阅读说明：本技术 一种用于预防或治疗溃疡性结肠炎的多肽 (Polypeptide for preventing or treating ulcerative colitis ) 是由 邢新会 何东 王怡 张翀 于 2020-04-08 设计创作，主要内容包括：本发明涉及一种用于治疗溃疡性结肠炎的多肽,所述多肽的氨基酸序列为选自PVLGPVRGPPLL(Seq ID No.1)、YQEPVLGPVR(Seq ID No.2)、WNMNMMTA(Seq ID No.3)、CCNFCMSS(Seq ID No.4)中的一种。所述多肽在DSS处理或不处理条件下均能显著提高紧密连接蛋白的表达,并能抑制DSS处理造成的NCM460细胞的单层细胞通透性增加,保持细胞屏障的完整性。(The invention relates to a polypeptide for treating ulcerative colitis, wherein the amino acid sequence of the polypeptide is one selected from PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) and CCNFCMSS (Seq ID No. 4). The polypeptide can obviously improve the expression of the tight junction protein under the condition of DSS treatment or no treatment, can inhibit the increase of the monolayer cell permeability of NCM460 cells caused by the DSS treatment, and can maintain the integrity of cell barriers.)

1. A polypeptide having the amino acid sequence: one selected from PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3), and CCNFCMSS (Seq ID No. 4).

2. Use of a polypeptide having an amino acid sequence of PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) or CCNFCMSS (Seq ID No.4) in the manufacture of a medicament for the prevention or treatment of ulcerative colitis.

3. Use of a polypeptide having amino acid sequence PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) or CCNFCMSS (Seq ID No.4) in the manufacture of a medicament for the alleviation of impaired colonic mucosal barrier function.

4. Use of a polypeptide having the amino acid sequence PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) or CCNFCMSS (Seq ID No.4) in the manufacture of a medicament for use in the repair of the intestinal mucosa.

5. Use of a polypeptide having the amino acid sequence PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) or CCNFCMSS (Seq ID No.4) in the manufacture of a medicament for maintaining intestinal mucosal homeostasis and balance.

6. Use of a polypeptide having amino acid sequence PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) or CCNFCMSS (Seq ID No.4) in the manufacture of a medicament for increasing the expression level of claudin.

7. A pharmaceutical composition or health product for preventing or treating ulcerative colitis, wherein the pharmaceutical composition or health product comprises the polypeptide of claim 1 as an active ingredient.

8. The pharmaceutical composition or health product of claim 7, wherein the pharmaceutical composition or health product further comprises a pharmaceutically acceptable carrier or excipient.

9. A pharmaceutical composition or health product for alleviating barrier function impairment of colonic mucosa, wherein the pharmaceutical composition or health product comprises the polypeptide of claim 1 as an active ingredient.

10. The pharmaceutical composition or health product of claim 9, wherein the pharmaceutical composition or health product further comprises a pharmaceutically acceptable carrier or excipient.

Technical Field

The invention relates to a polypeptide and application thereof in preparing a medicament for preventing or treating ulcerative colitis.

Background

Ulcerative Colitis (UC) is a kind of Inflammatory Bowel Disease (IBD), the pathological changes of which are limited to the mucosal layer of the colon, and the impairment of the barrier function of the colonic mucosa is one of the pathogenesis and has a higher risk of canceration. At present, the incidence of the disease is increased year by year in China, and no specific medicine exists in clinic due to unclear etiology and repeated attack, so that the disease has serious influence on physical and mental health and life quality of patients. The existing treatment medicines are few, mainly comprise aminosalicylic acid preparation, glucocorticoid, immunosuppressant and the like, generally have the problems of poor specificity, strong side effect, great difficulty in medication and the like, and have poor curative effect.

