Application of detection reagent of interleukin-22 in preparation of product for predicting radioactive intestinal injury sensitivity

Abstract

The invention discloses application of a detection reagent of interleukin-22 in preparation of a product for predicting radioactive intestinal injury sensitivity, and belongs to the technical field of medical detection. The expression level of IL-22 before radiation is related to the severity of intestinal injury after radiation, mice with high expression of IL-22 show more serious intestinal injury symptoms after radiation, IL-22 can be used as a biomarker for predicting sensitivity of radioactive intestinal injury, the risk of intestinal injury can be preliminarily evaluated by detecting the level of IL-22 before individual radiation, and the risk of intestinal injury can be further evaluated by detecting the level of IL-22 before individual radiation. Early warning is realized; the IL-22 can increase the radiosensitivity of intestinal tissues, the kit provided by the invention adopts a modern detection technology, such as enzyme-linked immunosorbent assay (ELISA), performs high-sensitivity and high-specificity detection on the IL-22, is simple and convenient to operate, and supports multiple sample types. The kit has important scientific research value and application prospect, and is expected to play a key role in prevention and treatment of radioactive intestinal injury along with the development of nuclear energy technology and radiotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection. Specifically, it relates to the application of a detection reagent for interleukin-22 in the preparation of a product for predicting the sensitivity of radiation-induced intestinal injury. Background Art

[0002] The intestine is a rapidly regenerating tissue organ and is extremely sensitive to radiation. Radiation-induced intestinal injury is not only a serious consequence of nuclear radiation disasters but also an important complication in the radiotherapy of pelvic and abdominal tumors, seriously affecting the treatment effect and the quality of life of patients. Therefore, identifying sensitive biomarkers for radiation-induced intestinal injury is of great significance for preventing the occurrence of radiation-induced intestinal injury and optimizing clinical radiotherapy.

[0003] The prevention and treatment of radiation-induced intestinal injury are complex multi-faceted pathophysiological conditions, affected by multiple factors, and remain a challenge in radiation medicine. Therefore, identifying sensitive biomarkers for radiation-induced intestinal injury is of great significance for preventing the occurrence of radiation-induced intestinal injury and optimizing clinical radiotherapy. Citrulline is a metabolic by-product of small intestinal cells and has been identified as a physiological biomarker for measuring and tracking radiation-induced small intestinal epithelial injury in intestinal tissue. Claudin-3 is a tight junction protein whose expression decreases in intestinal tissue after radiation, indicating its potential as a biomarker for evaluating radiation-induced intestinal barrier injury. MicroRNAs are also related to predicting the severity of radiation-induced intestinal injury. It has been reported that miR-122-5p increases radiosensitivity and exacerbates radiation-induced rectal injury. Changes in the intestinal flora and its metabolites provide a new direction for predicting radiation injury. For example, the intestinal bacterium Erysipelatocostridium and its related metabolite peptidosteroid A are related to the development of grade 2 radiation-induced intestinal injury and can be used as diagnostic biomarkers for such diseases. In addition, certain microorganisms and metabolites with radiation protection effects have been found to contribute to the survival of mice after radiation exposure. In addition to the above physiological indicators, reference indicators of changes in intestinal tissue morphology can also predict radiation-induced intestinal injury. The parameters of dynamic contrast-enhanced MRI (DCE-MRI) show moderate diagnostic performance in predicting severe acute radiation-induced rectal injury. Currently, most studies have focused on diagnostic or prognostic biomarkers for radiation-induced intestinal injury, and there is still a lack of sensitive biomarkers for radiation-induced intestinal injury. Studying sensitive biomarkers for radiation-induced intestinal injury in normal individuals helps to evaluate the susceptibility of individuals to radiation before exposure.

