Application of methyl mercaptan oxidase Selenbp1 in preparation of medicine for treating radiation colitis and proctitis

Abstract

The invention discloses an application of methyl mercaptan oxidase Selenbp1 in preparation of a medicine for treating radiation colitis and proctitis. The research finds that the expression of Selenbp1 in a clinical specimen of radiation enteritis is obviously reduced; animal experiments and organoid culture experiments show that after the Selenbp1 is knocked out, intestinal epithelium is repaired to be impaired; in-vitro experiments find that the lack of Selenbp1 can cause the intestinal epithelium to generate a proinflammatory factor TNF-a and promote the polarization of macrophage M1; on a molecular mechanism, after the Selenbp1 enters a cell nucleus, SUMO modification of an intestinal epithelium key molecule Satb2 is promoted, and intestinal epithelium repair is facilitated. After the expression of the methyl mercaptan oxidase Selenbp1 is increased, the histological damage of radiation colitis can be alleviated. Therefore, the methyl mercaptan oxidase Selenbp1 can be used for preparing the medicine for treating radiation colitis and proctitis.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to application of methyl mercaptan oxidase Selenbp1 in preparing a drug for treating radiation colitis. Background Art

[0002] Ionizing radiation can cause serious damage to human tissues. In radiotherapy for pelvic and abdominal tumors, the colorectal mucosal epithelium is highly sensitive to ionizing radiation due to its rapid proliferation and renewal. Therefore, radiation-induced colorectal injury is a common complication of radiotherapy for cervical cancer and other diseases. Accidental radiation exposure can also cause gastrointestinal syndrome with symptoms such as nausea, diarrhea, and dehydration.

[0003] Inflammatory cell infiltration is common in other types of enteritis (such as infectious enteritis and inflammatory bowel disease), but there are fewer inflammatory cells in the colorectal mucosal tissue of radiation enteritis. Current enteritis treatment is mainly based on biological agents, which can relieve inflammation but fail to improve goblet cell defects. There are limited treatment options for patients with radiation colorectal inflammation. Patients need surgical treatment because the mucosa cannot be repaired and the intestinal wall is fibrotic, which significantly affects their quality of life.

[0004] In radiation enteritis, goblet cells are abnormally enlarged and their functions are impaired. Goblet cells are key cells for maintaining the homeostasis of the colorectal epithelium. In recent years, the heterogeneity of goblet cells has gradually been recognized, and they can be further divided into typical, atypical, proliferative, and inter-crypt goblet cells, which are distributed in the entire crypt layer, the upper half of the crypt, the lower half of the crypt, and between the crypts. However, the functions of each subtype of goblet cells are currently unclear. Identifying key molecules that maintain goblet cell homeostasis may be more helpful in promoting colorectal mucosal repair. Selenbp1 is expressed in atypical goblet cells in the upper half of the crypt. In 2018, Selenbp1 was identified as methyl mercaptan oxidase, and the functional loss of Selenbp1 is associated with the onset of halitosis. So far, there has been no study on the mechanism of Selenbp1 in enteritis.

[0005] It is generally believed that the role of methyl mercaptan oxidase Selenbp1 is to decompose methyl mercaptan, which is a metabolite of methionine. Methionine is the only essential sulfur-containing amino acid in the body. After entering the cell, it first forms S-adenosyl methionine (SAM). SAM acts as a methyl donor and participates in regulating gene transcription. SAM is then demethylated to form S-adenosyl homocysteine, and finally hydrolyzed into adenosine and homocysteine. About half of homocysteine ​​can be methylated to form methionine again, which is the "methionine cycle"; the other half of methionine is decomposed into methyl mercaptan and metabolized by the intestinal mucosa. Selenbp1 has two localizations: cytoplasmic and nuclear. It plays the role of a classic methyl mercaptan oxidase in the cytoplasm, and its function in the nucleus is still unclear. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide an application of methyl mercaptan oxidase Selenbp1 in the preparation of drugs for treating radiation-induced colitis. The present invention has found that methyl mercaptan oxidase Selenbp1 can reduce the histological damage of radiation-induced colitis; the lack of Selenbp1 will cause the intestinal epithelium to produce the proinflammatory factor TNF-a, promote the polarization of macrophage M1; after entering the cell nucleus, Selenbp1 promotes the SUMO modification of the key intestinal epithelial molecule Satb2 and helps intestinal epithelial repair. Therefore, methyl mercaptan oxidase Selenbp1 can be used to prepare drugs for treating radiation-induced colitis.

[0007] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0008] The invention provides an application of methyl mercaptan oxidase Selenbp1 in preparing a medicine for treating radiation colitis.