Disclosure of Invention

The existing research finds that an alcohol extract derived from wheat germ and apple juice lactobacillus fermented fruit juice has a certain application in the aspect of preparing medicines for inhibiting diseases such as colitis, gouty arthritis and the like (CN 108244621A). After the alcohol-soluble component of the lactobacillus fermented fruit juice is subjected to component analysis, the lactobacillus fermented fruit juice is found to contain rich polypeptide components. The performance characterization and excavation of the polypeptides in the alcohol soluble component are found to relieve intestinal mucosa damage, repair intestinal mucosa of a DSS model-making colitis mouse, improve the expression level of corresponding tight junction protein, and can be used for preparing a medicine for preventing or treating ulcerative colitis.

The application aims to provide a polypeptide and application thereof in preparing a medicament for preventing or treating ulcerative colitis.

In particular, the invention relates to the following:

1. a polypeptide having the amino acid sequence: one selected from PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3), and CCNFCMSS (Seq ID No. 4).

2. Use of a polypeptide having an amino acid sequence of PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) or CCNFCMSS (Seq ID No.4) in the manufacture of a medicament for the prevention or treatment of ulcerative colitis.

3. Use of a polypeptide having amino acid sequence PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) or CCNFCMSS (Seq ID No.4) in the manufacture of a medicament for the alleviation of impaired colonic mucosal barrier function.

4. Use of a polypeptide having the amino acid sequence PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) or CCNFCMSS (Seq ID No.4) in the manufacture of a medicament for use in the repair of the intestinal mucosa.

5. Use of a polypeptide having the amino acid sequence PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) or CCNFCMSS (Seq ID No.4) in the manufacture of a medicament for maintaining intestinal mucosal homeostasis and balance.

6. Use of a polypeptide having amino acid sequence PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) or CCNFCMSS (Seq ID No.4) in the manufacture of a medicament for increasing the expression level of claudin.

7. A pharmaceutical composition or a health product for preventing or treating ulcerative colitis, wherein the pharmaceutical composition or the health product comprises the polypeptide of item 1 as an active ingredient.

8. The pharmaceutical composition or health product according to item 7, wherein a pharmaceutically acceptable carrier or adjuvant can be added to the pharmaceutical composition or health product.

9. A pharmaceutical composition or a health product for alleviating barrier function impairment of colonic mucosa, wherein the pharmaceutical composition or the health product comprises the polypeptide of item 1 as an active ingredient.

10. The pharmaceutical composition or health product according to claim 9, wherein a pharmaceutically acceptable carrier or adjuvant can be added to the pharmaceutical composition or health product.

11. A pharmaceutical composition or a health product for repairing an intestinal mucosa, wherein the pharmaceutical composition or the health product comprises the polypeptide of item 1 as an active ingredient.

12. The pharmaceutical composition or health product according to claim 11, wherein a pharmaceutically acceptable carrier or adjuvant can be added to the pharmaceutical composition or health product.

13. A pharmaceutical composition or health product for maintaining intestinal mucosa homeostasis and balance, wherein the pharmaceutical composition or health product comprises the polypeptide of item 1 as an active ingredient.

14. The pharmaceutical composition or health product according to item 13, wherein a pharmaceutically acceptable carrier or adjuvant may be added to the pharmaceutical composition or health product.

15. A pharmaceutical composition or health product for increasing the expression level of claudin, wherein said pharmaceutical composition or health product comprises the polypeptide of item 1 as an active ingredient.

16. The pharmaceutical composition or health product according to claim 15, wherein a pharmaceutically acceptable carrier or adjuvant can be added to the pharmaceutical composition or health product.

17. A method of preventing or treating ulcerative colitis, the method comprising administering to a subject an effective amount of a polypeptide having an amino acid sequence of: one selected from PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3), and CCNFCMSS (Seq ID No. 4).

18. A method for alleviating impaired colonic mucosal barrier function comprising administering to a subject an effective amount of a polypeptide having an amino acid sequence of: one selected from PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3), and CCNFCMSS (Seq ID No. 4).

