Oligopeptide for relieving radioactive intestinal injury as well as preparation method and application of oligopeptide

Abstract

The invention provides an oligopeptide for relieving radioactive intestinal injury. The amino acid sequence of the oligopeptide is SEQ ID NO: 1 ATXYXRTGR; wherein each X is independently selected from any one of natural amino acid residues. The invention also provides a preparation method and application of the oligopeptide. The oligopeptide has the effect of relieving intestinal tissue damage caused by ionizing radiation, the survival rate can be remarkably increased, and meanwhile the oligopeptide has the advantages of being stable in structure and good in curative effect; the medicine disclosed by the invention can also be used for preparing a medicine for treating radiation injury and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of radiation damage prevention and treatment and polypeptide synthesis, and particularly relates to a polypeptide capable of reducing radiation-induced intestinal injury by oral administration and a preparation method thereof. BACKGROUND

[0002] The mammalian intestine is a highly sensitive organ to radiation. High-dose ionizing radiation exposure (>10 Gy) can cause fatal intestinal injury in humans, with clinical manifestations mainly including diarrhea, hematochezia, and severe endogenous infection. Currently, there is no surviving case of intestinal acute radiation sickness worldwide. In addition, patients with abdominal and pelvic malignant tumors will also develop radiation-induced intestinal injury after receiving radiotherapy. Data shows that about 90% of patients will develop gastrointestinal symptoms of varying degrees within three months after radiotherapy, which not only affects the efficacy of tumor radiotherapy, but also significantly reduces the quality of life of patients (Redox Biol. 2023, 66: 102857). There is a severe shortage of drugs that can effectively alleviate radiation-induced intestinal injury in the current clinic, and it is urgent to develop prevention and treatment measures for this disease.

[0003] Studying the pathogenesis of radiation-induced intestinal injury is crucial for developing effective treatment strategies. Increasing evidence suggests that in addition to the inherent radiosensitivity of intestinal epithelial cells, the intestinal microbiota significantly affects the host's radiation response reaction (Science. 2020, 370: 6516). There are about 100 trillion microorganisms in the human intestine, and this complex ecosystem is essential for maintaining intestinal homeostasis. After the abdomen is exposed to ionizing radiation, the composition of the intestinal microbiota will change significantly, with typical features including reduced diversity, decreased content of probiotics (such as lactobacilli and bifidobacteria), and increased abundance of pathogenic bacteria (such as Escherichia coli and Staphylococcus) (J Radiat Res. 2024, 65: 194-204). Conversely, maintaining intestinal microbiota homeostasis is a potential strategy to alleviate radiation-induced intestinal injury (Gut Microbes. 2022, 17: 2489072).

[0004] As an important effector molecule in the intestinal defensin family, human defensin 5 (HD5) secreted by intestinal Paneth cells is essential for maintaining intestinal flora homeostasis (Nat Immunol. 2010, 11: 76-83). Since we first identified linear HD5 in humans (Sci Rep. 2016, 6: 22875), more and more studies have found that natural HD5 will be proteolytically processed to form shorter linear peptide segments, including AT28, TG26, CA23, and AT9, etc.; these peptide segments play an important role in maintaining the composition of the intestinal microbiome (Proc Natl Acad Sci U S A. 2019, 116: 3746-3751). In in vivo and in vitro experimental screening, it was found that HD5 (SEQ ID NO: 3 ATCYCRTGRCATRESLSGVCEISGRLYRLCCR) derived peptide segment AT9 can play a radioprotective role by regulating the intestinal flora. Compared with natural HD5 with complex spatial structure, AT9 oligopeptide (SEQ ID NO: 2 ATCYCRTGR) is easy to prepare by solid-phase chemical synthesis method, and the synthesis cost is low, which has great potential for drug conversion. However, the amino acid sequence of AT9 contains two cysteines (Cys 3 , Cys 5 ) carrying reduced sulfhydryl, which is easy to oxidize to form intramolecular or intermolecular disulfide bonds, weakening the radioprotective effect of the polypeptide. Under this premise, how to improve the stability of AT9 becomes the bottleneck of limiting the drug.