[0009] Starting from Satb2, a key molecule for colorectal homeostasis, the present invention identified that the number of goblet cells decreased significantly after Satb2 was lost, and the atypical goblet cell marker Selenbp1 was significantly reduced. Atypical goblet cells marked by Selenbp1 were significantly reduced in radiation-induced colitis. After Selenbp1 was reduced, intestinal epithelial cells produced more pro-inflammatory factor TNF-a, aggravating intestinal mucosal inflammation. The expression level of Selenbp1 is correlated with the repair of intestinal mucosal damage. Therefore, improving the homeostasis of atypical goblet cells marked by Selenbp1 may be one of the important ways to alleviate radiation-induced colitis.

[0010] This invention focuses on the reduction of atypical goblet cells after Satb2 deficiency, and experimentally simulates and identifies goblet cell subtypes involved in intestinal mucosal repair. Combined with human radiation colitis specimens, genetically modified mice, radiation intestinal damage repair models and molecular biology experiments, the effect of atypical goblet cell marker Selenbp1 in improving radiation colitis and its specific molecular mechanism are explored, in order to provide new options for the treatment of radiation colitis.

[0011] The methyl mercaptan oxidase Selenbp1 reduces histological damage in radiation-induced colitis.

[0012] The deficiency of the methyl mercaptan oxidase Selenbp1 causes the intestinal epithelium to produce the pro-inflammatory factor TNF-a and promote the polarization of macrophages M1.

[0013] The methyl mercaptan oxidase Selenbp1 directly enters the cell nucleus, promotes SUMO modification of the key intestinal epithelial molecule Satb2, and helps intestinal epithelial repair.

[0014] The methyl mercaptan oxidase Selenbp1 is used as the sole active ingredient to prepare a medicine for treating radiation colitis.

[0015] The methyl mercaptan oxidase Selenbp1 is used in combination with other drugs to prepare drugs for treating radiation colitis.

[0016] The medicine comprises methyl mercaptan oxidase Selenbp1 and pharmaceutically acceptable excipients.

[0017] The auxiliary materials include one or more of a wetting agent, an emulsifier, a diluent, an antioxidant, a preservative, a disintegrant or a binder.

[0018] The wetting agent is selected from at least one of water and ethanol.

[0019] The emulsifier is selected from at least one of Tweens, Spans, glycerol fatty acid esters, pectin, agar, sodium alginate or silicon dioxide.

[0020] The diluent is selected from at least one of starch, sugar, cellulose or inorganic salts.

[0021] The antioxidant is selected from at least one of ascorbic acid, sulfite, bisulfite, gallic acid and lipids thereof.

[0022] The preservative is selected from at least one of benzoic acid and its salts, sorbic acid and its salts or parabens.

[0023] The disintegrant is selected from at least one of starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked polyvinyl pyrrolidone.

[0024] The binder is selected from at least one of starch slurry, sodium carboxymethyl cellulose, povidone, hydroxypropyl cellulose, methyl cellulose or ethyl cellulose.

[0025] The dosage form of the medicine is capsule, granule, tablet, oral solution, injection or infusion.

[0026] The drug of the present invention can be administered in various known ways, such as oral administration, injection, and administration by inhalation spray. The drug of the present invention can be administered alone or in combination with other drugs. The oral composition can be in any oral acceptable dosage form, including but not limited to capsules, granules, tablets, suspensions and solutions.

[0027] Sterile injectable compositions can be formulated according to techniques known in the art using suitable dispersants or wetting agents and suspending agents. Pharmaceutically acceptable carriers and solvents that can be used include water, sodium chloride solution, and the like.

[0028] The actual dosage level of the active ingredient in the medicament of the present invention can be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response and is non-toxic to the patient for a particular patient, composition and mode of administration. The selected dosage level depends on a variety of factors, including the route of administration, time of administration, rate of excretion, duration of treatment, other drugs, compounds and / or materials used in combination with methyl mercaptan oxidase Selenbp1, age, sex, weight, general health and past medical history of the patient being treated, and similar factors well known in the medical field.

[0029] The present invention has found that low methionine helps retain atypical goblet cells marked by Selenbp1 and promotes intestinal mucosal repair. Methionine restriction helps repair colorectal mucosa after injury, because low methionine helps Selenbp1 enter the cell nucleus, and then binds to Satb2, a key molecule of colorectal epithelium, and promotes SUMO modification of Satb2. The low methionine refers to a methionine content of ≤0.12%.

[0030] Beneficial effects:

[0031] The methyl mercaptan oxidase Selenbp1 of the present invention can be used as an active ingredient for treating radiation colitis, opening up new uses for the methyl mercaptan oxidase Selenbp1 and providing a new option for preparing drugs for treating radiation colitis. The methyl mercaptan oxidase Selenbp1 of the present invention can also be used as a basis for developing drugs targeting Selenbp1 for the preparation of drugs for treating clinical radiation colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The changes of different subtypes of goblet cells in the colorectal mucosa after knocking out Satb2; Figure 1 A is the AB staining of the colorectal mucosa of Satb2IEC-KO mice, with goblet cells marked in blue; Figure 1 B is a statistical diagram of the expression differences of different subtypes of goblet cell markers in the RNA-seq data of the colorectal mucosa of Satb2 IEC-KO and control mice; Figure 1 C is a heat map of the correlation between Satb2 and differentially expressed goblet cell markers in the RNA-seq data of TCGA colorectal cancer adjacent tissues; Figure 1 D is the immunofluorescence staining of the typical goblet cell marker Tff3 and the atypical goblet cell Selenbp1 in the colorectal mucosa of Satb2 IEC-KO mice.