19. A method for repairing intestinal mucosa comprising administering to a subject an effective amount of a polypeptide having an amino acid sequence of: one selected from PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3), and CCNFCMSS (Seq ID No. 4).

20. A method for maintaining intestinal mucosal homeostasis and balance, the method comprising administering to a subject an effective amount of a polypeptide having an amino acid sequence: one selected from PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3), and CCNFCMSS (Seq ID No. 4).

21. A method for increasing the expression level of claudin, comprising administering to a subject an effective amount of a polypeptide having an amino acid sequence of: one selected from PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3), and CCNFCMSS (Seq ID No. 4).

Effects of the invention

The application provides an active polypeptide capable of relieving intestinal mucosa injury on the basis of an alcohol extract of lactobacillus fermented juice, and the amino acid sequence of the active polypeptide is analyzed and determined to be one of PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) and CCNFCMSS (Seq ID No. 4). Meanwhile, the polypeptide is found to be capable of remarkably improving the expression of the tight junction protein under the condition of DSS treatment or no treatment, inhibiting the increase of the monolayer cell permeability of NCM460 cells caused by the DSS treatment and keeping the integrity of cell barriers. In a DSS animal experiment, the polypeptide is used as a therapeutic drug, so that the clinical symptoms of UC mice can be improved, the damage of colon mucous membranes can be reduced, the weight loss condition of the mice can be reduced, the spleen weight index can be reduced, and the IL-6 expression level in colon tissues can be reduced.

Drawings

FIG. 1 is a schematic diagram of the determination of the cytotoxic activity of the polypeptide against NCM460 cells by CCK8 in example 2.

FIG. 2 is a schematic representation of the effect of different polypeptides on claudin in example 3.

FIG. 3 is a graphical representation of the effect of T24 on claudin expression under DSS treatment in example 3.

FIG. 4 is a graphical representation of the effect of T24 on claudin expression in the absence of DSS treatment in example 3.

FIG. 5 is a schematic diagram of the penetration of FITC dextran in the NCM460 cell layer in example 4.

FIG. 6 is a graph showing the physiological indicators obtained in the experiment of DSS-induced acute UC mice in example 5.

FIG. 7 is a photograph of a tissue section obtained from the experiment of the DSS-induced acute UC mouse in example 5.

Detailed Description

The following detailed description of the present application is provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the application, however, the description is made for the purpose of illustrating the general principles of the application and is not intended to limit the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

The application relates to a polypeptide, in particular to an amino acid sequence of the polypeptide selected from one of PVLGPVRGPPLL (Seq ID No.1), YQEPVLGPVR (Seq ID No.2), WNMNMMTA (Seq ID No.3) and CCNFCMSS (Seq ID No. 4). The polypeptide can be applied to preparing medicines for preventing or treating ulcerative colitis.

In a specific embodiment, the polypeptide is derived from an alcohol extract of a wheat germ and apple juice lactic acid bacteria fermented juice.

In a specific embodiment, the polypeptide can be obtained by the following preparation method: firstly, juicing and fermenting apples and wheat germs to obtain fresh wheat germ and apple fermented juice; dissolving the fermented juice in 75% ethanol, centrifuging, sterilizing, and freeze drying to obtain alcohol soluble component of fresh wheat germ apple fermented juice; dissolving the alcohol soluble components, sequentially performing ultrafiltration through ultrafiltration membranes (30, 10 and 3kDa) with different molecular weights, further separating and purifying components with the alcohol soluble components being less than 3kDa by using a circulating preparative liquid chromatography to collect 11 components, verifying that the F7 component has the best effect on relieving DSS-induced intestinal epithelial cell tight junction protein injury, and analyzing polypeptide sequences contained in the F7 component to obtain four target peptide sequences with the best effect, namely T24(PVLGPVRGPPLL (Seq ID No.1)), T26(YQEPVLGPVR (Seq ID No.2)), T27(WNMNMMTA (Seq ID No.3)) and T28(CCNFCMSS (Seq ID No. 4)).