[0004] Interleukin-22 (IL-22) belongs to the IL-10 cytokine family and is mainly produced by T cells and certain myeloid and non-hematopoietic cells, playing a key role in immune regulation, inflammatory responses, and tissue repair. Since its discovery, the biological functions of IL-22 in various tissues such as the intestine have been extensively analyzed. Research has shown that IL-22 promotes intestinal epithelial regeneration by stimulating the proliferation of Lgr5+ intestinal stem cells; in addition, IL-22 is also related to the maintenance of the intestinal barrier, including promoting the production of mucin and antimicrobial peptides (AMPs). Although the important role of IL-22 in intestinal injury has been widely recognized, there is no report on the research of IL-22 in existing techniques for predicting radiation-induced intestinal injury, and there is no patent or literature reporting interleukin-22 as a sensitive biomarker for radiation-induced intestinal injury. Summary of the Invention

[0005] Aiming at the problems in the prior art such as the lack of effective sensitive biomarkers for radiation-induced intestinal injury and the inability to effectively predict the occurrence of individual radiation-induced intestinal injury, the present invention aims to provide the application of interleukin-22 detection reagents in the preparation of products for predicting the sensitivity of radiation-induced intestinal injury.

[0006] To achieve the above object, the present invention is implemented by the following technical solutions: The present invention provides the application of interleukin-22 detection reagents in the preparation of products for predicting the sensitivity of radiation-induced intestinal injury.

[0007] The expression level of the interleukin-22 is directly proportional to the sensitivity of radiation-induced intestinal injury.

[0008] The product includes a chip, a kit, or a nucleic acid membrane strip capable of detecting the expression level of interleukin-22.

[0009] Further, the chip includes a gene chip and a protein chip. The gene chip includes oligonucleotide probes for interleukin-22 genes used to detect the transcriptional level of interleukin-22 genes, and the protein chip includes specific binders for interleukin-22 proteins; the kit includes a gene detection kit and a protein detection kit. The gene detection kit includes reagents or chips for detecting the transcriptional level of interleukin-22 genes, and the protein detection kit includes reagents or chips for detecting the expression level of interleukin-22 proteins.

[0010] The sample detected by the product is any one of the blood, serum, plasma, feces, or intestinal tissue specimens of a subject with radiation-induced intestinal injury.

[0011] The detection reagent includes reagents for detecting the expression product of interleukin-22 by nucleic acid hybridization technology, nucleic acid amplification technology, protein immunization technology, sequencing technology, chromatography technology, and mass spectrometry technology.

[0012] Furthermore, the detection reagent is a reagent for detecting the expression product of interleukin-22 by protein immunization technology.

[0013] Even further, the detection reagent is selected from a probe that specifically recognizes interleukin-22; or a primer that specifically amplifies interleukin-22; or a binder that specifically binds to the protein encoded by the interleukin-22 gene.

[0014] The detection reagent evaluates the responsiveness of a subject to IL-22 neutralizing antibody before receiving radiation treatment by detecting the expression product of interleukin-22, and predicts its sensitivity to radiation-induced intestinal injury.

[0015] The present invention also provides a kit for predicting the sensitivity to radiation-induced intestinal injury, which includes a reagent for detecting interleukin-22.

[0016] The kit includes reagents for detecting the expression level of the interleukin-22 by RT-PCR method, qRT-PCR method, biochip detection method, Southern blotting method, in situ hybridization method, immunoblotting method, and enzyme-linked immunosorbent assay.

[0017] The present invention provides a composition, which includes an interleukin-22 neutralizing antibody and an interleukin-22 inhibitor.

[0018] Compared with the prior art, the present invention achieves the following technical effects: The present invention provides the application of a detection reagent for interleukin-22 in the preparation of a product for predicting the sensitivity to radiation-induced intestinal injury, providing a new biomarker for the early prediction and risk assessment of radiation-induced intestinal injury. It not only expands the application of IL-22, but also provides new ideas for the clinical management and treatment strategies of radiation-induced intestinal injury; by exploring its application in the preparation of a product for predicting the sensitivity to radiation-induced intestinal injury, the severity of radiation-induced intestinal injury is related to the expression level of IL-22 before irradiation, providing a new prediction tool for the clinic. By detecting the IL-22 level before individual irradiation, the intestinal injury risk under the radiation environment can be preliminarily evaluated to achieve early warning, which has important preventive significance for people who need to receive radiation treatment or may be exposed to the radiation environment.

[0019] Even lower, it reveals the mechanism of action of IL-22 in radiation-induced intestinal injury, that is, high expression of IL-22 can increase the radiosensitivity of intestinal tissue, making it more vulnerable to radiation damage, providing a new perspective for understanding the mechanism of radiation-induced intestinal injury, contributing to the in-depth study of the pathophysiological process of radiation injury, and providing a theoretical basis for the development of new treatment strategies; modern detection techniques such as enzyme-linked immunosorbent assay (ELISA) can be applied to the detection of IL-22, accurately and reliably measuring the level of IL-22 in individuals, providing a solid foundation for clinical prediction and evaluation.