[0033] Figure 2 The expression of Selenbp1 in clinical radiation enteritis specimens; Figure 2A is the localization and semi-quantitative expression level of Selenbp1 in radiation-induced colitis and colorectal cancer adjacent tissues (control) detected by immunohistochemistry / immunofluorescence; Figure 2 B shows the localization and semi-quantitative expression levels of Satb2 and Selenbp1 in radiation enteritis specimens detected by immunohistochemistry. Figure 3 This is the result of the colorectal epithelial damage and repair model after intestinal epithelial-specific Selenbp1 knockout; Figure 3 A is a picture of the colorectal specimens of Selenbp1 IEC-KO mice and control mice before the irradiation damage and repair experiment, during the damage period - the third day after irradiation (3dpi), and during the repair period - the fifth day after irradiation (5dpi); Figure 3 B is a statistical graph of intestinal weight and intestinal length of the above mice's colorectum; Figure 3 C is the HE staining image of the above mice and the histological scoring image of crypt damage, inflammation depth and overall inflammation.

[0034] Figure 4 To reduce the expression changes of inflammatory factors in the intestinal epithelium and the effects on macrophages after Selenbp1; Figure 4 A: After knocking down Selenbp1 in SW480 and DLD, the expression levels of TNF-a and IL-10 were detected by RT-qPCR; Figure 4 B: After knocking down Selenbp1 in SW480 and DLD, the cells were co-cultured with M0 macrophages, and the expression levels of macrophage M1 polarization and M2 polarization markers were detected by RT-qPCR; Figure 4 C: After knocking down Selenbp1 in SW480 and DLD, the cells were co-cultured with M0 macrophages, and the expression levels of macrophage M1 polarization and M2 polarization markers were detected by immunoblotting; Figure 4 D is the organoid culture of the isolated colon crypts of Selenbp1 IEC-KO and control mice, showing the morphology of the organoids at 2 days after irradiation (2dpi), 6 days after irradiation (6dpi), and 10 days after irradiation (10dpi); Figure 4 E: Organoids were collected before irradiation and on the 10th day after irradiation to extract RNA, and the expression level of inflammatory factor TNF-a was detected by RT-qPCR.

[0035] Figure 5 The expression changes of atypical goblet cells marked by Selenbp1 and the repair of intestinal mucosal damage after methionine intervention; Figure 5 A is the HE staining image of the colorectal mucosa of mice after methionine restriction or supplementation diet intervention and the statistical graph of colorectal mucosal thickness; Figure 5B is the immunofluorescence detection of Muc2 and Selenbp1 in the colorectal mucosa of mice after methionine restriction or supplementation diet intervention, reflecting the number of atypical goblet cells; Figure 5 C is a HE staining image of the colorectum at the injury stage on day 3 after irradiation (3 dpi) in the irradiation damage repair model of mice with methionine restriction or supplementation diet intervention; Figure 5 D is the HE staining image of the colorectum at 5 days after irradiation (5 dpi) during the repair period, the immunofluorescence detection of Muc2 and Selenbp1, and the statistical graph of the number of atypical goblet cells.

[0036] Figure 6 Selenbp1 enters the nucleus when methionine is low and promotes SUMO modification of Satb2; Figure 6 A: The expression levels of SUMO2 and SUMO3 were detected by RT-qPCR in the colorectal mucosal tissue of Selenbp1 IEC-KO mice; Figure 6 B: Selenbp1 was knocked down in SW480 and DLD cells, and the expression levels of SUMO2 / 3, E3 ligase (ZNF451 and PIAS1), and Satb2 were detected by immunoblotting; Figure 6 C is the subcellular expression level of Selenbp1 detected by immunoblotting after nuclear-cytoplasmic separation after methionine intervention in the cell culture medium; Figure 6 D is the immunoprecipitation detection of the mutual binding ability of Satb2 with SUMO2 / 3, E3 ligase (ZNF451 and PIAS1) and Selenbp1 under methionine intervention; Figure 6 E: After knocking down Selenbp1 in SW480 and DLD cells and treating them with methionine-deficient medium for 24 hours, the expression levels of SUMO2 / 3, E3 ligase (ZNF451 and PIAS1) and Satb2 were detected by immunoblotting. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0038] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0040] Example 1 Intestinal epithelial-specific knockout of Satb2 (Satb2f / f ;Vil cre ) Establishment of mouse model

