Oligopeptide for treating colorectal polyp and preparation method thereof

Abstract

The invention relates to the technical field of biology, in particular to oligopeptide for treating colorectal polyp and a preparation method of the oligopeptide. The oligopeptide SEISNQLVG with a treatment effect on colorectal polyp is separated from astragalus membranaceus serving as a natural source of an innovative medicine, and the oligopeptide can be used for effectively treating or relieving the colorectal polyp and can be used for preventing the colorectal polyp from developing into colorectal cancer to a certain extent.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a short peptide for treating colorectal polyps and a preparation method thereof. Background Art

[0002] Colorectal polyps are proliferative lesions that protrude from the intestinal mucosal surface into the intestinal lumen, and their incidence increases significantly with age. According to pathological characteristics, polyps can be divided into adenomatous, inflammatory and proliferative types. Among them, adenomatous polyps (especially villous adenomas) are considered to be the main precancerous lesions of colorectal cancer (CRC). Globally, the incidence of colorectal cancer ranks third among malignant tumors, and about 80% of sporadic CRCs evolve from adenoma-carcinoma sequences. Although endoscopic polypectomy can effectively reduce the risk of canceration, the postoperative recurrence rate is still as high as 30%-50%, and some patients cannot receive invasive treatment due to multiple polyps, special locations or combined underlying diseases. Therefore, the development of new treatment strategies that are non-invasive, highly targeted and safe has become the focus of clinical research.

[0003] The limitations of traditional treatments have driven the exploration of molecular targeted therapies. In recent years, peptide drugs have shown potential in the fields of tumors and inflammatory diseases due to their unique biological activity. Peptides are biological macromolecules (typically containing 2-50 amino acid residues) composed of amino acids linked by peptide bonds. They have advantages such as small molecular weight, strong tissue penetration, low immunogenicity, and easy chemical modification. In the pathogenesis of colorectal polyps, abnormally activated Wnt / β-catenin signaling pathways, chronic inflammatory microenvironments (such as the IL-6 / STAT3 pathway), and intestinal dysbiosis are key factors driving adenoma formation. Peptides can precisely regulate these molecular targets by mimicking natural ligands or through competitive antagonism. For example, peptides targeting the β-catenin binding domain can inhibit its interaction with the TCF / LEF transcription complex, thereby blocking the expression of pro-proliferation genes; anti-inflammatory peptides (such as antimicrobial peptides derived from lactoferrin) can alleviate mucosal inflammatory responses by regulating macrophage polarization.

[0004] Plant-based peptide drugs hold great promise for development. However, their development is limited by the complex composition of plants, low peptide content, and a lack of effective isolation and purification methods. To address these limitations, a growing number of isolation, purification, and identification methods have emerged, achieving promising results. Currently, the isolation and purification of peptides and proteins still requires specialized treatments, such as urea extraction and enzymatic preparation, which are complex and require extensive post-processing. Therefore, developing an effective, simple, and rapid isolation and purification method is a highly valuable research topic. Summary of the Invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is that the cure rate of colorectal polyps has been improved, but the treatment effect is not ideal, and the serious adverse reactions greatly reduce the quality of life of patients.

[0006] To achieve the above objectives, the present invention provides a short peptide for treating colorectal polyps, wherein the amino acid sequence of the short peptide is SEISNQLVG.

[0007] The preferred polypeptide is derived from plants such as Astragalus, Codonopsis, etc.

[0008] Preferably, the polypeptide is a variant of SEISNQLVG obtained by substitution, addition or deletion of one amino acid, and the variant still has the efficacy of treating colorectal polyps.

[0009] Preferably, the polypeptide is prepared by recombinant expression and chemical synthesis.

[0010] In a preferred embodiment of the present invention, the present invention provides a polynucleotide that can encode the short peptide SEISNQLVG.

[0011] In another preferred embodiment of the present invention, the present invention provides an expression vector comprising a polynucleotide encoding the polypeptide.

[0012] In another preferred embodiment of the present invention, the present invention provides a cell comprising a polynucleotide or an expression vector encoding an Astragalus short peptide.

[0013] In a preferred embodiment of the present invention, the present invention provides a pharmaceutical composition comprising the short peptide.

[0014] In a specific embodiment of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a dressing.

[0015] In a specific embodiment of the present invention, the dosage form of the pharmaceutical composition includes but is not limited to injection, oral preparation, tablet, capsule, powder and the like.

[0016] Another object of the present invention is to provide an application of an astragalus short peptide in the preparation of a drug for treating colorectal polyps.

[0017] The present invention uses Astragalus as the natural source of an innovative drug, and isolates a short peptide SEISNQLVG from it that has a therapeutic effect on colorectal polyps. This peptide can effectively treat or alleviate colorectal polyps and, to a certain extent, prevent colorectal polyps from developing into colorectal cancer.

[0018] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 UPLC separation profile of chromatographic fraction P1.

[0020] Figure 2 Secondary mass spectrum of Astragalus short peptide.

[0021] Figure 3 The number of colorectal polyps and tumors in the drug-treated group and the control group, A: drug-treated group; B: control group.