The application also relates to the application of the polypeptide in obviously preventing or treating ulcerative colitis, and the polypeptide has wide application value in the development and application of pharmaceutical compositions or health products for preventing and treating ulcerative colitis.

In a particular embodiment, the polypeptide may alleviate impaired colonic mucosal barrier function and thereby prevent or treat ulcerative colitis. The damage of the barrier function of the colonic mucosa is considered as one of the pathogenesis of the ulcerative colitis, and the alleviation of the damage of the intestinal mucosa is one of the key factors for preventing and treating the ulcerative colitis.

In a particular embodiment, the polypeptide may repair the intestinal mucosa and thereby prevent or treat ulcerative colitis.

In a specific embodiment, the polypeptide can maintain intestinal mucosal homeostasis and balance, and thereby prevent or treat ulcerative colitis.

In a specific embodiment, the polypeptide can increase the level of claudin expression. The tight junction protein is one of basic structural substances of the intestinal epithelial barrier function, and the damage of the intestinal epithelial cell barrier can be directly caused by the reduction of the expression amount of the tight junction protein. Ulcerative colitis can be prevented or treated by increasing the expression level of claudin.

The application relates to a pharmaceutical composition or health product for preventing or treating ulcerative colitis, in particular to a pharmaceutical composition or health product which takes the polypeptide disclosed by the application as an active ingredient.

In one embodiment, a pharmaceutical composition or health product for repairing intestinal mucosa is provided, and specifically, the pharmaceutical composition or health product uses the polypeptide disclosed in the application as an active ingredient.

In one embodiment, a pharmaceutical composition or health product for maintaining intestinal mucosa homeostasis and homeostasis is provided, in particular, the pharmaceutical composition or health product comprises a polypeptide disclosed herein as an active ingredient.

In one embodiment, a pharmaceutical composition or health product for increasing the expression level of claudin is provided, in particular, the pharmaceutical composition or health product comprises the polypeptide disclosed in the present application as an active ingredient.

In a specific embodiment, a pharmaceutically acceptable carrier or adjuvant may be added to the pharmaceutical composition or health product described herein.

Specifically, the pharmaceutical composition may be prepared in the form of: mixing the polypeptide and a pharmaceutically acceptable carrier, for example, to obtain an oral preparation such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets), capsules (including soft capsules, microcapsules), granules, powders, troches, syrups, emulsions, suspensions, films (e.g., orally disintegrating films), and the like, a parenteral preparation such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, instillations), external preparations (e.g., skin preparations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), pills, nasal drops, respiratory preparations (inhalants), eyedrops, and the like. In addition, these formulations can be provided as controlled release formulations (e.g., sustained release microcapsules), such as immediate release formulations, sustained release formulations, and the like. Such formulations may be obtained by preparation methods conventionally used in the art.

Specifically, examples of the above pharmaceutically acceptable carriers include excipients (e.g., starch, lactose, sucrose, calcium carbonate, calcium phosphate, etc.), binders (e.g., starch, gum arabic, carmellose, hydroxypropylcellulose, crystalline cellulose, alginic acid, gelatin, polyvinylpyrrolidone, etc.), lubricants (e.g., magnesium stearate, calcium stearate, talc, etc.), disintegrants (e.g., carmellose calcium, talc, etc.), diluents (e.g., water for injection, saline, etc.), additives (e.g., stabilizers, preservatives, coloring agents, flavoring agents, dissolution aids, emulsifiers, buffers, isotonic agents, etc.), and the like.

A medicament or pharmaceutical composition comprising a polypeptide described herein may be administered to a mammal (e.g., human, mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey). The mode of administration may be oral or parenteral (e.g., intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, rectal, vaginal, intraperitoneal, etc.).

The amount of the polypeptide of the present application to be administered to a subject varies depending on the administration route, symptoms, age of the patient, and the like, and can be actually determined by a clinician.