[0005] In summary, radiation-induced intestinal injury not only reduces the survival rate of radiation-injured personnel, but also affects the efficacy of patients with abdominal and pelvic tumor radiotherapy, and there are limited treatment options for this disease. HD5 derived peptide AT9 can play a radioprotective role by regulating the flora, and has potential for conversion due to low synthesis cost and easy preparation. However, the intramolecular cysteine of AT9 is easy to form disulfide bonds, affecting the stability of linear peptides and limiting the drugability of the oligopeptide, which needs to be optimized to improve the application potential. SUMMARY

[0006] The purpose of the present application is to provide an oligopeptide which is easy to synthesize artificially, has stable structure, and can reduce radiation-induced intestinal injury by oral administration, so as to solve the defect of shortage of existing drugs.

[0007] To achieve the above-mentioned purpose, the present application first provides an oligopeptide for reducing radiation-induced intestinal injury, whose amino acid sequence is SEQ ID NO: 1 ATGYGRTGR.

[0008] The present application also provides a pharmaceutical composition for reducing radiation-induced intestinal injury, wherein the active ingredient comprises an oligopeptide with an amino acid sequence of SEQ ID NO. 1.

[0009] The present invention further provides the use of the above-described oligopeptides or pharmaceutical compositions in the preparation of medicaments for alleviating radiation-induced intestinal damage.

[0010] In one embodiment of the invention, the oligopeptide or pharmaceutical composition, upon administration, can alleviate small intestinal epithelial shedding caused by radiation-induced intestinal injury and maintain intestinal villus height and crypt number.

[0011] In one embodiment of the invention, the oligopeptide or pharmaceutical composition is capable of reducing the level of peripheral blood endotoxins caused by radiation-induced intestinal injury after administration.

[0012] In one embodiment of the invention, the oligopeptide or pharmaceutical composition, upon administration, can significantly reduce the levels of peripheral blood inflammatory factors IL-6 and IL-1β induced by radiation-induced intestinal injury.

[0013] In another aspect, the present invention provides a method for preparing the above-mentioned oligopeptide, comprising:

[0014] 1) Place an appropriate amount of 2-Cl(Trt)-Cl resin in a reactor and add dichloromethane (DCM) for soaking; preferably, the soaking time is 30 minutes;

[0015] 2) Fmoc-Arg(PBF)-OH (C-terminus) was dissolved in anhydrous DCM, and 0.5 mmol of N,N-diisopropylethylamine (DIEA) was added. After mixing, the mixture was added to the reactor and reacted for 90 minutes under nitrogen bubbling conditions. Then, a mixture of methanol and DCM was added and the reaction was continued for 20 minutes. The molar ratio of 2-Cl(Trt)-Cl resin to Fmoc-Arg(PBF)-OH was 10 g:3 mmol, and the ratio of resin to initial DCM and coupled DCM was 1 g:20 mL:20 mL.

[0016] 3) After the reaction is complete, remove the liquid, soak and wash the resin with industrial-grade dimethylformamide (DMF) for 30 seconds, then dry it. Repeat this process several times.

[0017] 4) Add piperidine / DMF solution to the resin and bubble it with nitrogen for 20 minutes to remove the Fmoc protecting group;

[0018] 5) Perform multiple washes as described in step 3), and switch to analytical grade DMF for the final wash;

[0019] 6) Condensation of the second amino acid: Dissolve Fmoc-Gly-OH and HOBT in DMF at a 1:1 molar ratio, add 5 equivalents of DIC, mix for 1 minute, and then add to the resin. Bubble the mixture with nitrogen for 1 hour. The ratio of Fmoc-Gly-OH to DMF is 1 mmol: 10 mL. After the reaction is complete, wash several times with industrial-grade DMF.

[0020] 7) Perform the subsequent amino acid condensation sequentially: Remove Fmoc sequentially and condense Fmoc-Thr-OH, Fmoc-Arg-OH, Fmoc-Gly-OH, Fmoc-Tyr-OH, Fmoc-Gly-OH, Fmoc-Thr-OH and Fmoc-Ala-OH in the manner described above;

[0021] 8) Wash according to the method in step 5);

[0022] 9) Wash the resin three times with methanol and vacuum dry it until the resin is dry and granular.

[0023] 10) Transfer the dried resin to a centrifuge tube, add the cutting solution, and react at room temperature for 1.5 hours;

[0024] 11) Filter to remove resin, wash the filtrate with ice-cold ether and centrifuge to obtain crude peptide precipitate;

[0025] 12) The crude product was purified by liquid chromatography to obtain high-purity oligopeptides;

[0026] Preferably, the process further includes resin testing after the washing step, the resin testing being performed by a method comprising the following steps:

[0027] Take a small amount of resin, add 2–3 drops of ninhydrin solution, and heat at 100°C until a blue color appears to confirm that the Fmoc group has been successfully removed.