[0020] Furthermore, by detecting the expression product of IL-22 to evaluate the sensitivity of the subject to IL-22 neutralizing antibody radiation, it provides a new means for evaluating the patient's response to the treatment plan and contributes to the formulation of personalized treatment plans.

[0021] The kit for predicting the sensitivity of radiation-induced intestinal injury provided by the present invention realizes the early diagnosis and risk assessment of intestinal injury after radiation by quantitatively detecting the expression level of IL-22 in the subject, providing a scientific basis for predicting the sensitivity of radiation-induced intestinal injury; with advantages such as simple operation and support for multiple sample types, it has important scientific research value and application prospects.

[0022] The composition provided by the present invention contains both IL-22 neutralizing antibody and IL-22 inhibitor. By inhibiting the activity of IL-22 and neutralizing its effects, the biological effects of IL-22 can be more effectively controlled, and the occurrence of radiation-induced intestinal injury can be more effectively alleviated or prevented, providing new strategies and methods for the treatment of diseases related to abnormal expression of IL-22. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Mice with high levels of IL-22 showed more severe intestinal injury after abdominal irradiation (TAI); among them, A is the expression level of serum IL-22 in mice before irradiation; B is the daily body weight change of mice after irradiation; C is the correlation between the body weight change of mice on the 5th day after irradiation and the serum IL-22 expression level before irradiation; D is the colon length of mice on the 5th day after irradiation, E is the statistical chart of the colon length of mice on the 5th day after irradiation, F is the HE staining map of small intestine tissue, G is the statistical chart of intestinal villus length, H is the statistical chart of crypt depth, and I is the statistical chart of the number of surviving crypts; Figure 2Effect of high-level IL-22 on the proliferation and apoptosis of crypt cells after abdominal irradiation; among them, A is the ki67-positive crypt cell diagram on the 5th day after irradiation, scale bar: 100 μm; B is the statistical chart of ki67-positive crypt cells on the 5th day after irradiation, C is the PCNA-positive crypt cell staining diagram on the 5th day after irradiation, scale bar: 100 μm; D is the statistical chart of PCNA-positive crypt cells on the 5th day after irradiation, E is the TUNEL staining diagram on the 5th day after irradiation, scale bar: 10 μm; F is the statistical chart of TUNEL-positive crypt cells on the 5th day after irradiation, the data represent three independent experiments, n = 3-8 in each group, the data are Mean±SEM, *p<0.05 and ***p<0.001; Figure 3 Effect of administering IL-22 before irradiation on intestinal injury in mice; among them, A is the experimental flow chart, B is the daily body weight change curve of mice after irradiation, C is the survival curve of each group of mice (n = 8 in each group), D is the colon length diagram of mice on the 5th day after irradiation, E is the statistical chart of colon length of mice on the 5th day after irradiation; F is the HE staining diagram of ki67-positive crypt cells and PCNA-positive crypt cells in intestinal tissue on the 5th day after irradiation, G is the statistical chart of villus length, H is the statistical chart of crypt depth, I is the statistical chart of the number of surviving crypts, scale bar: 200 μm, J is the ki67-positive crypt cells on the 5th day after irradiation, scale bar: 200 μm; K is the statistical chart of PCNA-positive crypt cells on the 5th day after irradiation, the data represent three independent experiments, n = 3-8 in each group; the data are Mean±SEM, ns, no