Polypeptides and Their Preparation and Applications

Abstract

This application relates to the field of biomedical technologies, and particularly relates to a polypeptide and its preparation and application. The amino acid sequence of the polypeptide is as shown in SEQ ID NO.1. Compared with the traditional technology, the beneficial effects of this application include: The small molecule polypeptide involved in this application is a novel bioactive polypeptide discovered by the applicant from the hydrolysate of breast milk protein of premature parturients with the help of peptidomics technology, and has a significant effect in preventing and treating neonatal necrotizing enterocolitis.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to a polypeptide and its preparation and application. Background Art

[0002] Protein is the most abundant component in breast milk. More than 400 proteins have been identified. In addition to nutritional value, small-molecule peptides are produced after digestion and hydrolysis in the gastrointestinal tract. They are easily absorbed by newborns and have important physiological activities. Studies have shown that small-molecule peptides produced by hydrolysis of breast milk proteins play an important role in the regulation of digestion, immunity, and nervous systems. For example, casein hydrolysates have the function of protecting gastric mucosa and inhibiting Helicobacter pylori; phosphopeptides have the function of regulating calcium ion uptake and function of intestinal epithelial cells; pentapeptides (WSVPQPK) have the effect of scavenging free radicals and anti-oxidation. The lactoferrin-derived peptide PRELP-I can protect the intestinal function of newborns by regulating intestinal bifidobacteria. These studies have shown that peptides produced by hydrolysis of breast milk proteins play an important role in protecting the digestive system function of newborns. At present, breast milk small-molecule peptides have become a hot spot in drug research and development due to their good biological functions and safety.

[0003] However, although there are many small molecule peptides currently available, only a limited number of them are used to protect the digestive system functions of newborns. In order to provide new technical strategies for preventing and treating neonatal digestive system diseases, it is necessary to develop new small molecule peptides. Summary of the invention

[0004] Based on this, one or more embodiments of the present application provide a polypeptide and its preparation and application.

[0005] One or more embodiments of the present application provide a polypeptide, the amino acid sequence of the polypeptide is shown as SEQ ID NO.1.

[0006] Yet another one or more embodiments of the present application provide a use of the polypeptide in the preparation of a drug for preventing and treating neonatal enterocolitis (NEC).

[0007] One or more embodiments of the present application also provide a drug, which includes the polypeptide and pharmaceutically acceptable excipients.

[0008] One or more embodiments of the present application also provide a method for producing the polypeptide. Optionally, the production method comprises chemical synthesis.

[0009] One or more embodiments of the present application also provide a nucleic acid molecule encoding the polypeptide.

[0010] One or more embodiments of the present application also provide a vector, which includes the nucleic acid molecule.

[0011] One or more embodiments of the present application also provide a cell, which includes the nucleic acid molecule, the vector or exogenously expresses the polypeptide.

[0012] One or more embodiments of the present application also provide the use of the polypeptide in preparing an inhibitor of the transformation of macrophages into M1 macrophages.

[0013] One or more embodiments of the present application also provide an inhibitor of the transformation of macrophages into M1 macrophages, wherein the inhibitor comprises the polypeptide.

[0014] One or more embodiments of the present application also provide a method for inhibiting the transformation of macrophages into M1 macrophages, the method comprising treating macrophages with the polypeptide to inhibit the transformation of macrophages into M1 macrophages.

[0015] Compared with the traditional technology, the beneficial effects of this application include:

[0016] The small molecule polypeptide involved in the examples of the present application is a new type of bioactive polypeptide discovered by the applicant from the hydrolyzate of breast milk protein of premature infants and mothers with the help of peptide omics technology, and has a significant effect in preventing and treating neonatal necrotizing enterocolitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0018] Figure 1 This is a diagram showing the results of H&E staining, colon length measurement, and NEC score evaluation of the colon of rats in each treatment group in one embodiment;

[0019] Figure 2 This is a graph showing the results of immunofluorescence analysis of the colon of rats in each treatment group in one embodiment;