[0022] Figure 4 The number and size of colorectal polyps in the treatment group and the control group. DETAILED DESCRIPTION

[0023] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0024] Example 1 Extraction of Astragalus polypeptide

[0025] (1) Take the dried Astragalus root, remove impurities, and grind it into 80-100 mesh fine powder using a grinder to increase the contact area and improve the extraction efficiency;

[0026] (2) Astragalus powder was mixed with phosphate buffered saline (PBS, pH 7.4, containing 1 mM EDTA) at a ratio of 1:15 (w / v). After stirring and extraction for 2 h, the mixture was placed in a 50°C water bath and subjected to intermittent ultrasound (200 W, 2 s on / 3 s off) to destroy the cell walls.

[0027] (3) Centrifuge at 10,000 × g for 20 minutes, collect the supernatant, repeat the extraction of the residue once, and combine the two supernatants.

[0028] (4) Flavor protease and alkaline protease were further added to the crude Astragalus extract, and enzymatic hydrolysis was carried out at 45°C for 35 minutes to remove the polysaccharides attached to the protein. After enzymatic hydrolysis, the hydrolyzate was ultrafiltered through 10 kDa and 3 kDa ultrafiltration membranes in sequence, and the filtrate with a molecular weight less than 3 kDa was collected and further separated and purified using a Sephadex G-25 gel column to obtain P1, P2, and P3.

[0029] The purified P1 fraction was further separated and purified using RP-HPLC using a C18 column. The RP-HPLC separation conditions were: isocratic elution, mobile phase A: deionized water containing 0.05% TFA; mobile phase B: acetonitrile containing 0.05% TFA. The flow rate was 1 mL / min, the injection volume was 10 μL, the sample concentration was 10 mg / mL, the column temperature was 20°C, and the elution peak was detected at 214 nm.

[0030] During the elution process of the RP-HPLC separation spectrum, three absorption peaks were collected, such as Figure 1 As shown, they are marked as F1, F2, and F3 in sequence, and each component is freeze-dried and stored.

[0031] The amino acid sequence of component F1 was analyzed by LC-MS / MS, and the Astragalus polypeptide was identified, and its amino acid sequence was SEISNQLVG. Figure 2 Its secondary mass spectrum.

[0032] Example 2 Construction of a colorectal polyp mouse model

[0033] Twenty 6- to 8-week-old C57BL / 6 mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were selected and maintained under constant temperature and humidity conditions with a light intensity of 12 h per day and a basal diet.

[0034] After the mice were acclimated to the environment for 1 week, AOM (10 mg / kg) was injected intraperitoneally once a week for 6 consecutive weeks.

[0035] Example 3 Peptide Effect Experiment

[0036] SEISNQLVG is difficult to obtain naturally. In order to reduce production costs, SEISNQLVG was prepared by organic solid phase synthesis.

[0037] Twenty mice were randomly divided into a control group and a drug-treated group. The mice were treated with the short peptide at a concentration of 400 mg / kg / d. The control group was given an equal amount of 0.9% sodium chloride solution.

[0038] At 16 weeks of the experiment, each group of mice was killed by cervical dislocation, and the colorectum of the mice was separated and removed. After longitudinal incision, the colon mucosa was washed and the polyp formation was observed. The number of colorectal polyps in each group of mice was recorded in detail, and the size was accurately measured (mm) with a caliper to two decimal places. After counting, each colorectal polyp was removed and fixed in 4% paraformaldehyde.

[0039] The results are as follows Figure 3 As shown, the control group had more polyps and tumors of varying sizes, while the polypeptide treatment group had only a small number of polyps and no tumor masses.

[0040] The number and size of colorectal polyps in each group are shown in Table 1 and Figure 4 shown.

[0041] Table 1 Number and size of colorectal polyps in each group (x±s)

[0042]

[0043] Note: * indicates P < 0.05 compared with the control group; ** indicates P < 0.01 compared with the control group

[0044] From Table 1 and Figure 4 As can be seen, there were statistically significant differences in the number and size of polyps in the drug-treated group compared to the control group (P < 0.05). This indicates that the SEISNQLVG short peptide of the present invention has a good effect in treating or alleviating colorectal polyps and preventing colorectal polyps from developing into colorectal cancer.

[0045] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A short peptide for treating colorectal polyps, characterized in that: The amino acid sequence of the short peptide is SEISNQLVG.

2. The polypeptide according to claim 1, characterized in that The polypeptide is a variant of SEISNQLVG obtained by substitution, addition or deletion of one amino acid, and the variant still has the efficacy of treating colorectal polyps.

3. The short peptide according to claim 1 or 2, characterized in that The polypeptide is prepared by recombinant expression or chemical synthesis.

4. A polynucleotide, characterized in that It encodes the short peptide described in any one of claims 1-3.

5. An expression vector, characterized in that It comprises the polynucleotide according to claim 4.

6. A cell, characterized in that It comprises the polynucleotide according to claim 4 or the expression vector according to claim 5.

7. A pharmaceutical composition, characterized in that The invention comprises the short peptide according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

8. The drug according to claim 8, wherein the specific dosage form includes but is not limited to injection, oral preparation, and tablet.

9. Use of the short peptide according to claims 1-3 and / or the pharmaceutical composition according to any one of claims 7-8 in the preparation of a drug for treating colorectal polyps.

10. Use of the short peptide according to claims 1-3 and / or the pharmaceutical composition according to any one of claims 7-8 in the preparation of a drug for preventing colorectal polyps from developing into colorectal cancer.

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