significant difference, *p<0.05, **p<0.01 and ***p<0.001; Figure 4 Concentrations of IL-22 in serum and intestine before TAI; among them, A is the concentration of IL-22 in serum, B is the concentration of intestinal IL-22, n = 6-8 in each group, the data are mean ± SEM, **p<0.01 and ****p<0.0001; Figure 5The severity of IL-22-induced intestinal injury is related to the apoptosis and DNA damage of crypt cells. Among them, A is the HE staining image of intestinal tissue on the 1st day after irradiation, scale bar: 200 μm; B is the statistical analysis of ki67-positive cells in crypts on the 1st day after irradiation, scale bar: 20 μm; C is the statistical analysis of PCNA-positive cells in crypts on the 1st day after irradiation, scale bar: 20 μm; D is the TUNEL staining image of intestinal tissue on the 1st day after irradiation, scale bar: 100 μm; E is the statistical graph of TUNEL staining of intestinal tissue on the 1st day after irradiation; F is the γ-H2AX staining of intestinal tissue on the 1st day after irradiation, scale bar: 100 μm; G is the statistical graph of γ-H2AX staining of intestinal tissue on the 1st day after irradiation; H is the relative expression level of Lgr5 in intestinal tissue on the 1st day after irradiation; I is the relative expression level of olmf4 mRNA in intestinal tissue on the 1st day after irradiation; J is ki67-positive cells in crypts on the 0th day before irradiation, scale bar: 20 mm; K is the statistical graph of ki67-positive cells in crypts on the 0th day before irradiation; L is the relative mRNA expression level of Zfp652 in mouse intestinal tissue on the 0th day before irradiation; M is the relative mRNA expression level of ki67 in mouse intestinal tissue on the 0th day before irradiation; N is the relative mRNA expression levels of Lgr5 and olmf4 (intestinal stem cell markers) in mouse intestinal tissue on the 0th day before irradiation; The data represent three independent experiments, n = 3 - 8 in each group, the data are expressed as Mean±SEM, ns, no significant difference, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001; Figure 6 IL-22 pretreatment reduces the survival rate of intestinal organoids after radiation exposure. Among them, A shows the effects of different concentrations of IL-22 (0, 0.1, 0.5, 1 ng / mL) on the growth of mouse intestinal organoids; B is the statistical analysis of the number and area of intestinal organoids with different concentrations of IL-22 on the 0th day before irradiation; C is the statistical analysis of the number and area of intestinal organoids with different concentrations of IL-22 on the 5th day after irradiation; D is the representative image of organoids on the 5th day after 6 Gy radiation and IL-22 pretreatment; E is the percentage of organoid survival. The data are Mean±SEM, ns, no significant difference, *p<0.05, **p<0.01; Figure 7Figure showing the effects of pretreatment with an IL-22 neutralizing antibody on radiation-induced physiological and pathological changes in mice. Among them, A is pretreatment with an IL-22 neutralizing antibody (50 μg / mouse / i.p.); B is the body weight change curve of different treatments; C is the picture of the colon of mice with different treatments; D is the statistical analysis of the colon length of different treatments; E is the small intestine tissue section (HE staining) of different treatments; F is the statistical analysis of the villus length of different treatments, G is the statistical analysis of the villus width of different treatments, H is the statistical analysis of the survival rate of different treatments, I is the immunohistochemical staining and quantitative analysis of Ki67 of different treatments, J is the immunohistochemical staining and quantitative analysis of PCNA of different treatments. The data are Mean±SEM, ns, no significant difference, *p<0.05, **p<0.01. Detailed implementation manners