The medicaments or pharmaceutical compositions referred to in this application can also be used together with other medicaments known in the art for the treatment of ulcerative colitis. When used together, there is no limitation on the administration time of the respective drugs, two or more different drugs may be administered simultaneously, and the respective drugs may be administered at different times. The dose of a known drug can be determined in accordance with the number of administration clinically used and appropriately selected depending on the administration patient, the administration route, and the like.

Examples

EXAMPLE 1 isolation, purification and measurement of the polypeptide

Carrying out probiotic fermentation on apples and wheat germs according to the following process conditions: 200 g of apples and 200 g of wheat germs are crushed for 5 minutes by a juicer, the juice is squeezed and the residue is dissolved in 4L of water. Heating the mixture at 70 deg.C for 1h, extracting with ultrasonic wave for 30min, and centrifuging at 8450 × g for 10min to obtain raw wheat germ apple juice. The pasteurized raw wheat germ apple juice is inoculated with 6% (v/v) mixed lactic acid bacteria (lactobacillus bulgaricus: lactobacillus casei: lactobacillus plantarum: lactobacillus helveticus ═ 1:1:1, OD600 ═ 1.9), and is kept stand and cultured at 37 ℃ for 24 hours to prepare the fresh wheat germ apple fermented juice.

Dissolving the fermented juice of fresh wheat germ and apple in 75% ethanol for 30min, and centrifuging at 8450 × g speed for 10 min. Filtering, sterilizing, and freeze drying the supernatant to obtain the alcohol soluble component of the fermented juice.

Dissolving the alcohol soluble components of the fresh wheat germ apple fermented juice, sequentially performing ultrafiltration through ultrafiltration membranes (30, 10 and 3kDa) with different molecular weights, finally respectively collecting protein hydrolysate solutions with different molecular weights, concentrating and drying through a rotary evaporator to respectively obtain 4 components (30 kDa, 10-30kDa, 3-10kDa and 3kDa) with different molecular weights, and refrigerating for later use. The alleviation effect of the four components on DSS-induced intestinal epithelial cell tight junction protein damage is detected by using a western blot method, and the component with the alcohol-soluble component of less than 3kda of the fermented juice of the fresh wheat germ apples is found to have the best effect.

Alcohol-soluble component of fermented juice of fresh wheat germ and apple<The 3kDa fraction was further isolated and purified by preparative liquid chromatography on cycles (Japan analytical industries, Inc.) with the parameters set to: the UV detector was set at λ 214nm in room temperature aqueous solution (10 mg/mL)^-1) In the flow rate of 2.0mg/mL^-1The amount of the sample was 3 mL. 11 components are collected from the circulating preparation liquid phase, and the F7 component is proved to have the best effect on relieving the DSS-induced intestinal epithelial cell tight junction protein injury by western blot verification. The biomedical test center of the university of Qinghua, delegated to analyze the sequence of the polypeptide contained in F7, wherein the analysis of molecular weight was performed using matrix-assisted laser desorption time-of-flight mass spectrometer MALDI-TOF-TOF 4800Plus (ABI, USA); the analysis of the polypeptide sequence was carried out using an Orbitrap Fusion LUMOS LC MS (Thermo Fisher Co., USA). Four most effective targeting peptide sequences were obtained by analysis, namely T24(PVLGPVRGPPLL (Seq ID No.1)), T26(YQEPVLGPVR (Seq ID No.2)), T27(WNMNMMTA (Seq ID No.3)), and T28(CCNFCMSS (Seq ID No. 4)).

Experiments will be carried out in the following examples using the above-mentioned T24, T26, T27, T28 sequence polypeptides obtained in example 1.

EXAMPLE 2 Effect of the Polypeptides on NCM460 intestinal epithelial cells

The CCK-8 method is adopted to detect the cytotoxicity of the polypeptide with different concentrations on NCM460 cells after 24 hours of treatment.

