Retinaldehyde dehydrogenase activating peptide and application thereof in prevention and treatment of radiotherapy intestinal side reaction

Abstract

The invention discloses application of retinaldehyde dehydrogenase activating peptides derived from three euphausia superba in prevention or treatment of radiotherapy intestinal side reactions, and belongs to the technical field of bioactive peptides. The euphausia superba-derived retinaldehyde dehydrogenase activating peptides have three kinds of amino acid sequences, and the amino acid sequences of the retinaldehyde dehydrogenase activating peptides are respectively shown as SEQ ID NO.1-3. The euphausia superba-derived retinaldehyde dehydrogenase activating peptide is applied to prevention or treatment of radiotherapy intestinal side reactions. According to the invention, three potential retinaldehyde dehydrogenase activated peptides are identified from the antarctic krill protein peptide sequence by using high-resolution mass spectrometry, and the effect of the retinaldehyde dehydrogenase activated peptides on preventing or treating radiotherapy intestinal side reactions is verified by using animal experiments. Results show that three small molecule peptides can improve weight loss and intestinal structure damage caused by radiation, thereby repairing intestinal mucosal barriers and improving side effects of radiotherapy intestinal tracts.

Description

Technical Field

[0001] This invention relates to retinaldehyde dehydrogenase-activating peptide and its application in the prevention and treatment of intestinal side effects of radiotherapy, belonging to the field of bioactive peptide technology. Background Technology

[0002] Radiation intestinal injury (RII) is a serious side effect caused by radiotherapy for pelvic / abdominal tumors. Clinical manifestations include diarrhea, abdominal pain, and rectal bleeding. 90% of patients experience gastrointestinal side effects, significantly impacting their quality of life. Current treatments are primarily symptomatic, lacking targeted repair methods.

[0003] In the prevention and treatment of intestinal side effects from radiotherapy, retinoic acid (RA)-mediated intestinal mucosal immune repair plays a crucial role. The biosynthesis of RA is highly dependent on the catalytic action of retinal dehydrogenase (ALDH1A1). When ALDH1A1 activity is low, it directly leads to insufficient RA synthesis, which in turn prevents the effective initiation of repair processes such as intestinal mucosal immune cell differentiation, tight junction protein expression, and gut microbiota homeostasis regulation. This is the fundamental mechanism by which intestinal side effects from radiotherapy struggle to achieve self-repair. Based on this pathological logic, targeting and activating ALDH1A1 activity to restore the endogenous RA synthesis capacity has become a highly promising therapeutic target for the prevention and treatment of intestinal side effects from radiotherapy.

[0004] In recent years, the development of molecular docking virtual screening technology has provided a new pathway for the efficient discovery of target peptides. By simulating ligand-receptor interactions using computers, large-scale peptide library screening and mechanism analysis can be completed within weeks, significantly improving research and development efficiency. Therefore, this invention identifies and screens three retinaldehyde dehydrogenase-activating peptides from Antarctic krill protein peptides based on molecular docking virtual screening technology, and clarifies their application value in the prevention and treatment of retinitis repens (RII) through animal experiments. Summary of the Invention

[0005] This invention utilizes molecular simulation technology to successfully obtain three novel retinaldehyde dehydrogenase activating peptides through virtual screening from a self-built database of Antarctic krill protein peptides. The binding sites of these peptides with retinaldehyde dehydrogenase were further analyzed, and their efficacy was verified through animal experiments, providing a novel solution for the prevention and treatment of retinaldehyde-induced irritation (RII).

[0006] This invention is achieved through the following technical solution:

[0007] It includes the following three types, with the following amino acid sequences:

[0008] FEEF, as shown in SEQ ID NO.1;

[0009] FGREE, as shown in SEQ ID NO.2;

[0010] DFLAAT, as shown in SEQ ID NO.3.

[0011] The above three Antarctic krill bioactive peptides are used as or in the preparation of retinaldehyde dehydrogenase activators; and as or in the preparation of foods or drugs that improve intestinal side effects of radiotherapy.

[0012] The three bioactive peptides of this invention were identified and screened from Antarctic krill protein peptides using high-resolution mass spectrometry and molecular docking virtual screening technology, as follows:

[0013] (1) The peptide sequences of Antarctic krill protein peptides were identified by directly detecting peptides present in the sample using high-resolution mass spectrometry, and a peptide database was established.

[0014] (2) Retinaldehyde dehydrogenase (ALDH1A1) docks with oligopeptides in the peptide library.