[0024] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0025] For those experimental steps or conditions not specified in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not specified by the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0026] 1. Experimental materials Healthy adult male and female C57BL / 6J mice aged 6 - 8 weeks (20 - 22 g) were used. Under a 12 h dark / light cycle, the mice had free access to water and food, were raised under normal light and dark cycles, and the bedding was changed regularly to ensure a clean breeding environment. All experimental operations in this study were carried out in accordance with the relevant regulations of the Experimental Animal Center and Management Committee of the Air Force Medical University.

[0027] 2. Experimental procedures According to the IL-22 content in the mouse serum, the mice were divided into low IL-22 or high IL-22 groups. For the IL-22 protein treatment experiment, the mice were randomly divided into the following groups: (1) control group, (2) IL-22 group, (3) 10 Gy irradiation group, and (4) 10 Gy + IL-22 group. Before abdominal irradiation (TAI), IL-22 was intraperitoneally injected continuously for three days at a dose of 4 μg / mouse.

[0028] Irradiation and IL-22 and IL-22 antibody treatment: Mice were subjected to single abdominal irradiation of 10 Gy (1 Gy / min) using an RS-2000 XE X-ray irradiator (Rad Source Technologies, Alpharetta, GA, USA). For IL-22 treatment, 4 μg / mouse of IL-22 (HY-P72793, Med Chem Express, USA) was intraperitoneally injected into the mice 50 hours, 26 hours, and 2 hours before abdominal irradiation. For IL-22 antibody treatment, intraperitoneal injection of IL-22 monoclonal antibody (IL22JOP) (16-7222-82, eBiosciences, USA) and rat IgG2a-kappa isotype control antibody (eBR2a) (16-4321-38, eBiosciences, USA) was started two days before abdominal irradiation at a dose of 50 μg / mouse once a day.

[0029] Histological analysis: Mouse intestinal tissues were fixed with 10% neutral formalin, embedded in paraffin, and cut into 4-μm thick cross-sections. Subsequently, the samples were stained with H&E. The villus length and crypt depth were observed and measured under a microscope as histological and pathological references. For immunohistochemistry, the samples were dewaxed and subjected to antigen retrieval treatment. After washing with PBS, the tissues were blocked with an endogenous peroxidase blocker (PV-9001-1, ZSGB-BIO, China) for 30 minutes. The primary antibodies used in this invention were anti-ki67 antibody (1:200, ab16667, Abcam, UK) and anti-PCNA antibody (1:1000, ab18197, Abcam, UK). The secondary antibodies included goat anti-rabbit IgG (PV-9001-3, ZSGB-BIO, Peking, China). Images were captured using a microscope (Nikon, C2+, Japan) and a slide scanning microscope (Olympus, VS200, Japan), and analyzed using Image J software (NIH, Image J, USA).

[0030] For immunofluorescence staining, the tissue sections were first hydrated and then subjected to antigen retrieval by boiling in citrate buffer (pH 6.0) for 20 minutes. After standing at room temperature for 30 minutes, the tissues were treated with 1xPBS + 1% Triton X-100 to increase their permeability. 3% BSA solution was dropped onto the tissues and incubated for 30 minutes to prevent non-specific binding. Subsequently, the tissues were incubated overnight at 4°C with anti-γH2AX antibody (1:200, GB111841, Servicebio, China). The next day, after rinsing the samples, they were incubated with secondary antibody donkey anti-rabbit IgG(H+L)-Alexa fluor 488 (1:500, A-21206, Invitrogen, US) at room temperature for 1 hour. After staining the cell nuclei with DAPI (P0131, Beyotime, China), observation and photography were performed using a fluorescence microscope (EVOS M5000, ThermoFisher).

[0031] Enzyme-linked immunosorbent assay (ELISA): Mouse serum and supernatant of intestinal tissue homogenate were collected, and the content of IL-22 was detected using a mouse IL-22 ELISA kit (E-MSEL-M0040, Elabscience Biotechnology, China) according to the instructions of the kit.

[0032] TUNEL apoptosis detection: A TUNEL detection kit (T2195, Solarbio, China) was used to identify the presence of apoptotic cells. After hydrating the tissue sections, they were permeabilized with PBS containing 20 μg / mL proteinase K, and then the tissue samples were incubated with the TUNEL reaction mixture at 37°C for 1 hour. After counterstaining the cell nuclei with DAPI, the counting of TUNEL-positive cells was observed using a fluorescence microscope.

[0033] Intestinal organoid culture: Intestinal organoids were cultured with reference to the literature ("Establishment of intestinal organoid cultures modeling injury-associated epithelial regeneration", M. Qu, L. Xiong, Cell Research, 31(3) (2021) 259-271). The small intestine tissue was rinsed with PBS, longitudinally incised, and then divided into ~0.5 cm segments. The tissue fragments were resuspended in 10 mL of cell dissociation reagent (#100-0485, stemcell, Canadian), and dissociated on a shaker (200 rpm) at room temperature for 20 minutes. Then the tissue was rinsed with PBS, gently pipetted 5 times in 10 mL of PBS, and then passed through a 70 μm (BD biosciences) cell strainer. The above operations were repeated twice. The obtained cell suspension was centrifuged at 290 RCF for 5 minutes, the cells were collected and resuspended in 5 mL of PBS, and centrifuged at 200 RCF for 5 minutes. The obtained cells were resuspended in Matrigel Matrix (#356231, Corning, US) matrix gel and plated in a 24-well plate at a density of 50 μL / 200 crypts / quantity. The cell culture plate containing the matrix gel was placed in a cell culture incubator. After 30 minutes, 500 μL of mouse intestinal organoid growth medium (#06005, stemcell, Canadian) was added, and the cells were cultured in a 5% carbon dioxide incubator at 37°C. The medium was changed every three days.