[0020] Figure 3 This is a graph showing the expression evaluation results of macrophage pro-inflammatory factors (IL-1β, IL-6 and TNF-α) corresponding to cells in each treatment group in an embodiment;

[0021] Figure 4 This is a flow cytometry analysis result diagram of CD86 and CD206 corresponding to cells in each treatment group in an embodiment. DETAILED DESCRIPTION

[0022] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0024] the term

[0025] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0026] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0027] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0028] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0029] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0030] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0031] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0032] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0033] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0034] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0035] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0036] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0037] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0038] One or more embodiments of the present application provide a polypeptide, the amino acid sequence of the polypeptide is shown as SEQ ID NO.1.

[0039] "Polypeptide" is a compound with more than 10 peptide bonds (-CONH-) formed by the condensation of multiple molecules of α-NH2 and -COOH. It contains a variety of compounds with certain physiological activities in the body. It can be extracted from animal tissues or synthesized artificially.

[0040] Yet another one or more embodiments of the present application provide a use of the polypeptide in the preparation of a drug for preventing and treating neonatal enterocolitis (NEC).

[0041] In this application, "prevention" and "prevention and / or treatment" have the same meaning and can be used interchangeably. In the present invention, "prevention" includes prevention, treatment, adjuvant therapy and other aspects. As used herein, "prevention" means to alleviate, delay the progression, attenuate, prevent, or maintain an existing disease or condition. "Prevention" also includes curing one or more symptoms of a disease or condition, preventing its development, or alleviating it to a certain extent.

[0042] One or more embodiments of the present application also provide a drug, which includes the polypeptide and pharmaceutically acceptable excipients.

[0043] In this application, "drug" includes any agent, compound, composition or mixture that provides a pharmacological effect in vivo or in vitro, and often provides a beneficial effect. The scope of the pharmacological effect of the "drug" in vivo is not particularly limited, and it can be a systemic effect or only a local effect. The activity of the "drug" is not particularly limited, and it can be an active substance that can interact with other substances, or it can be an inert substance that does not interact.

[0044] As used herein, "pharmaceutically acceptable" refers to those ligands, materials, compositions, and / or dosage forms that are suitable for administration to a patient within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0045] In this application, "pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. As used herein, the phrase "pharmaceutically acceptable excipient" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with drug administration. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Suitable examples include, but are not limited to: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch, potato starch and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc. Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffered saline; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0046] In the present application, "excipients" include but are not limited to mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium ethylenediaminetetraacetic acid (disodium EDTA), calcium sodium EDTA, carbonates, acetates, phosphates or aqueous solutions of monovalent alkali metals, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, oligofructose, dextran, glycine, starch, sucrose, dextrin (such as maltodextrin), lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, and magnesium stearate.

[0047] One or more embodiments of the present application also provide a method for producing the polypeptide. The present application does not specifically limit the method for producing the polypeptide, which may be a chemical synthesis method or a biosynthesis method, such as fermentation preparation by constructing an engineered bacterium that exogenously expresses the polypeptide.

[0048] One or more embodiments of the present application also provide a nucleic acid molecule encoding the polypeptide.

[0049] One or more embodiments of the present application also provide a vector, which includes the nucleic acid molecule.

[0050] One or more embodiments of the present application also provide a cell, which includes the nucleic acid molecule, the vector or exogenously expresses the polypeptide.

[0051] One or more embodiments of the present application also provide the use of the polypeptide in preparing an inhibitor of the transformation of macrophages into M1 macrophages.

[0052] One or more embodiments of the present application also provide an inhibitor of the transformation of macrophages into M1 macrophages, wherein the inhibitor comprises the polypeptide.

[0053] One or more embodiments of the present application also provide a method for inhibiting the transformation of macrophages into M1 macrophages, the method comprising treating macrophages with the polypeptide to inhibit the transformation of macrophages into M1 macrophages.

[0054] One or more embodiments of the present application also provide a method for preventing and treating neonatal enterocolitis, comprising administering a therapeutically effective amount of the polypeptide of the present application to a subject.