[0015] (3) Animal experiments were used to verify the effect of the above-screened bioactive peptides on intestinal side effects of radiotherapy.

[0016] Using the above method, this invention screened three potential marine-derived ALDH1A1 activating peptides. Animal experiments were conducted to verify the effects of these three Antarctic krill active peptides on intestinal side effects of radiotherapy in mice. The results showed that the three oligopeptides exhibited significant improvement effects. These oligopeptides have the potential to serve as functional products for improving intestinal side effects of radiotherapy and can be used to prepare foods or drugs with efficacy in improving intestinal side effects of radiotherapy.

[0017] This invention revolutionizes the traditional, cumbersome method for screening ALDH1A1 activating peptides. By utilizing molecular informatics technology to perform virtual screening of marine-derived bioactive peptides from the entire protein sequence, it significantly improves the screening efficiency of ALDH1A1 activating peptides. Animal experiments have verified that the ALDH1A1 activating peptides screened using this invention do indeed have application value in preventing intestinal side effects of radiotherapy.

[0018] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0019] Figure 1 Molecular docking results of FEEF and ALDH1A1. A: Surface map; B: Secondary structure map; C: Binding site map.

[0020] Figure 2 Molecular docking results of FGREE and ALDH1A1. A: Surface map; B: Secondary structure map; C: Binding site map.

[0021] Figure 3 Molecular docking results of DFLAAT and ALDH1A1. A: Surface map; B: Secondary structure map; C: Binding site map.

[0022] Figure 4 Effects of three ALDH1A1 activating peptides on the general condition of irradiated mice.

[0023] Figure 5 Effects of three ALDH1A1 activating peptides on intestinal morphology and structure in irradiated mice.

[0024] Figure 6 The effects of three ALDH1A1 activating peptides on improving intestinal barrier function in irradiated mice.

[0025] Figure 7 The effect of three ALDH1A1 activating peptides on improving zonulin leakage in irradiated mice. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0027] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0028] Example 1: Three retinaldehyde dehydrogenase activating peptides identified by high-resolution mass spectrometry were docked with ALDH1A1.

[0029] ALDH1A1 was docked with selected oligopeptides from a bioactive peptide database. ChemDraw 19.0 and AutoDock Vina were used for peptide plotting and docking, respectively. The docking box was set to center x = 18.1, center y = 149.5, center z = 23.7, size x = 51.8, size y = 67.9, and size z = 53.1 (PDB ID: 7JWV). Other parameters were kept at their default values. For each ligand, nine optimal poses were generated using the docking software, and the degree of binding was determined by the affinity value (peptide sequences are shown in Table 1). Three strongly activating ALDH1A1 oligopeptides (FEEF, FGREE, and DFLAAT) were selected.

[0030] Table 1 Information on retinaldehyde dehydrogenase-activating peptides

[0031] polypeptide ALDH1A1 Affinity (kcal / mol) toxicity FEEF -8.81±0.42 Non-Toxin FGREE -8.02±0.22 Non-Toxin DFLAAT -8.29±0.43 Non-Toxin

[0032] Example 2 This example involves an experiment on the effects of three ALDH1A1 strongly activating oligopeptides (FEEF, FGREE, and DFLAAT) on radiotherapy-induced intestinal adverse reactions in mice. To verify the effect of Antarctic krill peptides on radiotherapy-induced intestinal adverse reactions, a mouse model of radiotherapy-induced intestinal adverse reactions was constructed using 12 Gy (400 cGy / min) X-rays. The changes in the intestinal barrier and tissue structure of mice after intervention with the three Antarctic krill active peptides were observed. The specific experimental steps are as follows:

[0033] (1) Establishment and grouping of animal models of intestinal side effects of radiotherapy