[0028] In one embodiment of the present invention, the ninhydrin solution is obtained by dissolving ninhydrin in anhydrous ethanol, and its concentration is 0.05 g / mL.

[0029] In one embodiment of the invention, the piperidine / DMF solution in step 4) is prepared by mixing piperidine and DMF in a volume ratio of 1:4.

[0030] In one embodiment of the present invention, the cutting fluid is prepared by mixing trifluoroacetic acid (TFA), triisopropylsilane, 1,2-ethylenedithiol and ultrapure water in a volume ratio of 95:2:2:1; the recommended ratio of cutting fluid to resin is 10 mL:1 g.

[0031] Preferably, the amount of icy ether added is 10 times the volume of the cutting fluid.

[0032] The beneficial effects of the above-mentioned technical solution of the present invention are as follows:

[0033] The anti-radiation oligopeptide C3GC5G-AT9 described in this invention consists of only 9 natural amino acids and can effectively reduce radiation-induced intestinal damage. Compared with the original peptide AT9 of the same sequence length, C3GC5G-AT9 does not contain cysteine, which is easily oxidized, resulting in better stability, a stronger anti-radiation intestinal damage effect, and higher medicinal value. Attached Figure Description

[0034] Figure 1 The liquid chromatogram of the C3GC5G-AT9 oligopeptide of this invention is shown.

[0035] Figure 2 This is the mass spectrometry identification diagram of the C3GC5G-AT9 oligopeptide of the present invention.

[0036] Figure 3 The results of the present invention, namely the C3GC5G-AT9 and AT9 oligopeptides, improve the survival rate of mice with radiation-induced intestinal injury.

[0037] Figure 4 The results of the present invention regarding the reduction of small intestinal epithelial shedding in mice with radiation-induced intestinal injury by C3GC5G-AT9 and AT9 oligopeptides are presented.

[0038] Figure 5 The results of the present invention regarding the maintenance of intestinal villus height and crypt number in mice with radiation-induced intestinal injury by the C3GC5G-AT9 and AT9 oligopeptides are presented.

[0039] Figure 6 The results of the present invention regarding the reduction of peripheral blood endotoxin levels in mice with radiation-induced intestinal injury by C3GC5G-AT9 and AT9 oligopeptides are shown.

[0040] Figure 7 The results of the present invention regarding the reduction of peripheral blood inflammatory factors IL-6 and IL-1β levels in mice with radiation-induced intestinal injury by the C3GC5G-AT9 and AT9 oligopeptides are presented. Detailed Implementation

[0041] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0042] Unless otherwise specified, all reagents used in this embodiment are of analytical grade, and the progress of all chemical reactions is detected by thin-layer chromatography.

[0043] Example 1: Preparation of C3GC5G-AT9 Oligopeptide

[0044] 1. Accurately weigh 0.5g of 2-Cl(Trt)-Cl resin (Kanglong Biotechnology, Changzhou) onto an electronic balance, place it in a 25×250mm reactor, and add an appropriate amount of dichloromethane (DCM, Tianquan Chemical, Chengdu) to soak for 30 minutes.

[0045] 2. Take 0.15 mmol of Fmoc-Arg(PBF)-OH (Jier Biochemical, Shanghai, as the C-terminal amino acid) in a centrifuge tube, dissolve it in anhydrous DCM, and add 0.5 mmol of N,N-diisopropylethylamine (DIEA, Aladdin Biochemical, Shanghai). Mix well and transfer to the reactor through a disposable pipette. React under nitrogen bubbling for 90 minutes, then add 1 mL of a mixture of methanol (Aladdin Biochemical) and 5 mL of DCM, and continue the reaction for 20 minutes.

[0046] 3. After the reaction is complete, use a circulating water vacuum pump to remove the reaction solution, add industrial-grade dimethylformamide (DMF) to immerse the resin, stir and wash for 30 seconds, then dry. Repeat this washing process a total of 4 times.

[0047] 4. Add 20% piperidine (Sinopharm Reagent, Shanghai) / DMF solution to completely cover the resin, and bubble the reaction under nitrogen for 20 minutes to remove the Fmoc protecting group.

[0048] 5. Perform four DMF washes as described above, with the final wash using analytical grade DMF.

