Polypeptides for treating ulcerative colitis

Abstract

The present application relates to the technical field of prevention and treatment of intestinal inflammatory diseases, and in particular to a polypeptide for treating ulcerative colitis. The polypeptide provided by the present application is an active heptapeptide derived from a natural protein, has a clear chemical structure and a reproducible preparation process. Research results show that the polypeptide can significantly improve the symptoms of ulcerative colitis induced by dextran sodium sulfate (DSS) in mice, including reducing body weight loss, reducing disease activity index (DAI), restoring colon length, improving colon tissue pathological damage and inhibiting inflammation-related signaling pathways, thereby verifying its improvement effect on intestinal inflammation. By preparing the polypeptide into a pharmaceutical composition, a comprehensive effect of prevention and treatment, inflammation improvement and barrier repair in parallel can be achieved, thereby providing a new strategy and material basis for the prevention and treatment of ulcerative colitis and other intestinal inflammatory diseases.

Description

Technical Field

[0001] This invention relates to the field of prevention and treatment of intestinal inflammatory diseases, and in particular to a polypeptide for treating ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a common chronic inflammatory bowel disease. Clinical symptoms mainly include persistent or recurrent diarrhea, abdominal pain, and bloody stools. Some patients also experience neurological, respiratory, and urinary system complications. Recent epidemiological surveys show that this disease has affected the health of millions of people worldwide over the past 20 years. Poor dietary habits, pathogen infections, and food contamination can all cause damage to the colonic mucosa, including congestion, edema, necrosis, and ulceration, severely impairing the intestinal barrier and leading to UC.

[0003] The intestinal mucosal barrier is a functional structural system with highly selective barrier effects. The mechanical barrier is the most important component of this system, with tight junction protein-1 (ZO-1) and mucin 2 (MUC2) playing crucial roles in maintaining the integrity of the intestinal mucosal mechanical barrier. Abnormal secretion or overexpression of inflammatory cytokines is considered an important pathogenic mechanism leading to the occurrence and development of colitis. The nuclear transcription factor / NOD-like receptor thermoprotein domain 3 (NF-κB / NLRP3) signaling pathway is an important pathway involved in apoptosis and inflammation, playing a significant role in the development and progression of colitis. NF-κB participates in the initial stage of inflammation by initiating the transcription of pro-inflammatory factors, while the NLRP3 inflammasome amplifies the inflammatory response by activating caspase-1, mediating the maturation and release of interleukin-1β (IL-1β) and interleukin-18 (IL-18). Currently, clinical treatment mainly relies on anti-inflammatory and biological immunomodulatory agents. However, long-term use leads to problems such as drug dependence, drug resistance, decreased immune function, and unsatisfactory efficacy. Furthermore, the high cost and high relapse rate after discontinuation pose significant challenges to treatment. Finding economical, effective, and safe treatments for the prevention and treatment of UC remains a hot research topic in the academic community.

[0004] Macadamia nuts are highly nutritious and have an excellent taste, rich in monounsaturated fatty acids. Macadamia protein (MP) is a protein extracted from the mealy pulp left after cold-pressing for oil extraction. It contains all eight essential amino acids for the human body, including lysine, tryptophan, and phenylalanine, in a balanced manner, making it a high-quality source of plant protein. In recent years, with the deepening research on the bioactivity of nut protein peptides, nut protein peptides have shown various activities such as antioxidation, antitumor activity, blood pressure reduction, brain health, and immune regulation. The physiological activity of nut protein peptides is related to the molecular weight and amino acid sequence composition of the peptides, and the isolation and purification of bioactive peptides from nuts has become a current research hotspot. Currently, there is a lack of research reports on the application of macadamia nut peptides in the prevention and treatment of ulcerative colitis (UC). Summary of the Invention

[0005] This invention covers the following technical solutions:

[0006] One aspect of the present invention relates to a polypeptide having the amino acid sequence shown in SEQ ID NO: 1.