[0041] Intestinal epithelium-specific Satb2 knockout mice (Satb2 f / f ;Vil cre The protocol for establishing intestinal epithelium-specific Satb2 knockout (Satb2 IEC-KO) mice is as follows:

[0042] Satb2 + / - FLP mice were hybridized with FLP tool mice (both purchased from Guangzhou Saiye Company) to obtain FLP + / - Satb2 floxp / - mice, and then FLP + / - Satb2 floxp / - Mice mate with each other to obtain Satb2 floxp / floxp Mouse (referred to as Satb2 f / f ). Subsequently, based on the Cre-LoxP recombinase principle, Satb2 floxp / floxp After crossing mice with Villin-Cre mice (purchased from Guangzhou Saiye Company), heterozygous mice with Satb2 knockout in intestinal epithelium were obtained. floxp / + ;Vil Cre Mouse, Satb2 floxp / + ;Vil Cre Mice were mated with each other to obtain Satb2 f / f ;Vil Cre Mouse.

[0043] The mouse genotype was identified by taking the number of the cut toes. The DNA extraction reagent dBiozol was Bioflux, catalog number: BSC16M1. After extracting DNA from toe tissue, PCR reaction was performed to identify the mouse genotype. The enzyme (2xTaq Plus MasterMix ll) was Vazyme, catalog number: P213. Satb2 floxp / floxp Genotype and Villin cre The primer sequences and target gene band sizes for genotype identification are shown in Table 1. The primers are from Genewise Biotechnology. The PCR reaction system is shown in Table 2.

[0044] The PCR reaction program was set up as follows:

[0045] ①Satb2loxP / loxP reaction procedure: 94°C for 5 min; 94°C for 30 s, 56°C for 30 s, 72°C for 30 s, for a total of 35 cycles; 72°C for 10 min; maintain at 4°C.

[0046] ②Villin-Cre reaction program: 94℃3min; 94℃20s, 64℃30s, 72℃35s, 25 cycles in total; 72℃10min; maintain at 4℃.

[0047] Table 1 Primers for identifying mouse genotypes

[0048]

[0049] Table 2 PCR system for DNA identification

[0050]

[0051] Example 2 Intestinal epithelial-specific knockout of Selenbp1 (Selenbp1 f / f ;Vil creER ) Establishment of mouse model

[0052] CRISPR / Cas9 technology was used to anchor Loxp sequences on both sides of exons 2-7 of Selenbp1, and Selenbp1 was successfully obtained. f / f The mice were produced by Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0053] Selenbp1 f / f Mouse identification primers are:

[0054] F:5'-TCC AGC CCC AGT CAT TGC TCT TAA-3';

[0055] R:5'-CAG CTG TGT GTC CTT AGG CTT CTT TC-3'.

[0056] The wild-type fragment size is 279 bp; the fragment size after flox anchoring is 384 bp.

[0057] Thanks to Selenbp1 f / f ;Vil cre The mice died after birth. f / f Mice and Villin creER Mice were caged to obtain Selenbp1 f / f ;Vil creER Mice, subsequent experiments used Selenbp1 f / f ;Vil creER Mice as a Selenbp1 intestinal epithelium-specific knockout mouse model.

[0058] Tamoxifen powder was dissolved in corn oil at a concentration of 20 mg / mL, and then placed on a mixer at 37°C overnight to fully dissolve, and then divided into 1.5 mL EP tubes and stored at -20°C to avoid repeated freezing and thawing. 100 μL of 20 mg / mL tamoxifen corn oil solution was injected intraperitoneally for 5 consecutive days, once a day. One month after the injection, Selenbp1 was knocked out in the intestinal epithelium.

[0059] Example 3 Knockout of Satb2 leads to a significant decrease in atypical goblet cells marked by Selenbp1

[0060] The Satb2 IEC-KO mice constructed in Example 1 and the control Satb2 floxp / floxp AB staining of mice showed that knockout of Satb2 in the intestinal epithelium led to a significant decrease in the number of goblet cells in the colorectal mucosa ( Figure 1 A).

[0061] Using CASAVA software, Satb2 f / f and Satb2 f / f ;Vil cre The RNA-seq data of mouse colorectal tissue (the same as the previously published article Journal of Crohn's and Colitis, 2021) were converted into raw reads, quality controlled, aligned using TopHat2 software (v2.1.1), and mapped to the mouse reference genome (mm10). The alignment file was input into Cufflinks software (v2.1.1) for gene assembly, and the differentially expressed genes were analyzed by the R software DESeq2 package. The screening criteria were |log2 Fold Change|>1 and P value <0.05. Finally, the pheatmap package in R software was used to visualize the differentially expressed genes. The markers of different subtypes of goblet cells are from Science, 2021, doi:10.1126 / science.abb1590. The analysis results showed that after Satb2 knockout, the expression of atypical and inter-crypt goblet cell markers was significantly downregulated in the four goblet cells (typical goblet cells, atypical goblet cells, proliferating goblet cells, and inter-crypt goblet cells) ( Figure 1 B).