[0049] 6. Take 5–10 resin samples, add 2–3 drops of ninhydrin solution, and heat at 100°C until a blue color appears, indicating that the Fmoc group has been successfully removed.

[0050] 7. Condensation of the second amino acid (Fmoc-Gly-OH, Jier Biochemical): Weigh 0.5 mmol of the amino acid and 0.5 mmol of HOBT (Aladdin Biochemical) into centrifuge tubes, dissolve in 5 mL of DMF, then add 2.5 mmol of DIC (Aladdin Biochemical), mix for 1 minute, and then add to the resin system. Bubble under nitrogen for 1 hour. After the reaction, wash four times with industrial-grade DMF, take a sample, add ninhydrin solution, and heat at 100°C for 2 minutes. If the solution turns colorless, the condensation is complete.

[0051] 8. Perform subsequent amino acid condensation sequentially: After removing Fmoc as in step 4, repeat step 7 to condense 0.5 mmol of Fmoc-Thr-OH, Fmoc-Arg-OH, Fmoc-Gly-OH, Fmoc-Tyr-OH, Fmoc-Gly-OH, Fmoc-Thr-OH and Fmoc-Ala-OH (all from Jier Biochemical).

[0052] 9. Perform resin testing according to the method described in step 7. If the sample is colorless, it indicates that all amino acids are completely linked.

[0053] 10. Perform the final wash as described in step 5.

[0054] 11. Wash the resin three times with methanol and vacuum dry it until the resin is in loose granular form.

[0055] 12. Transfer the dried resin to a centrifuge tube, add the cutting solution, and react at room temperature for 1.5 hours.

[0056] 13. Filter to remove resin, wash the filtrate with ice-cold ether (Chuandong Chemical, Chongqing) and centrifuge to collect the crude peptide precipitate.

[0057] 14. The crude product was purified by liquid chromatography with daisogel as the stationary phase, mobile phase A being 0.1% TFA aqueous solution (v / v) and mobile phase B being acetonitrile solution containing 0.1% TFA (v / v) (analytical grade, Aladdin Biochemistry). The target oligopeptide was finally purified (sequence see SEQ ID NO:1).

[0058] SHIMADZU high-performance liquid chromatography (HPLC) was used to analyze the purity of pure C3GC5G-AT9 oligopeptide. The analytical column was an Inertsil ODS-SP (4.6 × 250 mm × 5 μm), the injection volume was 30 μL, the mobile phase A was 0.1% TFA / water, and the mobile phase B was 0.1% TFA / acetonitrile. The flow rate was 1 mL / min, and the detection wavelength was 220 nm. Figure 1 As shown, the oligopeptide of this invention elutes at 9.194 min with a purity of 95.745%. Analysis using a SHIMADZU LC-MS2010 liquid chromatography-mass spectrometry system revealed the molecular weight of the eluting product to be 937.65 Da. Figure 2 The difference between the theoretical molecular weight of 938 Da and the actual molecular weight is less than 1.

[0059] Example 2: Effect of C3GC5G-AT9 oligopeptide on survival rate of mice with radiation-induced intestinal injury

[0060] 1. Male C57BL / 6J mice (weighing 20-22g) aged 6-8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and were cared for and treated in accordance with the National Institutes of Health's guidelines for the care and use of laboratory animals. The animal experiments were approved by the Animal Experimentation Ethics Committee of Army Medical University.

[0061] 2. Experimental grouping: C57BL / 6J mice were randomly divided into six groups (n=10 per group). (1) Control group (Sham); (2) AT9 (SEQ ID NO: 2ATCYCRTGR) administration group alone; (3) C3GC5G-AT9 administration group alone; (4) Whole abdomen irradiation group (TAI) group; (5) AT9+TAI group; (6) C3GC5G-AT9+TAI group.

[0062] 3. Radiation treatment: using 60Mice were subjected to a single whole-abdomen irradiation with a Coγ-ray source at a dose of 12 Gy and a dose rate of 0.4 Gy / min.

[0063] 4. Administration method and dosage: The drug was administered by gavage. Mice in the AT9-only and C3GC5G-AT9 groups were administered 35 μM peptide by gavage for 10 consecutive days. Mice in the AT9+TAI and C3GC5G-AT9+TAI groups were administered 35 μM peptide by gavage for 7 consecutive days, followed by whole-abdominal irradiation of 12 Gy, and then continued gavage for 3 days after irradiation.