Wherein, the CCK-8 solution is purchased from Beijing Soilebao Biotechnology Co., Ltd, and the NCM460 cell is purchased from Shanghai Guangdi bioengineering Co., Ltd. Specific measurement methodComprises the following steps: cell suspensions (400. mu.L/well) were seeded in 24-well plates at a cell number of 10⁴Perwell, and adding 100ug/ml final volume of polypeptide, pre-culturing the plate in an incubator for 12h (37 ℃, 5% CO)₂). To each well 40. mu.L of CCK-8 solution was added. The plates were incubated in an incubator for 2 hours. Absorbance at 450nm was measured with a microplate reader. The vitality calculation is based on the following formula: cell viability (%) ([ a (medicated) -a (blank)]/[ A (0 dosing) -A (blank)]X 100; wherein, A (adding medicine): absorbance of wells with cells, CCK-8 solution and drug solution; a (blank): absorbance of wells with medium and CCK-8 solution without cells; a (0 dosing): absorbance of wells with cells, CCK-8 solution, but no drug solution. Cell viability: cell proliferation activity or cytotoxic activity.

The results of the experiment are shown in FIG. 1.

As can be seen in FIG. 1, at various concentrations ranging from 10 to 200. mu.g/mL, the four polypeptides all exhibited similar cell viability, i.e., similar cell viability, as the blank. The morphology and growth of the cells of each group were observed under a microscope, and no abnormality was observed. This indicates that the polypeptide at a concentration of 10-200. mu.g/mL is not cytotoxic to NCM460 cells and is suitable for further experiments.

EXAMPLE 3 Effect of the Polypeptides on the amount of expression of Claudin

In order to investigate the effect of the polypeptides T24, T26, T27 and T28 on the expression of claudin, experiments were carried out on the effect of the addition of DSS on the expression levels of ZO-1, claudin 1 and occludin1 using western blot. NCM460 cells were washed with 1 Xprotein loading buffer (50mm Tris-HCl-pH6.8, 2% SDS, 10% glycerol, 1% beta-mercaptoethanol, 12.5mm EDTA, 0.02% bromophenol blue) by secondary lysis of PBS and trituration with a grinding rod for 5 min. The lysate was collected, heated at 100 ℃ for 15min, centrifuged at 13000 Xg for 5min, and the supernatant was subjected to protein electrophoresis using 15% SDS-PAGE. The gel was transferred to a PVDF membrane (Millipore, MA, USA) for 2 hours at a constant current of 300 mA. The blotting membrane was blocked with 5% (w/v) skim milk in PBST and incubated with 1:1000 diluted antibody for 2 hours. All major antibodies were purchased from CST biotechnology (MA, USA). The antigen-antibody complex was detected with peroxidase-conjugated secondary antibody diluted 1:5000, and the signal of the antibody was detected using ECL chemiluminescent reagent. The results are shown in FIG. 2.

As can be seen, the expression of ZO-1, claudin 1 and Occludin-1 was down-regulated in the NCM460 cells treated with DSS alone, as compared with the control group. The 4 peptides T24, T26, T27 and T28 (100. mu.g/mL) all significantly reduced the loss of ZO-1, claudin-1 and Occludin-1 (p < 0.05).

To investigate the effect of different concentrations of T24 polypeptide treatment on claudin expression, western blot was used to determine the effect of T24 on the expression levels of ZO-1, claudin 1 and occludin1 with and without DSS treatment. The experimental results are shown in fig. 3 and 4, respectively.

As can be seen from FIG. 3, in the experiment of adding DSS, the T24 polypeptide with the concentration of 20-200ug/mL can significantly increase the expression level of ZO-1 (P <0.05), with the trend of increasing first and then slightly decreasing; the expression level of claudin 1 increases with the increase of the concentration of T24 polypeptide, while the expression level of occludin is significantly increased at low concentration of T24(20ug/mL) but decreased at high concentration (50-200ug/mL), indicating that T24 polypeptide can increase the expression level of intestinal epithelial cell tight junction overall, but the effect on each tight junction protein is not consistent. The effect of T24 polypeptide at concentrations of 20-1000ug/mL on claudin was also examined in experiments without DSS treatment. The experimental result in FIG. 4 shows that the T24 polypeptide increases the expression level of ZO-1, ZO-2 and Claudin 4, the expression level is increased and then decreased along with the increase of the concentration, but the expression level of Claudin 1 is slightly increased and is not obviously increased.