[0062] The normalized expression data of RNA-seqV2 in the TCGA (The Cancer Genome Atlas) colorectal cancer (COADREAD) dataset were downloaded, and the RNA-seq data of paracancerous tissues were extracted for analysis. The Pearson correlation coefficient between different genes was calculated using the corr package in R software, and the correlation results were visualized by corrplot. The results showed that Satb2 was highly positively correlated with the atypical goblet cell marker Selenbp1 (r=0.69, P<0.001) ( Figure 1 C).

[0063] Co-localization and semi-quantitative analysis of the typical goblet cell marker Tff3 and the atypical goblet cell marker Selenbp1 in Satb2 IEC-KO and control mice were performed. The specific experimental plan is as follows:

[0064] After dewaxing, hydration, antigen repair and removal of endogenous peroxidase, paraffin tissue sections were circled with a special histochemical pen, and 50 μl of immunofluorescence blocking solution (PBS containing 5% normal goat serum (Aibixin, Catalog No.: abs933) and 0.3% tritonX-100 (Solebo, Catalog No.: p1080)) was added to the tissue, placed in a humidified box, and incubated at 37°C for 1 hour. After removing the blocking solution, 50 μl of an antibody mixture in an antibody diluent containing 1% BSA (BBI, Catalog No.: A600332) and 0.3% Triton X-100 was added, including Tff3 (Thermo Fisher Scientific, 14-4758-82, 1:1000) and Selenbp1 (Abcam, ab272692, 1:400), and incubated at 4°C overnight. Then, PBS was used to wash 3 times, 3 minutes each time. Add 50 μl of a mixture of goat anti-mouse IgG (CST, Catalog No.: 4408) and goat anti-rabbit IgG (CST, Catalog No.: 4413) at a dilution ratio of 1:1000, and incubate at 37°C in the dark for 1 hour. Add 50 μl of DAPI working solution (1:1000) diluted with PBS, incubate at 37°C in the dark for 20 minutes, and wash 3 times with PBS, 3 minutes each time. Finally, add PBS containing 50% glycerol to seal the slide, store in the dark, and observe using a fluorescence microscope (Leica, model: DMi8).

[0065] Immunofluorescence co-localization assay showed that after Satb2 knockout, the expression of the classic goblet cell marker Tff3 was not significantly different, while the atypical goblet cell marker Selenbp1 was significantly decreased ( Figure 1 D) Thus, the atypical goblet cell marker Selenbp1 is significantly reduced after Satb2 knockdown.

[0066] Example 4: Selenbp1 expression is reduced in chronic radiation enteritis

[0067] The experimental steps of immunohistochemistry (IHC) are as follows: dewaxing and hydration of sections, high-pressure antigen repair, blocking of endogenous peroxidase, serum blocking, primary antibody incubation (antibodies and dilution ratios are the same as those described in Example 3 for immunofluorescence), biotin secondary antibody (Zhongshan Jinqiao, catalog number: PV-6001) incubation, DAB color development, hematoxylin counterstaining, ethanol gradient dehydration, xylene transparent and neutral resin sealing. Among them, antigen repair adopts high temperature and high pressure repair method in 0.01M sodium citrate, pH 6.0 buffer. The antibody of Selenbp1 is the same as that in Example 3, and the other reagents and operation steps are carried out according to the kit (Zhongshan Jinqiao, catalog number: SP-9001). The experimental steps of immunofluorescence are the same as those described in Example 3. Immunofluorescence has higher specificity than immunohistochemistry, but due to the quenching of fluorescence, image information is difficult to preserve for a long time. Immunohistochemical sections are preserved for a long time after scanning, but the specificity is slightly lower. Therefore, immunohistochemistry or immunofluorescence detection is selected according to different experimental purposes.

[0068] Immunohistochemistry / immunofluorescence was used to detect the localization and semi-quantitative expression level of Selenbp1 in chronic radiation colitis specimens (from the Department of Pathology of the Sixth Affiliated Hospital of Sun Yat-sen University) and colorectal cancer adjacent tissue specimens (control specimens, from the Department of Pathology of Nanjing Drum Tower Hospital). In normal colorectal mucosa, Selenbp1 is mainly expressed in the cytoplasm and nucleus of the upper half of the crypts, and Selenbp1 expression is significantly reduced in chronic radiation colitis ( Figure 2 A). In chronic radiation enteritis, partial crypt rupture and loss of Satb2 expression were observed, while Selenbp1 expression was significantly decreased ( Figure 2 B).

[0069] Example 5 Knockout of Selenbp1 leads to decreased repair ability of colorectal mucosa after injury

[0070] As shown in Example 2, we constructed an intestinal epithelium-specific knockout of Selenbp1 (Selenbp1 f / f ;Vil creER ) mice, and then used a radiation intestinal injury repair model to elucidate the role of Selenbp1 in colorectal mucosal injury repair.