[0007] Another aspect of the present invention relates to a nucleic acid capable of expressing the polypeptide as described above.

[0008] Another aspect of the present invention relates to a carrier containing the nucleic acid described above.

[0009] Another aspect of the present invention relates to a host cell containing the nucleic acid as described above or the vector as described above.

[0010] Another aspect of the present invention relates to a pharmaceutical composition comprising the polypeptide as described above and a pharmaceutically acceptable excipient.

[0011] Another aspect of the present invention relates to the use of the polypeptide described above in the preparation of a medicament for treating ulcerative colitis.

[0012] The polypeptide provided by this invention is an active heptapeptide derived from a natural protein, possessing a well-defined chemical structure and a reproducible preparation process. Research results show that this polypeptide can significantly improve the symptoms of DSS-induced ulcerative colitis in mice, including reducing weight loss, decreasing the disease activity index (DAI), restoring colon length, and improving colonic tissue pathological damage, thus verifying its ameliorative effect on intestinal inflammation. By formulating this polypeptide into a pharmaceutical composition, a comprehensive effect of prevention and treatment, and simultaneous inflammation improvement and barrier repair can be achieved, providing a new strategy and material basis for the prevention and treatment of ulcerative colitis and other inflammatory bowel diseases. Attached Figure Description

[0013] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0014] Figure 1 Isolation, identification, and synthesis of MPAF from macadamia nuts. A: Amino acid sequence and structure of MPAF; B: LC-MS / MS chromatogram of MPAF isolation and identification; C: HPLC chromatogram of MPAF chemical synthesis purity detection; D: LC-MS chromatogram of MPAF molecular weight detection.

[0015] Figure 2Effects of MPAF on liver biochemical parameters and tissue morphology in UC mice. A: ALT; B: AST; C: AKP; D: Liver morphology and structure.

[0016] Note: Compared to group CON P < 0.01, P < 0.05; compared with the DSS group, ##P < 0.01, #P < 0.05, the same below.

[0017] Figure 3 Effects of MPAF on body weight, DAI score, colon length, and histological structure in UC mice. A: Mouse body weight, B: DAI score, CD: Colon length, E: Colon morphology and structure.

[0018] Figure 4 Effects of MPAF on the intestinal barrier in UC mice. A: PAS-stained colonic tissue; B: Goblet cell count; C: ZO-1 protein expression in the colon; D: IOD value of ZO-1 protein; E: Relative expression level of ZO-1 mRNA; F: Relative expression level of MUC-2 mRNA.

[0019] Figure 5 Effects of MPAF on the activity of SOD (A), GSH-pX (B), and CAT (C) in the colon of UC mice.

[0020] Figure 6 KEGG pathway enrichment analysis and molecular docking of NF-κB and NLRP3 proteins; A: Top 20 pathways in the KEGG database NAFLD; B: Volcano plot generated using fold change values ​​and p-value correction values; Red dots represent genes with significant fold change values ​​and p-values, blue dots represent genes with significant p-values, and gray dots represent genes with neither significant fold change values ​​nor p-values; Differentially expressed genes have been annotated; C: Molecular docking pattern of MPAF and NF-κB; D: Molecular docking pattern of MPAF and NLRP3.

[0021] Figure 7 Effects of MPAF on the NF-κB / NLRP3 pathway in the colon of UC mice; A: NF-κB protein expression in the colon; B: NF-κB protein level statistics; C: WB detection of NLRP3 and NF-κB (p-p65 / p65) proteins in the colon; D: NLRP3 and NF-κB (p-p65 / p65) protein expression statistics; F: Relative expression levels of NF-κB, NLRP3, ASC, Caspase-1, IL-1β, and IL-18 mRNA.