[0055] In some embodiments, the administration of the drug includes, but is not limited to, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection, and topical administration, inhalation.

[0056] In some of these embodiments, the drug may be administered orally, by enema, or parenterally.

[0057] In some of these embodiments, the drug administration cycle can be intermittent administration, periodic administration, continuous administration or long-term administration.

[0058] Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffer. Solid dosage forms such as tablets, sugar pills, capsules, pills and granules may be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active ingredient in such compositions may be delayed in a certain part of the digestive tract. Examples of embedding components that may be used are polymeric substances and waxes. If necessary, the active ingredient may also be formed into microcapsules with one or more of the above-mentioned excipients.

[0059] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, specifically, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices. For example, the suspension may contain a suspending agent, specifically, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitan esters, microcrystalline cellulose, aluminum methylate and agar or mixtures of these substances.

[0060] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0061] Dosage forms for topical administration include ointments, powders, patches, sprays and inhalants, which are prepared by mixing the active ingredient with pharmaceutically acceptable excipients and any preservatives, buffers, or propellants that may be required under sterile conditions.

[0062] It is to be understood that the drugs of the embodiments of the present invention can be added with different pharmaceutically acceptable carriers to prepare suitable clinical dosage forms, which include but are not limited to the dosage forms described above.

[0063] In the present application, "subject" refers to a patient who takes a polypeptide or a pharmaceutical composition.

[0064] In the present application, "subject" is an animal, preferably a mammal, more preferably a human, and the subject includes but is not limited to consumers of health products and patients with diseases, disorders and / or symptoms. The subject in the present invention is preferably a mammal. The term "mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: such as cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, humans, etc., preferably primates, more preferably humans.

[0065] In some embodiments, the drug is suitable for humans or other mammals. In the present invention, "other mammals" are not humans, and non-limiting examples of "other mammals" include cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, etc., and further include mice or rats; non-limiting examples of "other mammals" include primates.

[0066] In some embodiments, the subject is a mammal.

[0067] In some embodiments, the subject is a human, rat, or mouse.

[0068] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0069] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0070] We collected breast milk (colostrum, mature milk) from premature infants at different stages. Referring to the reported research scheme, we added pepsin and trypsin to breast milk and mixed it at 37°C to simulate gastrointestinal digestion. The obtained product was separated by ultrafiltration interception (10KDa) to separate small molecule peptides in the product and quantified after freeze-drying. Then we performed peptide omics analysis and identified a total of 6182 small molecule peptides. Further analysis found that the molecular weight of these peptides was mainly concentrated in 1000Da-3000Da, and the number of amino acids contained was mainly between 5-25. Given that colostrum contains more abundant nutritional value and active molecules, and previous experiments have found that the peptide mixture obtained by hydrolysis of colostrum protein has more significant biological functions than mature milk. Therefore, further analysis of colostrum polypeptides revealed that 52 polypeptides (numbered ANECP1 to ANECP52) were significantly higher than those in mature milk (>6 times, p<0.05). This embodiment of the application used "① the most significant difference; ② high polypeptide abundance" as the screening criteria to screen out peptide No. 1 (SEQ ID NO.1: DLENLHLPLPLLQPLM).

[0071] Newborn SD rats (1 day old) were fed with Abbott puppy formula every 3 hours through an orogastric feeding tube (the concentration of the milk powder was based on the official recommended concentration of Abbott puppy formula). To induce necrotizing enterocolitis (NEC), these newborns were fed 3 times a day and exposed to 5% O after each feeding. 2 and 95% N 2 atmosphere for 10 minutes, thereby withstanding the pressure. During the experiment, the feeding amount started from 0.1 mL and gradually increased to 0.25 mL. The healthy control group consisted of naturally born newborn rats, which were fed by their mothers. The newborn rats were injected intraperitoneally with peptide No. 1 (200 mg / kg) once a day during the establishment of the NEC model, and the healthy control group was injected with control peptide (Scr) (200 mg / kg) accordingly. At the end of the experiment on the 5th day, the animals were killed and the colon was collected for H&E staining, colon length measurement, and NEC score evaluation.