[0071] Construction of radiation-induced intestinal injury and repair model:

[0072] 6-8 week old mice were selected, and 10-12 week old mice were subjected to a radiation intestinal injury repair experiment 4 weeks after intraperitoneal injection of tamoxifen (same as Example 2). The mice were placed in a plastic mouse holder and placed in a biological X-ray radiator (Radsource, RS-2000Pro). The radiation conditions were: source-skin distance 40 cm, absorbed dose rate 1.225 Gy / min, whole body radiation 392 seconds, and single total radiation dose of 8 Gy. According to the experimental needs, the mice were sacrificed at different time points, and the colorectal tissue was taken for subsequent experiments.

[0073] The histological scoring scheme for inflammation in mouse colorectal HE sections is shown in Table 3.

[0074] Table 3 Scoring criteria for inflammation after intestinal mucosal injury

[0075]

[0076] Intestinal epithelium-specific Selenbp1 knockout (Selenbp1 f / f ;Vil creER The ratio of colorectal weight to intestinal length of mice before and after irradiation was higher than that of control Selenbp1 f / f The colorectal length of the mice group was shorter and the weight was heavier, suggesting that knocking out Selenbp1 can lead to intestinal mucosal edema, severe inflammatory response, and intestinal epithelial repair disorder ( Figure 3 A. Figure 3 B) Histological analysis showed that Selenbp1 f / f ;Vil creER The colorectal epithelium of mice was damaged, and more inflammatory cells infiltrated the lamina propria. During the irradiation injury period, both the Selenbp1 knockout group and the control group showed crypt damage, intestinal epithelial shedding and mucus overflow, and an increase in inflammatory cells in the lamina propria. During the repair period, the intestinal epithelial cell nuclei of the control group mice were significantly enlarged, showing a state of repair, while the Selenbp1 f / f ;Vil creER The mice still showed obvious intestinal mucosal damage. The mice in the Selenbp1 knockout group showed higher crypt damage, inflammation depth and overall inflammatory histological scores during the repair period ( Figure 3 C) These results indicate that Selenbp1 knockout leads to a significant decrease in the repair capacity of colorectal mucosa.

[0077] Example 6: Decreased Selenbp1 promotes intestinal epithelial cells to secrete the pro-inflammatory factor TNF-α and induces macrophage M1 polarization

[0078] After DLD and SW480 cell lines were transfected with Selenbp1 siRNA (purchased from Guangzhou Ruibo Company), cell RNA was collected and fluorescent quantitative PCR experiments were performed. The reverse transcription system is shown in Table 4, the primer sequence is shown in Table 5, the PCR reaction system is shown in Table 6, and the program settings are shown in Table 7.

[0079] Table 4 Reverse transcription system

[0080]

[0081]

[0082] Table 5 Primers for fluorescence quantitative PCR detection of inflammatory factors

[0083]

[0084] Table 6 Fluorescence quantitative PCR system

[0085]

[0086] Table 7 Real-time fluorescence quantitative PCR program

[0087]

[0088] Our experiments found that after knocking down Selenbp1 in DLD and SW480 cells, the expression of the pro-inflammatory factor TNF-α increased significantly, while the anti-inflammatory factor IL-10 did not change significantly ( Figure 4 A). Selenbp1-knockdown DLD or SW480 cells were co-cultured with THP-1 macrophages. Quantitative PCR and protein analysis showed that after Selenbp1 knockdown, the macrophage M1 markers CD64 and CD86 were significantly increased at both RNA and protein levels, while the M2 markers CD163 and CD206 were significantly decreased ( Figure 4 B. Figure 4 C).

[0089] Example 7 Low methionine helps preserve atypical goblet cells marked by Selenbp1 and promotes intestinal mucosal repair

[0090] (1) Colon organoid culture protocol

[0091] 2-week-old Selenbp1 f / f ;Vil creER Four weeks after tamoxifen treatment, mice were injected with 20 mg / mL tamoxifen corn oil solution at 75 mg / kg body weight, intraperitoneally once a day for 5 consecutive days. f / fColon tissue was extracted from control mice for organoid culture. After the mouse abdomen was disinfected, the abdomen was opened, colon tissue was cut and placed in a culture dish containing pre-cooled PBS. PBS was drawn up with a syringe to rinse the intestinal segment, the mesenteric tissue was removed, the intestinal tube was cut longitudinally, and the residual contents were gently rinsed. The cleaned intestinal tube was cut into 2mm fragments, placed in a 15ml centrifuge tube containing 10ml pre-cooled DPBS, and gently blown with a pipette. After standing for 30 seconds, the sediment was precipitated by gravity, the supernatant was sucked off, and clean DPBS was added for washing. Repeat 15 times until the supernatant was clear and transparent. 0.5% EDTA DPBS solution was added and digested on ice for 20 minutes. After digestion, the tissue fragments were resuspended in DPBS containing 0.1% BSA, blown 5 times and then stood for 30 seconds. Take 10μl of supernatant and observe the crypts under a microscope. If the crypts are abundant, filter through a 70μm filter to obtain the first fraction. This step was repeated 4 times to obtain 4 fractions. After centrifugation (300g, 4°C, 5 minutes), the supernatant was removed and pre-cooled DMEM / F12 was added to resuspend the crypts, transferred to a 15 ml centrifuge tube, and centrifuged again (200g, 4°C, 3 minutes). The supernatant was removed, 10 ml of pre-cooled DMEM / F12 was added to resuspend the crypts, and counted.