[0064] 5. Experimental observation indicators: The mortality rate of mice was observed for 30 days after radiation injury.

[0065] 6. Testing showed that administration of C3GC5G-AT9 before and after irradiation significantly improved the survival rate of mice with total abdominal radiation injury (see [link to study]). Figure 3 In experimental mice, the 30-day survival rate after radiation injury was only 10%; after administration of C3GC5G-AT9, the survival rate of radiation-injured mice increased to 60%.

[0066] Example 3: Effect of C3GC5G-AT9 oligopeptide on intestinal mucosal integrity in mice with radiation-induced intestinal injury

[0067] 1. Animal model construction and drug administration: Refer to steps 1-4 in Example 2 to establish a mouse model of whole-abdominal radiation injury. Mice were orally administered 35 μM peptide for 7 consecutive days, and then irradiated with 12 Gy to the whole abdomen. After irradiation, they were continued to be administered by gavage for 3 days.

[0068] 2. Identification of Intestinal Tissue Damage: Three days after whole-abdomen irradiation, mice were euthanized by cervical dislocation. The ileum was removed and rinsed thoroughly with pre-cooled PBS. The small intestine was then rolled up with a small wooden stick, villi facing inwards, and fixed with a pin. After overnight fixation with Carnoy's fixative (G1120-500mL, Seville, Hubei), the tissue was dehydrated with gradient concentrations of alcohol, and the alcohol in the tissue was replaced with xylene. Clear tissue blocks were placed in melted paraffin and incubated in a paraffin bath. The embedded paraffin blocks were fixed in a microtome, cut into 3μm sections, and stained with hematoxylin and eosin (HE, C0105S, Beyotime, Shanghai) for observation.

[0069] 3. HE staining:

[0070] Paraffin was removed from mouse small intestinal tissue sections using xylene. The sections were then passed through high-concentration to low-concentration alcohol, and finally transferred to distilled water. The sections were removed, stained with hematoxylin for 2 minutes, rinsed with tap water for 1 minute, differentiated with 0.6% hydrochloric acid alcohol for 15 seconds, rinsed with tap water for 15 minutes, and then placed in distilled water briefly. They were then transferred to 1% alcohol solution for eosin staining for 30 seconds. The sections were dehydrated with alcohol, cleared with xylene, and covered with neutral resin and sealed with a coverslip.

[0071] 4. Pathological analysis

[0072] Pathological changes in small intestinal tissue observed using a Precipoint M8 scanning microscope revealed that a single 12 Gy whole-abdominal γ-ray irradiation severely damaged the mucosal barrier of the small intestine in mice, with significant shedding of intestinal epithelial cells and atrophy of intestinal villi. Administration of C3GC5G-AT9 before and after irradiation significantly alleviated the radiation-induced intestinal epithelial shedding, and the intestinal villi maintained a relatively good morphology (see...). Figure 4 The scale shows 200μm.

[0073] 5. Counting the height of intestinal villi and the number of crypts.

[0074] Following steps 3-4 in "Example 3", the mouse small intestinal tissue was stained with hematoxylin and eosin (HE) and observed. The height of the small intestinal villi was measured using ViewPoint software (version setup_70). Six well-oriented full-length crypt villi units were measured in each field of view. The average height of the ileal villi in normal mice, AT9-treated mice, and C3GC5G-AT9-treated mice were 284.24, 284.64, and 283.87 μm, respectively; the average number of crypts per millimeter was 17 in all three mice. After a single 12 Gy whole-abdomen irradiation with gamma rays, the intestinal villi decreased to 130.34 μm; the average number of crypts per millimeter was 3. Treatment with C3GC5G-AT9 maintained an intestinal villi height of 259.45 μm; the average number of crypts per millimeter was 12, showing a significant difference compared to irradiated mice (see [link to relevant documentation]). Figure 5 Among them, ***, p<0.001, which indicates a statistically significant difference.

[0075] Example 4. Effect of C3GC5G-AT9 oligopeptide on peripheral blood endotoxin levels in mice with radiation-induced intestinal injury

[0076] 1. Animal model construction and drug administration: Refer to steps 1-4 in Example 2 to establish a mouse model of total abdominal radiation injury and administer 35 μM peptide orally to the mice.

[0077] 2. Sample preparation: Blood was collected from one eye of mice 3 days after irradiation. The samples were left at room temperature for 1 hour, then centrifuged at 3500 rpm for 15 minutes, and the supernatant was collected for later use.