[0034] With reference to the literature ("Interleukin 22 Expands Transit-Amplifying Cells While Depleting Lgr5(+) Stem Cells via Inhibition of Wnt and Notch Signaling", J.-M. Zha, Cellular and Molecular Gastroenterology and Hepatology, 7(2) (2019) 255-274), the organoids were treated with IL-22 at 0, 0.1, 0.5, 1 ng / mL for 24 hours before irradiation, and the medium without IL-22 was changed 4 hours after irradiation.

[0035] Quantitative real-time RT-PCR (RT-qPCR) Total RNA was isolated from intestinal tissues using Trizol reagent (Invitrogen, USA), reverse transcribed into cDNA by PrimerScript RT Master Mix (#RR036A, Takara, Japan), and finally, qPCR was performed using TB Green Master Reagents (#RR820A, Takara, Japan). GAPDH was used as an internal reference gene, and the relative gene expression levels were calculated by 2-ΔΔCT (Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, K.J. Livak, T.D. Schmittgen, Methods, 25(4) (2001) 402-408). The detailed primer sequences are shown in Table 1.

[0036] Table 1: Primer sequences used in real-time PCR

[0037] 3. Experimental results All data in the experimental analysis were generated using GraphPad Prism 9.2 software (San Diego, CA, USA). Unpaired t-tests or one-way ANOVA were used. All values are expressed as Mean±SD. P<0.05 was considered statistically significant.

[0038] 3.1 Effect of IL-22 on radiation-induced intestinal injury The expression level of IL-22 in the serum of normal mice was detected by ELISA. Mice were divided into a low IL-22 group and a high IL-22 group according to the IL-22 level. The mice were subjected to 10 Gy of abdominal irradiation (TAI). The degree of body weight loss in the mice was monitored. The colon length, intestinal villus length, number of surviving crypts, and crypt depth were evaluated by histopathological examination. The experimental results are shown in the appendix. Figures 1 - 2 as shown.

[0039] ELISA results showed that the expression levels of IL-22 varied among individuals ( Figure 1 A). Mice were divided into a low IL-22 group and a high IL-22 group according to the IL-22 level in the serum ( Figure 1 A). After 10 Gy of TAI, it was found that the degree of body weight loss in the high IL-22 group of mice was more obvious ( Figure 1 B), and the degree of body weight loss in mice was positively correlated with the IL-22 expression level before irradiation (Figure 1 C). In addition, the histopathological results also showed that the colon length and intestinal villus length of the mice in the high IL-22 group were significantly shortened ( Figure 1 D, E), with fewer surviving crypts and no significant difference in crypt depth ( Figure 1 F-I). The above results indicate that the expression level of IL-22 in serum before irradiation is closely related to the severity of radiation-induced intestinal injury in mice. Mice with high IL-22 levels showed more severe intestinal injury after TAI. The proliferation of intestinal tissue was evaluated by detecting ki67 and PCNA (cell proliferation indicators). The results showed that the high IL-22 group showed fewer ki67- and PCNA-positive crypt cells on the 5th day after TAI (Figure 2A-D). In addition, the apoptosis of crypt cells was detected by TUNEL staining, and it was found that the number of TUNEL-positive crypt cells in the high IL-22 group increased sharply compared with the low IL-22 group ( Figure 2 E, F). The above results indicate that high levels of IL-22 reduce the proliferative ability of intestinal crypt cells after irradiation and induce apoptosis of intestinal crypt cells.

[0040] 3.2 Effect of IL-22 on the sensitivity of individual radiation-induced intestinal injury To further verify the effect of IL-22 on the sensitivity of individual radiation-induced intestinal injury, mice were treated with IL-22 protein for three consecutive days before 10 Gy TAI. After administration of IL-22, the IL-22 levels in the serum and intestinal tissue of the mice were detected, and the body weight loss, survival rate, colon length, villus length, crypt depth, and intestinal crypt survival rate of the mice were monitored after TAI. The number of ki67- and PCNA-positive crypts in the intestinal tissue was detected by immunohistochemistry. The specific results are shown in the appendix Figures 3 - 4 as follows.