[0092] Take the organoid growth medium and return to room temperature, add penicillin / streptomycin. Thaw Matrigel and preheat the 48-well plate. Obtain the crypt fraction according to the above steps, resuspend it with DMEM / F12 and mix it with Matrigel. Use a pipette to take 30μl of the mixture, add it to the preheated 48-well plate to form a dome, and place it in the incubator for 10 minutes to allow the Matrigel to solidify. Then add 200μl of organoid growth medium to each well, add PBS to other wells that are not inoculated with domes, and place them in the incubator for culture. Observe and photograph the morphological changes of organoids in the Selenbp1 knockout and control groups.

[0093] (2) Injury and repair experiments of colon organoid culture

[0094] In the organoid culture of Selenbp1 knockout and control colon crypts, methionine-deficient medium (RPMI-1640 medium, Sigma-aldrich, catalog number: R7513) was used for 24 hours, and the colon organoids were irradiated. The 48-well plate inoculated with colon organoids was placed in a biological X-ray irradiator, with a source plate distance of 40 cm, an irradiation dose rate of 1.225 Gy / min, irradiation for 98 seconds, and a total irradiation dose of 2 Gy. After irradiation, the morphological changes of the organoids were continuously observed, the survival rate (number of surviving organoids / total number of organoids) was recorded, and the number of buds of each surviving organoid was counted.

[0095] Ten days after irradiation, organoids were collected and RNA was further extracted. The RT-qPCR experimental protocol was the same as described in Example 6.

[0096] like Figure 4 As shown in D, Selenbp1 knockout colon crypts were isolated for organoid culture and irradiated with 2Gy. The Selenbp1 knockout group showed a significant decrease in organoid budding. The inflammatory factor TNF-α was significantly increased in Selenbp1 knockout organoids before and after irradiation ( Figure 4 E). Therefore, the loss of Selenbp1 leads to increased secretion of the pro-inflammatory factor TNF-α by the intestinal epithelium, aggravated intestinal epithelial damage, and impaired repair.

[0097] (3) Methionine dietary intervention model

[0098] Taking C57BL / 6 mice as an example, the mice were purchased from Shanghai Slake Experimental Animal Co., Ltd., and the 4-week-old mice were divided into two groups, which were given a methionine-restricted diet and a methionine-supplemented diet, respectively. Methionine is an essential amino acid, and the normal diet contains 0.6-0.8% (w / w) methionine. The methionine content in the methionine-restricted diet (Research Diet: A11051301B) is ≤0.12%. The methionine-supplemented diet group was given a normal diet and drinking water with a concentration of 0.5% L-methionine. In the end, the methionine intake of the methionine-supplemented diet group was about 10 times that of the restricted group. After 8 weeks of intervention, colon tissue was taken from some mice, and some mice underwent radiation intestinal damage repair experiments.

[0099] After 8 weeks of methionine restriction or supplementation in C57BL / 6 mice, the colorectal mucosa of the methionine restriction (MR) group was thinner than that of the methionine supplementation (MS) group ( Figure 5 A), which is consistent with the role of methionine as an essential amino acid for the human body. Although the colorectal mucosa of mice in the MR group was thinner and the number of goblet cells was less, the immunofluorescence colocalization of Muc2 and Selenbp1 showed that there was no significant difference in the number of atypical goblet cells between the two groups of mice ( Figure 5 B), indicating that the proportion of atypical goblet cells in the methionine-restricted diet group was higher. When mice were irradiated after methionine diet intervention, a large amount of mucus overflow was observed in the MS group mice during the injury period ( Figure 5 C); in the repair period, the intestinal mucosa did not recover well, goblet cell rupture was still observed, and the number of atypical goblet cells was significantly lower than that in the MR group ( Figure 5 D) Therefore, atypical goblet cells marked by Selenbp1 are affected by methionine dietary intake. After methionine restriction, atypical goblet cells increase, which is beneficial to the repair of intestinal mucosa after injury.

[0100] Example 8 Selenbp1 promotes SUMO modification of Satb2 by interacting with Satb2

[0101] We are at Selenbp1 f / f ;Vil creER In the mouse model, RT-qPCR detected the expression level of SUMO2 / 3 in colorectal tissue. The primer sequences of SUMO2 and SUMO3 are shown in Table 8. The detection method is the same as Example 6.