[0034] This embodiment used 60 SPF-grade 8-week-old male C57BL / 6 mice (20±2g) purchased from Vital River Pharmaceuticals, Beijing. All animal experiments were conducted in accordance with the "Guidelines of the Animal Experiment Ethics Review Committee of Qingdao University," and every effort was made to minimize animal suffering. The mice were allowed free access to food and water for one week at a temperature of (24±2) ℃ and a humidity of (50±5)%. Then, they were randomly divided into the following groups using a random number table: normal control group (CON), radiation model group (RII), FEEF oligopeptide intervention group (FEEF), FGREE oligopeptide intervention group (FGREE), DFLAAT oligopeptide intervention group (DFLAAT), and positive control glutamine group (Gln), with 10 mice in each group. Mice in the FEEF group were administered FEEF oligopeptide (0.5 g / kg·bw) by gavage daily; mice in the FGREE group were administered FGREE oligopeptide (0.5 g / kg·bw) by gavage daily; mice in the DFLAAT group were administered DFLAAT oligopeptide (0.5 g / kg·bw) by gavage daily; mice in the Gln group were administered glutamine (0.5 g / kg·bw) by gavage daily; and mice in the CON and RII groups were administered the same volume of ddH2O by gavage. One week after gavage, mice were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital before irradiation. After anesthesia, the mice were placed in a 6-MV linear accelerator for X-ray abdominal irradiation, exposing the entire abdomen with a source-skin distance of 100 cm and an irradiation field from below the xiphoid process of the sternum to the anus. Irradiation was performed at a dose rate of 400 cGy / min, with a total irradiation dose of 12 Gy. The normal control group underwent sham radiation.

[0035] (2) Effects of Antarctic krill peptides on body weight and intestinal structural damage in radiation-damaged mice

[0036] During the experiment, the mice were weighed daily, and the results were as follows: Figure 4 As shown in Figure A. Mice in each group were sacrificed on day 3 after radiation, and blood and intestinal tissue were collected. Colon length was measured, and the results are shown below. Figure 4 As shown in B.

[0037] There were no significant differences in baseline body weight among the groups of mice; however, activity decreased significantly after 3 days, food and water intake decreased, and some mice essentially stopped eating. Simultaneously, the radiation-treated mice exhibited slower responses to external stimuli, decreased shine, and showed signs of shedding or aggregation. The changes in body weight among the groups are shown below. Figure 4 As shown in Figure A, compared with the healthy control group, the body weight of mice in the RII group was significantly reduced 3.5 days after radiation (P < 0.05). From Figure 4 B shows that radiation did indeed lead to a significant reduction in colon length in mice, and the intervention of the three oligopeptides had a significant improvement effect (P < 0.05).

[0038] from Figure 5 As shown in Figure A, the jejunum of mice in the RII group exhibited irregular, atrophied, loose, and reduced-number intestinal villi with inconsistent shape and size, showing signs of breakage and necrosis. Intervention with the three oligopeptides significantly improved the intestinal morphology and structure of the mice, resulting in more regular and numerous villi. From... Figure 5 As can be seen from BD, the intestinal villus height in the RII group was significantly reduced compared to the CON group. The intervention of the three oligopeptides significantly improved the intestinal villus height in mice, and the villus height / crypt depth ratio was also significantly increased.

[0039] (3) Effects of three Antarctic krill bioactive peptides on improving intestinal barrier function in irradiated mice

[0040] from Figure 6 It can be seen that intervention with the three oligopeptides can improve the expression of tight junction proteins (ZO-1 and Occludin) regulating the intestinal mucosal barrier. Immunohistochemical staining of tight junction proteins is shown in the figure. Figure 6 As shown in Figure A, three days after radiation, the expression of ZO-1 and Occludin decreased in the RII group, and Antarctic krill bioactive peptides effectively restored the expression of tight junction proteins. These results indicate that radiation damages the intestinal mucosal barrier, and Antarctic krill bioactive peptides can repair the intestinal mucosal barrier. High expression of zonulin in the intestine causes structural loosening of the intestinal epithelial cell barrier, increasing intestinal permeability. Figure 7 It can be seen that intervention with Antarctic krill bioactive peptides can significantly reduce the relative level of zonulin and improve intestinal permeability.

[0041] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A retinaldehyde dehydrogenase-activating peptide, characterized in that, Its amino acid sequence is: FEEF, as shown in SEQ ID NO.

1.

2. A retinaldehyde dehydrogenase-activating peptide, characterized in that, Its amino acid sequence is: FGREE, as shown in SEQ ID NO.

2.

3. A retinaldehyde dehydrogenase-activating peptide, characterized in that, Its amino acid sequence is: DFLAAT, as shown in SEQ ID NO.

3.

4. The use of the retinaldehyde dehydrogenase activating peptide according to any one of claims 1 to 3 as or in the preparation of a retinaldehyde dehydrogenase activator.

5. The use of the retinaldehyde dehydrogenase activating peptide according to any one of claims 1 to 3 in the preparation of food or medicine for improving intestinal side effects of radiotherapy.

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