[0072] Pathologists performed histological evaluation of H&E-stained intestinal sections to assess ileal injury using a previously published NEC scoring system. This system evaluates the degree of intestinal injury on a scale of “0 to 4” as follows: 0: no histological injury; 1 (mild): slight separation of the submucosa and / or lamina propria; 2 (moderate): moderate separation of the submucosa and / or lamina propria and / or edema in the submucosa and muscle layers; 3 (severe): severe separation of the submucosa and / or lamina propria and / or severe edema in the submucosa and muscle layers, with localized villus loss; 4 (necrosis): loss of villi and necrosis. A score ≥2 was defined as NEC.

[0073] The results are as follows Figure 1 As shown, neonatal rats were intraperitoneally injected with peptide 1 once a day during the establishment of the NEC model (N=6), and the results showed that ANECP1 could significantly improve NEC intestinal damage.

[0074] Colons were collected and subjected to immunofluorescence analysis, with F4 / 80 in green and iNOS in red. The H&E staining steps included preparation, dewaxing of the sections and blocking with 5% BSA for 1 h at room temperature. Subsequently, they were incubated with the designated primary antibodies at a dilution of 1:500 at 4 °C overnight. After washing with PBS, the cells were incubated with Alexa Fluor 488 or 568-conjugated secondary antibodies at a dilution of 1:500 for 1 h while avoiding exposure to light. Cell nuclei were stained with DAPI. The results of immunofluorescence staining were observed using an Olympus BX53 microscope.

[0075] The results are as follows Figure 2 As shown, the results showed that ANECP1 inhibited the transformation of intestinal macrophages into M1 pro-inflammatory macrophages.

[0076] During the experiment, macrophages were pretreated with ANECP1 at the indicated concentration (i.e., treatment concentration) for 1 hour and then exposed to 10 ng / mL lipopolysaccharide for 6 hours. Subsequently, macrophages were collected for qPCR analysis to evaluate the levels of inflammatory factors.

[0077] qPCR reaction system (20μL): 10μL SYBR, 7.2μL DEPC water, 0.4μL primer-F+0.4μL primer-R+2μL cDNA.

[0078] Table 1. Detection primers

[0079]

[0080]

[0081] The reaction procedure of qPCR was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s (40 cycles from denaturation to extension steps).

[0082] The results are as follows Figure 3 As shown, the results showed that ANECP1 significantly inhibited the expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) in macrophages.

[0083] During the experiment, bone marrow-derived macrophages were pretreated with ANECP1 at the indicated concentration (i.e., 50 μM, i.e., treatment concentration) for 1 hour and then exposed to 10 ng / mL lipopolysaccharide for 24 hours. Subsequently, cells were collected for flow cytometric analysis of CD86 and CD206; the collected cells were suspended in FACS buffer (2% FCS in PBS). Subsequently, cells were treated with anti-Fc receptor antibodies at 4°C for 15 minutes. Afterwards, Alexa Fluor was used to detect the expression of Alexa Fluor. Cells were stained with 488 anti-mouse F4 / 80 antibody, APC anti-mouse CD86 antibody, and PE anti-mouse CD206 (MMR) antibody. Samples were analyzed using a BD FACSCanto II, and data were analyzed using FlowJo software.

[0084] The results are as follows Figure 4 As shown, the results showed that treatment with ANECP1 resulted in a decrease in the proportion of CD86-positive cells (M1 macrophages) from about 18% to about 10% compared to cells exposed to LPS alone. However, ANECP1 treatment did not show any significant effect on the proportion of M2 macrophages.

[0085] The study in this example found that ANECP1 can improve NEC intestinal damage by inhibiting the transformation of macrophages into M1 macrophages. This discovery provides a new strategy and method for the prevention and treatment of NEC.

[0086] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Use of a polypeptide in the preparation of a drug for preventing and treating neonatal enterocolitis; the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

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