[0078] 3. Peripheral blood endotoxin detection: A commercially available detection kit (endotoxin detection kit, BET, EC64405S) was used for detection. One vial of bacterial endotoxin working standard (1 EU / vial) was added to 1 mL of dedicated diluent (BET water), and the sample was vortexed at 2000 rpm for 5 min to obtain a 1 EU / mL stock solution. This stock solution was then serially diluted with water for bacterial endotoxin testing to prepare a series of standard solutions ranging from 0.03125 to 1 EU / mL. Endotoxin-free microplates were incubated at 37°C for 5 min. 50 μL of standard solutions, blood supernatant, and water for bacterial endotoxin testing (as controls) of different concentrations were added to each well. 50 μL of Limulus amebocyte lysate (LAL) reagent was then added to each well using a pipette, mixed thoroughly, and incubated at 37°C for 8 min. 100 μL of chromogenic matrix solution was added, mixed thoroughly, and then incubated at 37°C for 6 min. Finally, add 50 μL of reaction terminator, mix thoroughly, and read the absorbance value at 405 nm. Calculate the endotoxin content in the sample based on the standard curve.

[0079] 4. Testing showed that C3GC5G-AT9 significantly reduced endotoxin levels in the peripheral blood of irradiated mice, exhibiting a significant difference compared to the positive control group (mice irradiated throughout the abdomen). (See [link to relevant documentation]). Figure 6 Among them, ***, p<0.001, which indicates a statistically significant difference.

[0080] Example 5. Effects of C3GC5G-AT9 oligopeptide on the levels of peripheral blood inflammatory factors IL-6 and IL-1β in mice with radiation-induced intestinal injury.

[0081] 1. Animal model construction and drug administration:

[0082] Refer to steps 1-4 in Example 2 to establish a mouse model of total abdominal radiation injury and orally administer 35 μM peptide to the mice.

[0083] 2. Sample preparation: Blood was collected from one eye of mice 3 days after irradiation. The samples were left at room temperature for 1 hour, then centrifuged at 3500 rpm for 15 minutes, and the supernatant was collected for later use.

[0084] 3. Detection of peripheral blood inflammatory factors IL-6 and IL-1β: Commercially available detection kits were used for mouse interleukin 6 (IL-6) enzyme-linked immunosorbent assay kit (Elabscience, E-EL-M0044) and mouse interleukin 1β (IL-1β) enzyme-linked immunosorbent assay kit (Elabscience, E-EL-M0037).

[0085] The specific steps are as follows:

[0086] (1) Preparation of washing solution: Mix concentrated washing solution with ultrapure water at a volume ratio of 1:20.

[0087] (2) Add 1000 μL of diluent to one lyophilized standard, and after it is completely dissolved, let it stand for 15 min and mix well. Then dilute it proportionally to prepare a series of standards with different concentrations: 500, 250, 125, 62.5, 31.25, 15.625, and 0 pg / mL.

[0088] (3) Add 100 μL of the standard and blood supernatant to each well of a 96-well plate. Seal the plate tightly with adhesive and incubate at 37°C for 90 min.

[0089] (4) Wash the plate 4 times: Add 350μL of washing solution to each well, gently shake the plate left and right 10 times, and pat it dry on filter paper.

[0090] (5) Add 100 μL of biotinylated antibody working solution, attach the sealing adhesive tightly, incubate at 37°C for 60 min, and wash the plate 4 times.

[0091] (6) Add 100 μL of enzyme conjugate working solution, attach the sealing glue tightly, incubate at 37°C for 30 min, and wash the plate 4 times.

[0092] (7) Add 100 μL of colorimetric solution to each well, and incubate at 37°C for 15 min in the dark.

[0093] (8) Add 100 μL of stop solution to each well, mix well and immediately read the absorbance values ​​at 450 nm and 630 nm.

[0094] (9) When calculating the concentration of inflammatory factors, the absorbance value at 450nm is subtracted from the absorbance value at 630nm.

[0095] 4. Testing showed that C3GC5G-AT9 significantly reduced the levels of the inflammatory factors IL-6 and IL-1β in the peripheral blood of irradiated mice, with a significant difference compared to the positive control group (irradiated mice). (See [link to relevant documentation]). Figure 7 Among them, ***, p<0.001, which indicates a statistically significant difference.