[0102] Table 8 Primers for fluorescence quantitative PCR detection of inflammatory factors

[0103]

[0104] After Selenbp1 knockout, SUMO3 expression was significantly decreased ( Figure 6 A). After knockdown of Selenbp1 in DLD and SW480 cells, the expressions of SUMO2 / 3, E3 ligase ZNF451 and PIAS1 were significantly reduced ( Figure 6 B) Therefore, upon depletion of Selenbp1, SUMO2 / 3 modification of Satb2 is reduced.

[0105] After 24 hours of replacing the culture medium of DLD and SW480 cells with low methionine culture medium (Sigma-aldrich, catalog number: R7513), the cytoplasmic and nuclear proteins of the cell lines were extracted using a nuclear protein and cytoplasmic protein extraction kit. Selenbp1 was then detected by immunoblotting, and the experimental method was the same as before. We found that Selenbp1 was expressed in both the cytoplasm and the nucleus. Through nuclear-cytoplasmic separation experiments, we found that the expression of Selenbp1 in the nucleus increased when treated with low methionine ( Figure 6 C).

[0106] Next, we further explored the interaction between Selenbp1 and Satb2. Immunoprecipitation experiments were used to detect the binding of Selenbp1 to Satb2, and the binding of SUMO-modified E3 ligase or Sumo2 / 3 to Satb2. Taking DLD cells as an example, after being treated with methionine-deficient medium or normal 1640 medium for 24 hours, the cells were lysed and centrifuged at 12000g for 10 minutes at 4℃; after centrifugation, 5% of the supernatant was aspirated, and an equal volume of 2× Loading buffer was added as a total protein control (Input), and boiled at 108℃ for 8 minutes. 90% of the supernatant was mixed with 10μl protein A / G agarose and incubated on a shaker at 4℃ for 30 minutes to remove non-specifically bound proteins. After centrifugation, the supernatant was transferred to a new centrifuge tube, 10μl of Satb2 antibody was added and incubated on a shaker at 4℃ for 2 hours, and then 30μl protein A / G agarose was added and incubated on a shaker at 4℃ for 1 hour. Then centrifuge at 2000rpm for 5 minutes, carefully discard the supernatant, wash the beads at least 3 times with ice-cold cell lysis buffer, 5 minutes each time, and centrifuge at 2000rpm for 5 minutes to collect the beads. Finally, add an appropriate amount of 2×SDS loading buffer and denature at 108℃ for 8 minutes. Use SDS-PAGE gel for electrophoresis, and after antibody incubation, develop and detect the required protein. In addition to adding protease inhibitors, 10mM N-maleimide (N-ethylmaleimide, NEM) should also be added to the immunoprecipitation solution, which is a deSUMOylation inhibitor. Satb2 protein was immunoprecipitated with Satb2 antibody, and the SUMOylation level of Satb2 was detected using Sumo2 / 3 antibody.

[0107] Immunoprecipitation experiments showed that Selenbp1 binds to Satb2, and their interaction is enhanced under low methionine conditions, accompanied by enhanced SUMO2 / 3 modification of Satb2. At the same time, it was observed that the E3 ligase ZNF451 and PIAS1 bind to Satb2 more ( Figure 6 D). Further findings revealed that knocking down Selenbp1 reduced the SUMO2 / 3 modification of Satb2. Under low methionine conditions, Selenbp1 expression was restored and the SUMO2 / 3 modification of Satb2 was also significantly restored ( Figure 6 E).

[0108] In summary, Selenbp1 can enter the cell nucleus and interact with Satb2, and the SUMO2 / 3 modification of Satb2 is enhanced, which is beneficial to the repair of intestinal epithelium.

[0109] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. Application of methyl mercaptan oxidase Selenbp1 in the preparation of drugs for the treatment of radiation-induced colitis.

2. The use according to claim 1, characterized in that: The methyl mercaptan oxidase Selenbp1 reduces histological damage in radiation-induced colitis.

3. The use according to claim 1, characterized in that: The deficiency of the methyl mercaptan oxidase Selenbp1 causes the intestinal epithelium to produce the pro-inflammatory factor TNF-a and promote the polarization of macrophages M1.

4. The use according to claim 1, characterized in that: The methyl mercaptan oxidase Selenbp1 directly enters the cell nucleus, promotes SUMO modification of the key intestinal epithelial molecule Satb2, and helps intestinal epithelial repair.

5. The use according to claim 1, characterized in that: The methyl mercaptan oxidase Selenbp1 is used as the sole active ingredient to prepare a medicine for treating radiation colitis.

6. The use according to claim 1, characterized in that: The methyl mercaptan oxidase Selenbp1 is used in combination with other drugs to prepare drugs for treating radiation colitis.

7. The use according to claim 1, characterized in that: The medicine comprises methyl mercaptan oxidase Selenbp1 and pharmaceutically acceptable excipients.

8. The use according to claim 1, characterized in that: Low methionine helps preserve Selenbp1-marked atypical goblet cells and promotes intestinal mucosal repair.

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