[0022] Figure 8Effects of MPAF on inflammatory factors in the colon of UC mice; A: Immunohistochemical detection of TNF-α expression in the colon; B: Statistical analysis of TNF-α protein levels; C: ELISA detection of TNF-α content in the colon; D: ELISA detection of IL-6 content in the colon. Detailed Implementation

[0023] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0024] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0025] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, immunology-related terms and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0026] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0027] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0028] As used in this invention, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5%, of a given value or range. It also includes specific numbers, such as about 20 including 20.

[0029] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0030] The concentration values ​​involved in this invention include fluctuations within a certain range. For example, they can fluctuate within a corresponding precision range.

[0031] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0032] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0033] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0034] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0035] In this invention, the terms "treatment," "therapeutic," or "method of treatment" refer to the administration of an active ingredient to produce any beneficial physiological or pathological effect, including but not limited to: preventing the occurrence of a disease or pathological state; reducing, alleviating, or inhibiting the development of an existing disease or the worsening of its symptoms; and improving or eliminating an existing disease, lesion, or its symptoms. Therefore, the "treatment" described in this invention includes both prophylactic and therapeutic administration, which can be used in subjects who have not yet developed the disease to reduce the risk of disease, or in subjects who have developed the disease to alleviate, inhibit, or reverse the disease progression.

[0036] In this invention, the term "polypeptide" or "peptide" refers to a linear or cyclic molecule formed by two or more amino acid residues linked by peptide bonds (amide bonds). The polypeptide can be a peptide composed of natural amino acid residues, or it can contain non-natural amino acids, D-type amino acids, modified amino acids, or derivatives thereof. The polypeptide can be a product obtained from a natural source, through chemical synthesis, recombinant expression, or in vitro translation system. The polypeptide may further include its pharmaceutically acceptable salts, isotope labels, protecting derivatives, prodrug forms, fragments, analogs, or modifications, provided that they maintain a substantially identical structural backbone to the original polypeptide or have similar biological activity. In some embodiments, the polypeptide may be an oligopeptide (typically containing 2–20 amino acid residues) or a high molecular weight polypeptide.

[0037] In this invention, the term "nucleic acid" refers to a polymer molecule formed by nucleotide units linked by phosphodiester bonds, including both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleic acid can be single-stranded or double-stranded, linear or circular, and can be a naturally occurring sequence or a sequence that has been artificially modified, synthesized, mutated, or optimized; its nucleotide units may contain natural or non-natural bases, modified nucleotides, isotopic labels, or protecting groups. The nucleic acid described in this invention can be obtained through chemical synthesis, in vitro transcription, recombinant expression, or other biotechnological means, and includes pharmaceutically acceptable salts, complementary strands, antisense sequences, or mRNA forms encoding a target polypeptide (e.g., SEQ ID NO: 1).

[0038] This invention relates to polypeptides, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0039] This polypeptide can be obtained through chemical synthesis, recombinant expression, or enzymatic hydrolysis purification, and has high purity and stability.

[0040] In some embodiments, the polypeptide is a linear heptapeptide with a molecular weight of approximately 793 Da. This polypeptide is stable under in vivo or in vitro conditions and can be prepared and delivered using conventional drug carriers.

[0041] The polypeptide may further include its pharmaceutically acceptable salts, derivatives, isotope labels, protecting derivatives, prodrug forms, or analogs having the same amino acid backbone structure, provided that they maintain the same or similar biological activity as the polypeptide defined in SEQ ID NO: 1.

[0042] In a preferred embodiment, the polypeptide exhibits antioxidant, anti-inflammatory, and intestinal mucosal barrier-maintaining activities, and can be used to prevent or treat intestinal diseases caused by inflammatory responses or oxidative stress, especially ulcerative colitis.

[0043] According to one aspect of the invention, nucleic acids capable of expressing the polypeptides described above are also involved.

[0044] According to one aspect of the invention, a vector containing nucleic acids as described above is also involved.

[0045] To improve expression efficiency in different hosts, the nucleic acid sequence of the present invention can be codon optimized according to the codon usage preferences of the target host (such as Escherichia coli, yeast, insect cells or mammalian cells). The optimized nucleic acid is still within the protection scope of the present invention without changing the encoded amino acid sequence.