[0041] Before 10 Gy TAI, mice were treated with IL-22 protein for three consecutive days ( Figure 3 A). After administration of IL-22, the IL-22 levels in the serum and intestinal tissue of the mice increased significantly ( Figure 4 ). Administration of IL-22 protein before irradiation exacerbated the body weight loss of the mice on the 5th day after TAI ( Figure 3 B), and reduced the survival rate of the mice, with a survival time of only 5 days ( Figure 3 C). In addition, compared with the irradiated mice without pretreatment, the colon length ( Figure 3 D, E), villus length ( Figure 3 F, G), and crypt depth ( Figure 3 F, H) of the mice pretreated with IL-22 were significantly shortened. In addition, compared with the irradiation group, the intestinal crypt survival rate of the mice pretreated with IL-22 was reduced ( Figure 3In the intestinal tissue of group F and I, the number of ki67- and PCNA-positive crypts also decreased significantly ( Figure 3 F, J, K). These results indicate that high levels of IL-22 before irradiation lead to more severe radiation-induced intestinal injury, further verifying the previously observed positive correlation between IL-22 levels and the severity of radiation-induced intestinal injury.

[0042] 3.3 Correlation between the severity of radiation-induced intestinal injury induced by high levels of IL-22 and crypt cell apoptosis and DNA damage Ki67 and PCNA were detected on the first day after irradiation, and it was found that the number of ki67- and PCNA-positive cells in the intestinal crypts of mice pre-treated with IL-22 decreased significantly ( Figure 5 A-C). TUNEL staining showed that compared with the 10 Gy group, there were more TUNEL-positive cells in the group pre-treated with IL-22 ( Figure 5 D, E). Meanwhile, the expression of γ-H2AX in crypt cells was consistent with TUNEL staining ( Figure 5 F, G). The mRNA expression of the intestinal stem cell (ISCs) markers Lgr5 and Olmf4 was detected on the first day after irradiation, and it was found that these markers were significantly decreased in mice pre-treated with IL-22 ( Figure 5 H, I). To detect the effect of IL-22 on intestinal epithelial regeneration, the status of TA cells and ISCs in the crypts was determined, and it was found that on day 0 before TAI, the number of ki67-positive cells in the intestinal crypts increased ( Figure 5 J, K), while the mRNA expression of the transit amplifying cell marker Zfp652 ( Figure 5 L) and the proliferation marker ki67 ( Figure 5 M) also increased significantly. In addition, the mRNA expression levels of Lgr5 and Olmf4 also increased ( Figure 5 N).

[0043] The above results indicate that IL-22 enhances the proliferative ability of intestinal epithelial cells before radiation, making crypt cells more prone to apoptosis and DNA damage after radiation, thus increasing the radiation sensitivity of intestinal tissue. IL-22 plays a dual role in the pathological process of radiation-induced intestinal injury: on the one hand, it promotes intestinal epithelial proliferation before radiation, and on the other hand, it increases cell damage and apoptosis after radiation. This provides a new perspective for understanding the mechanism of radiation-induced intestinal injury and may provide potential targets for the prevention and treatment of radiation-induced intestinal injury.

[0044] 3.4 IL-22 reduces the survival rate of intestinal organoids after radiation exposure To clarify the effect of IL-22 on intestinal organoids after radiation, intestinal organoids were treated with different concentrations of IL-22 protein (0, 0.1, 0.5, 1 ng / mL) for 24 hours before radiation. See AppendixFigure 6 . The results showed that there were no significant differences in the number and size of organoids between the non-irradiated group after 24 hours ( Figure 6 A, B). However, on the 5th day, the organoids treated with 0.5 ng / ml IL-22 began to enlarge ( Figure 6 A, C). After exposure to 6 Gy of X-rays, IL-22 pretreatment significantly reduced the surviving number of organoids ( Figure 6 D, E). These results indicate that administration of IL-22 before irradiation can lead to a decrease in the viability of crypt cells after radiation exposure.

