromethyl) carbonate (4.79 g, 53.82 mmol) (Energy Chemical, Shanghai, China) in anhydrous tetrahydrofuran (35 mL) were added thiazolidine (4 g, 44.94 mmol) (Energy Chemical, Shanghai, China) in parts and triethylamine (8.1 mL) dropwise, the mixture was stirred under nitrogen atmosphere and in ice water bath for 1 h and then at room temperature for 10 h. The reaction mixture was filtered, and the residue was washed with dichloromethane (10 mL) for three times. The filtrate phase was combined and evaporated in vacuo. The resultant residue was used directly for the next step.
To a solution of 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one (1.6 g, 5.3 mmol) (Shuya Chemical Science and Technology, China) in DMF (30 mL) were added triethylamine (7 mL), 4-dimethylaminopyridine (192 mg, 1.57 mol), and thiazolidine-3-carbonyl chloride, the mixture was stirred under 0°C for 1 h and at room temperature for 10 h. The reaction mixture was poured into MeOH/H2O/DMSO 8/1.5/0.5
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 in the Institute of Radiation Medicine (IRM) of Chinese Academy of Medical Sciences (CAMS).
All experimental procedures were carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Institute of Radiation Medicine (IRM), Chinese Academy of Medical Sciences (CAMS) (Permit Number 2017053). The animals were cared for in accordance with the dictates of the National Animal Welfare Law of China.
The mice were exposed to ionizing radiation by using a 137Cs 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 9.0 Gy and 11.0 Gy TBI in the survival experiments (
In the remaining experiments, the animals were divided randomly into three groups (
At three days after IR, the mice were sacrificed, and the small intestines were collected and stained with hematoxylin-eosin (H&E) and analyzed under a microscope. For morphological analysis, six circular transverse sections were analyzed per mice in a blind manner from coded digital HE-stained photographs to measure the villi length and crypt number by using the ImageJ 1.37 software.
The 4
The 3
The method of isolating intestinal crypts was described [
Protein was extracted from small intestinal crypt cells with ice-cold lysis buffer (Solarbio Science and Technology, Beijing, China). Protein concentration was quantified using the bicinchoninic acid protein assay kit (Beyotime, Shanghai, China), and equal amounts of protein were resolved by SDS-PAGE gel. The blocked membrane was incubated using anti-Bax antibody (1 : 1000 dilution, Ruiying Bio, Suzhou, China) and antibodies against
The paraffin-embedded sections of the small intestine were subjected to antigen retrieval as described above and then washing thoroughly with PBS. The sections were blocked with 5% goat serum for 30 min at room temperature and then incubated with anti-caspase-8 (1 : 100 dilution, CST, MA, USA), anti-caspase-9 (1 : 1000 dilution, CST, MA, USA), anti-
Mice survival curves were analyzed by Kaplan-Meier method using GraphPad Prism 6.0 software for Mac. The data were expressed as mean ± standard deviation (SD). Analysis of variance (ANOVA) test was used to analyze differences among the groups, and
Based on design concept, we designed and prepared the compound XH-103, of which the synthetic routes were shown in Figure
Synthesis and structure of TZ (XH-103). (a) The thiazolidine 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 TZ.