[0096] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An oligopeptide for alleviating radiation-induced intestinal damage, the amino acid sequence of which is SEQ ID NO:1ATGYGRTGR.

2. A pharmaceutical composition for alleviating radiation-induced intestinal injury, wherein the active ingredient comprises an oligopeptide with the amino acid sequence SEQ ID NO.

1.

3. The use of the oligopeptide of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for alleviating radiation-induced intestinal injury.

4. The application as described in claim 3, wherein, After application, it can reduce the shedding of small intestinal epithelium caused by radiation-induced intestinal damage and maintain the height of intestinal villi and the number of crypts.

5. The application as described in claim 3 or 4, wherein, After application, it can reduce the content of peripheral blood endotoxins caused by radiation-induced intestinal damage.

6. The application as described in any one of claims 3-5, wherein, After application, it can significantly reduce the levels of peripheral blood inflammatory factors IL-6 and IL-1β caused by radiation-induced intestinal injury.

7. The method for preparing the oligopeptide according to claim 1, comprising: 1) Place an appropriate amount of 2-Cl(Trt)-Cl resin in a reactor and add dichloromethane (DCM) for soaking; preferably, the soaking time is 30 minutes; 2) Fmoc-Arg(PBF)-OH (C-terminus) was dissolved in anhydrous DCM, and 0.5 mmol of N,N-diisopropylethylamine (DIEA) was added. After mixing, the mixture was added to the reactor and reacted for 90 minutes under nitrogen bubbling conditions. Then, a mixture of methanol and DCM was added and the reaction was continued for 20 minutes. The molar ratio of 2-Cl(Trt)-Cl resin to Fmoc-Arg(PBF)-OH was 10 g:3 mmol, and the ratio of resin to initial DCM and coupled DCM was 1 g:20 mL:20 mL. 3) After the reaction is complete, remove the liquid, soak and wash the resin with industrial-grade dimethylformamide (DMF) for 30 seconds, then dry it. Repeat this process several times. 4) Add piperidine / DMF solution to the resin and bubble it with nitrogen for 20 minutes to remove the Fmoc protecting group; 5) Perform multiple washes as described in step 3), and switch to analytical grade DMF for the final wash; 6) Condensation of the second amino acid: Dissolve Fmoc-Gly-OH and HOBT in DMF at a 1:1 molar ratio, add 5 equivalents of DIC, mix for 1 minute, and then add to the resin. Bubble the mixture with nitrogen for 1 hour. The ratio of Fmoc-Gly-OH to DMF is 1 mmol: 10 mL. After the reaction is complete, wash several times with industrial-grade DMF. 7) Perform the subsequent amino acid condensation sequentially: Remove Fmoc sequentially and condense Fmoc-Thr-OH, Fmoc-Arg-OH, Fmoc-Gly-OH, Fmoc-Tyr-OH, Fmoc-Gly-OH, Fmoc-Thr-OH and Fmoc-Ala-OH in the manner described above; 8) Wash according to the method in step 5); 9) Wash the resin three times with methanol and vacuum dry it until the resin is dry and granular. 10) Transfer the dried resin to a centrifuge tube, add the cutting solution, and react at room temperature for 1.5 hours; 11) Filter to remove resin, wash the filtrate with ice-cold ether and centrifuge to obtain crude peptide precipitate; 12) The crude product was purified by liquid chromatography to obtain high-purity oligopeptides; Preferably, the process further includes resin testing after the washing step, the resin testing being performed by a method comprising the following steps: Take a small amount of resin, add 2–3 drops of ninhydrin solution, and heat at 100°C until a blue color appears to confirm that the Fmoc group has been successfully removed.

8. The preparation method according to claim 7, wherein, The ninhydrin solution was obtained by dissolving ninhydrin in anhydrous ethanol, and its concentration was 0.05 g / mL.

9. The preparation method according to claim 7 or 8, wherein, Step 4) The piperidine / DMF solution is prepared by mixing piperidine and DMF in a volume ratio of 1:

4.

10. The preparation method according to any one of claims 7-9, wherein, The cutting fluid is prepared by mixing trifluoroacetic acid (TFA), triisopropylsilane, 1,2-ethylenedithiol and ultrapure water in a volume ratio of 95:2:2:1; the recommended ratio of cutting fluid to resin is 10 mL:1 g. Preferably, the amount of ice-cold ether added is 10 times the volume of the cutting fluid.

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