[0045] 3.5 Antagonizing IL-22 before radiation exposure alleviates the severity of radiation-induced intestinal injury To reverse-verify the feasibility of IL-22 as a predictive biomarker for radiosensitivity, pre-irradiation antagonistic treatment was performed using an IL-22 neutralizing antibody (Anti-IL-22) in a mouse model. An irradiation group (only receiving radiation treatment) and an IL-22 neutralizing antibody pretreatment group (administered IL-22 neutralizing antibody before receiving radiation) were established. The mice in the pretreatment group were injected with the IL-22 neutralizing antibody and then subjected to radiation treatment. The effects on the physiological and pathological changes of the irradiated mice were observed, including changes in mouse body weight, colon length, small intestine pathology (observed by HE staining), and small intestine proliferation (observed by Ki67 and PCNA immunohistochemical staining). The specific experiments and results are shown in the appendix Figure 7 as shown. From the appendix Figure 7 data, it can be seen that the degree of body weight reduction in the mice of the IL-22 neutralizing antibody pretreatment group after irradiation was smaller than that in the irradiation group ( Figure 7 B), indicating that the pretreatment alleviated the negative impact of radiation on mouse body weight; at the same time, the colon length was longer than that of the mice in the irradiation group ( Figure 7 C, D), suggesting that the IL-22 neutralizing antibody helped protect the colon from radiation damage; further, HE staining was used to observe the small intestine pathology. The results showed that the villus length of the small intestine in the neutralizing antibody pretreatment group was longer than that in the radiation group ( Figure 7 E, F), the crypt depth was shorter than that in the radiation group ( Figure 7 E, G), and the number of surviving crypts was more than that in the radiation group ( Figure 7 E, H), indicating that IL-22 neutralizing antibody pretreatment helped alleviate the small intestine pathological damage caused by radiation; Ki67 and PCNA immunohistochemical staining were used to observe small intestine proliferation. The results showed that the number of Ki67- and PCNA-positive crypts in the small intestine of the mice in the IL-22 neutralizing antibody pretreatment group was more than that in the radiation group ( Figure 7E, I, J), indicating that pretreatment with the IL-22 neutralizing antibody promoted the proliferation and repair of small intestinal cells. Based on the above results, pretreatment with the IL-22 neutralizing antibody could alleviate the weight loss of irradiated mice, protect the colon length, reduce the pathological damage of the small intestine, and promote the proliferation and repair of small intestinal cells, supporting the feasibility of IL-22 as a predictive marker for radiosensitivity and suggesting the potential mechanism of action of IL-22 in radiation injury. This experiment provided strong evidence for the feasibility of IL-22 as a predictive marker for radiosensitivity.

[0046] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. Use of an interleukin-22 detection reagent in the preparation of a product for predicting the sensitivity to radiation-induced intestinal injury.

2. The application according to claim 1, wherein The expression level of interleukin-22 is directly proportional to the sensitivity to radiation-induced intestinal injury.

3. The application according to claim 1, wherein The product includes a chip, a kit, or a nucleic acid membrane strip capable of detecting the expression level of interleukin-22.

4. The application according to claim 3, wherein The sample detected by the product is any one of blood, serum, plasma, feces, or intestinal tissue specimens of a subject with radiation-induced intestinal injury.

5. The application according to claim 1, characterized in that The detection reagent includes a reagent for detecting the expression product of interleukin-22 by nucleic acid hybridization technology, nucleic acid amplification technology, protein immunization technology, sequencing technology, chromatography technology, or mass spectrometry technology.

6. The application according to claim 5, wherein The detection reagent is selected from a probe that specifically recognizes interleukin-22; or a primer that specifically amplifies interleukin-22; or a binder that specifically binds to the protein encoded by the interleukin-22 gene.

7. The application according to claim 1, characterized in that The detection reagent evaluates the responsiveness of a subject to an IL-22 neutralizing antibody before radiation treatment by detecting the expression product of interleukin-22, and predicts the sensitivity of the subject to radiation-induced intestinal injury.

8. A kit for predicting the sensitivity of radiation-induced intestinal injury, characterized in that, It includes a reagent for detecting interleukin-22.

9. The kit for predicting the sensitivity of radiation-induced intestinal injury according to claim 8, wherein, The kit includes reagents for detecting the expression level of interleukin-22 by RT-PCR method, qRT-PCR method, biochip detection method, DNA blotting method, in situ hybridization method, immunoblotting method, or enzyme-linked immunosorbent assay.

10. A composition, characterized in that, The composition includes an interleukin-22 neutralizing antibody and an interleukin-22 inhibitor.

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