From the above results, the T24 polypeptide was able to increase the expression level of intestinal epithelial cell claudin both with and without DSS treatment.

Example 4 detection of the Barrier Effect of the Polypeptides on cell membranes

In order to study the influence of the polypeptide on the tight junction barrier under the DSS treatment condition, a fluorescence transmittance experiment of FITC-dextran is carried out to determine the permeability of a single-layer membrane formed by the human intestinal epithelial cells NCM460, and further determine the protective effect of the addition of the polypeptide on the integrity of the intestinal mucosa. The specific experimental method comprises the following steps:

NCM460 cell monolayers at 5X 10⁵Per each was inoculated in a 24-well Transwell chamber, 100. mu.L and 500. mu.L of 1640 medium were added to the top and bottom chambers, respectively, and the medium was changed every other day. After 4 days, the apical chamber was treated with a further 3% DSS and polypeptide at a final concentration of 200. mu.g/mL for 48 hours. The PBS group alone was used as a negative control, and the DSS group alone without the polypeptide was used as a positive control. The permeability of the intestinal epithelial cell monolayer of the different treatment groups was examined by FITC-dextran (FITC-dextran, molecular weight 4000 Dalton) fluorescent tracer. The detection method comprises the following steps: the Transwell top and bottom chambers were washed 2 times with pre-warmed PBS buffer, and FITC-dextran (1mg/mL), PBS 250. mu.L, 37 ℃ and 5% CO were added to the top and bottom chambers₂Incubate under conditions for 12 hours. Taking the bottom chamber liquid, detecting the fluorescence length of the sample, the laser wavelength of 480nm and the generation light wavelength of 520nm in an enzyme-labeling instrument M200-PRO, and repeatedly measuring for 3 times to obtain an average value. The results of the experiment are shown in FIG. 5.

It can be seen that the fluorescence across the monolayer membrane was significantly increased in the DSS control group compared to the PBS control group, indicating a significant increase in the monolayer membrane permeability (P < 0.001). The addition of the 4 polypeptides T24, T26, T27 and T28(200, 1000. mu.g/mL) significantly reduced the fluorescence across the monolayer and the barrier permeability was correspondingly reduced compared to the DSS control group, indicating that the addition of the above polypeptides significantly protected the integrity of the intestinal epithelial cell monolayer.

Example 5 animal experiments with polypeptide relief from ulcerative colitis

In order to research the relieving effect of the polypeptide on the mouse ulcerative colitis, an acute UC mouse model is constructed for a comparison experiment, and the specific method comprises the following steps:

the 60B 6 mice were randomly divided into 4 groups of 15 mice each, namely a normal control group, a UC model control group, a T24 polypeptide gavage group and a T24 polypeptide subcutaneous injection group. Wherein the content of the first and second substances,

normal control group: the daily drinking water of the mice is distilled water, and 250 mu L of PBS is drenched once a day.

UC model control group: the daily drinking water for the mice was 3% DSS solution, 250 μ L PBS was administered once daily.

T24 polypeptide gavage group: the daily drinking water of the mice is 3% DSS solution, and the mice are subjected to intragastric administration once a day by using T24 polypeptide aqueous solution with the dosage of 100 mg/kg.

T24 subcutaneous injection group: the daily drinking water of the mice is 3% DSS solution, and the mice are injected subcutaneously once a day with T24 polypeptide water solution with the dosage of 100 mg/kg.