To determine the protective effect of XH-103 on mice exposure to radiation, we first observed the survival rate of mice after 9.0 Gy TBI (Figure
XH-103 enhances the survival rate of mice after TBI. Kaplan-Meier survival analysis of mice exposed to 9.0 Gy or 11.0 Gy TBI. (a) Three doses of XH-103-treated mice show reduced mortality following lethal doses of TBI (9.0 Gy) within 13 days, compared with IR + vehicle group 100% mortality within 5 days (
To determine the effect of XH-103 on radiation-induced intestinal injuries, the morphological changes of mouse small intestine are shown in Figure
XH-103 protects the intestinal morphology of mice after TBI. (a) Representative images showing the structure in cross sections of the small intestine with H&E stain. (b) Histogram showing the number of crypts. (c) Immunohistochemistry images showing the expression of villi. (d) Histogram demonstrating villus length in intestinal section from the control group, NS-treated group, and XH103-treated group. The results are represented as mean ± SEM,
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 intestinal regeneration following radiation [
XH-103 increases the proliferation and differentiation of the Lgr5+ small intestine after TBI. The small intestinal sections were analyzed by IHC. (a) Photomicrograph of Lgr5 immunostaining section of the control, IR + vehicle, and IR+ 103 group. (b) Histogram showing Lgr5-positive cells that were quantified in five crypts per section. (c) Immunostaining images showing quantitative analysis of Ki67 expression of intestinal crypts. (d) Histogram demonstrating Ki67-positive cells that were counted in five crypts per section. The results are represented as mean ± SEM,
To analyze the role of XH-103 in small intestine apoptosis after TBI, we evaluated the apoptosis by TUNEL assay (Figures
XH-103 reduces the apoptosis of the small intestine after TBI. (a) Apoptosis was assayed by TUNEL staining. (b) The number of TUNEL-positive cells was quantified per field. The paraffin-embedded sections of the small intestine were analyzed by immunofluorescence. (c) Representative DAPI and caspase-8-staining images of the small intestine (red, caspase-8; blue, DAPI). (d) Caspase-8-positive cells in a single field of view were quantified. (e) Photomicrograph of caspase-9-staining images of the small intestine (red, caspase-9; blue, DAPI). (f) Bar graph showing quantitative analysis of caspase-9-positive cells per field of view. The results are represented as mean ± SEM,
To determine whether XH-103 treatment could reduce TBI-induced DNA damage, we analyzed the expression of histone H2AX phosphorylation, which has been widely used as a marker for DNA double-strand breaks (DSBs). As demonstrated in Figure
XH-103 attenuates DNA damage of mice after TBI. The small intestines of control mice, vehicle-treated mice, and XH-103-treated mice were obtained at 3 d after 9.0 Gy TBI. (a) Representative immunofluorescence images for the expression of
To investigate the mechanisms on how XH-103 protects against the radiation-induced intestinal injuries, we determine the expression of p53 by immunofluorescence analysis (Figure
XH-103 decreases the expression of p53 and Bax after TBI. The small intestinal sections of the control, IR + vehicle, and IR+ 103 mice were gained at 3 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-103-treated mice at 3 d after 9.0 Gy TBI. The results are represented as mean ± SEM,
The development of the effective method and drug to mitigate the radiation-induced intestinal injuries is an important area in cancer therapy, nuclear accident, and terrorism. Many studies have reported that Chinese herbal medicine or extracts may reduce TBI-induced injuries in the brain, esophagus, and hematopoietic system of irradiated animals [
In this study, we observe that XH-103 improved the survival rate of mice exposed to the lethal dose TBI, which indicates that XH-103 could protect the mice from irradiation. Under physiological conditions, epithelial homeostasis is maintained by proliferative cells in crypts, and the small intestinal crypt cells are particularly sensitive to IR due to their high proliferative rate [
Apoptosis is programmed cell death that involves the controlled breakdown of intracellular components. Many studies have shown that IR induced tissue damage, such as small intestinal injuries, with increasing apoptosis cells [
Radiation induces DNA damages [
It is well known that p53 activates genes that regulate cell cycle checkpoints, DNA damage and repair, and apoptosis [
Our studies synthesize a new compound XH-103 and show protective effects of XH-103 against radiation-induced intestinal injury. The results also suggest that XH-103 may attenuate radiation-induced intestinal damage via the p53 pathway. However, XH-103 is a novel compound that needs further optimization.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.
Deguan Li and Hongqi Tian conceived and designed the experiments. Ying Cheng and Hongqi Tian designed and synthesized the new compound XH-103. Deguan Li, Yinping Dong, Qinlian Hou, and Jing Wu carried out the follow-up experiments, analyzed the data, and interpreted the results. Deguan Li and Yinping Dong contributed to data analysis and manuscript preparation. Yinping Dong and Ying Cheng contributed equally to this work and are co-first author.
This study was supported by the National Natural Science Foundation of China (no. 81673106, 81601411); the Natural Science Foundation of Tianjin (15JCZDJC35200); Tianjin Health Industry Key Research Projects (15KG138); the innovation team funding (1649) from the Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, and Fundamental Research Fund for CAMS&PUMC (2016ZX310199, 2016ZX310070); and CAMS Innovation Fund for Medical Sciences (CIFMS, 2017-I2M-3-019) from the Chinese Academy of Medical Sciences and Peking Union Medical College.
Figure S1a: 1H NMR spectra of TZ. 1H NMR (400 MHz, DMSO-d6)