The experimental time is 8 days, the general conditions of the mice such as spirit, activity, hair, diet and the like are observed every day in the experimental process, and the weight change and the stool character of each mouse are recorded in detail. After the experiment, the mice were sacrificed, the whole colon (from ileocecal part to anus) was taken out and fixed overnight at 4 ℃ in 4% paraformaldehyde solution, and the pathological injury of the colon tissue was observed by conventional tissue dehydration, paraffin embedding, sectioning and HE staining. After HE staining of the sections, they were observed under a microscope and photographed. Observing the infiltration degree of the mouse colon and intestinal mucositis cells, the change of gland structures and goblet cells, the presence or absence of crypt abscess formation, the falling off of colon mucous epithelium and the repair degree under a light microscope. The experimental results are shown in fig. 6 and 7.

The experiment shows that the normal control group mice are relatively active, the hair is bright, the body weight is generally kept, the stool is normal, and the colon length is normal. And the UC model control group mice begin to roll over and do not move, have anorexia, weight sharply reduced, body hair messy, loose stool and bloody stool from the 4 th day after administration, even have fresh blood at the anus, the colon is seriously atrophied and shortened, the spleen weight is enlarged, and the histological score is obviously increased. Compared with a UC model group, the T24 polypeptide gavage group and the subcutaneous injection group have certain alleviation on the symptoms of the colitis. Among them, the effect of the subcutaneous injection group was more significant. The clinical symptoms such as weight loss, hematochezia and the like of the mice in the group are reduced to different degrees, the colon length atrophy is relieved, and the histological score is obviously reduced (P < 0.05). The expression level of IL-6 in colon tissues of each group of mice is detected by ELISA, and the result shows that: compared with a normal control group, the expression level of IL-6 in the UC model group is obviously increased (P < 0.001). Compared with the UC model group, the T24 subcutaneous injection group significantly reduced the IL-6 expression level. The observation result of HE staining of the tissue section in FIG. 7 shows that the epithelial cells of the colon mucosa of the mice in the normal group are complete, the glands are arranged regularly, the mucosa, the blood vessels in the lamina propria and the fiber stroma are normal, and the muscle layer is not abnormal; the colon mucosal epithelium of the mouse in the model group is incomplete, epithelial cells are necrosed and shed, partial serious defects are caused, partial glands are disorganized, atrophy and even completely disappear, goblet cells are reduced, a mucous layer and a submucosa are infiltrated by a large amount of inflammatory cells, a small number of tunica muscularis can be reached, congestion and edema of partial submucosa are obvious, ulcer and crypt abscess can be formed, and lymph follicles and macrophages can be seen; compared with a model group, the T24 intragastric group has a certain relieving effect but is not as obvious as a subcutaneous injection group, the mucosal injury of the subcutaneous injection group is lighter, the mucosal injury is basically close to normal tissues except local inflammatory cell infiltration, the submucosa and the muscular layer are slightly edematous, inflammatory cells are reduced, granulation tissues participate in repair, epithelial repair is already generated on most surfaces, and the mucosal gland is recovered to be close to normal. In conclusion, the addition of the T24 polypeptide can effectively relieve the symptoms of the mice with the ulcerative colitis, and the effect of subcutaneous injection is better than that of oral administration.

The above description is only for the preferred embodiment of the present application and should not be taken as limiting the present application in any way. Any person skilled in the art may, using the teachings disclosed above, change or modify the equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application still belong to the protection scope of the technical solution of the present application.

Sequence listing

<110> Qinghua university

<120> a polypeptide for preventing or treating ulcerative colitis

<130> PD00849

<141> 2020-04-08

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 1

Pro Val Leu Gly Pro Val Arg Gly Pro Pro Leu Leu

1 5 10

<210> 2

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 2

Tyr Gln Glu Pro Val Leu Gly Pro Val Arg

1 5 10

<210> 3

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 3

Trp Asn Met Asn Met Met Thr Ala

1 5

<210> 4

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 4

Cys Cys Asn Phe Cys Met Ser Ser

1 5