[0046] The nucleic acid can be a single-stranded or double-stranded structure, a linear or circular molecule, and may include its complementary strand, antisense sequence, splice variant, fragment, fusion sequence, leader sequence, tag sequence (such as His tag, FLAG tag), or functional elements such as promoter, enhancer, terminator, etc. that can regulate its expression.

[0047] Furthermore, the nucleic acids of this invention also include their chemically modified or non-natural nucleotide forms, such as those containing modified bases (e.g., methylcytosine, pseudouridine), modified ribose (e.g., 2′-O-methylribose), phosphorylation-thioyl bonds, locked nucleic acid (LNA) units, isotopically labeled nucleotides, or other structural modifications to improve stability and translation efficiency. These modified or optimized nucleic acids, as long as they can express the polypeptide defined in SEQ ID NO: 1, are all within the scope of protection of this invention.

[0048] The term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences).

[0049] The present invention also relates to host cells containing the nucleic acids described above or the vectors described above.

[0050] The present invention also relates to pharmaceutical compositions comprising the polypeptides described above and pharmaceutically acceptable excipients.

[0051] Specific examples of substances that can be used as pharmaceutically acceptable excipients include phosphoric acid, citric acid, and other organic acids; antioxidants (e.g., ascorbic acid and methionine); antibacterial agents (e.g., octadecyl dimethylbenzene ammonium chloride, hexachlorocyclohexane quaternary ammonium chloride, benzalkonium chloride, phenol, butanol or benzyl alcohol, alkylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, or m-cresol); low molecular weight (less than about 10 kDa) peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; and hydrophilic polymers. The pharmaceutical composition may contain, for example, polyvinylpyrrolidone; amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including, for example, glucose, mannose, or dextran); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions; metal complexes; and / or nonionic surfactants (e.g., including TWEENTM, PLURONICS™, or polyethylene glycol). Furthermore, depending on the formulation method, commonly used fillers, diluents, binders, humectants, disintegrants, and / or surfactants may be appropriately selected by those skilled in the art. The pharmaceutical composition may be in solid, semi-solid, or liquid form, preferably in liquid form.

[0052] In some embodiments, the pharmaceutical composition further comprises a peptide stabilizer or a nucleic acid stabilizer.

[0053] In some embodiments, the pharmaceutical composition is an oral formulation, a rectal formulation, or an enteric-coated formulation for targeted release to the colon.

[0054] In some embodiments, the oral formulation is selected from tablets, capsules, granules, oral solutions, or oral emulsions. Appropriate amounts of diluents, disintegrants, lubricants, binders, solubilizers, or preservatives may be added as needed. For applications requiring action in the colon, enteric coating or pH-responsive polymer encapsulation techniques can be used to ensure drug stability in the stomach and small intestine, releasing the peptide only in the colonic environment, thereby achieving targeted delivery and high local efficacy.

[0055] In another embodiment, the pharmaceutical composition may further comprise a delivery carrier to improve the stability and bioavailability of the peptide. Suitable delivery carriers include, but are not limited to, liposomes, solid lipid nanoparticles, polylactic-co-glycolic acid (PLGA) nanoparticles, or chitosan nanogels. These carriers can bind to the peptide through encapsulation, adsorption, or covalent linkage to form a sustained-release or targeted delivery system, thereby avoiding enzymatic degradation of the peptide in the gastrointestinal tract and increasing the effective concentration and duration of action in the intestinal tract. The pharmaceutical composition may also be formulated as a solution, suspension, emulsion, or lyophilized powder as needed, and can be administered orally or rectally to prevent or treat ulcerative colitis.

[0056] The present invention also relates to the use of the polypeptides described above in the preparation of medicaments for the treatment of ulcerative colitis.

[0057] The present invention also relates to a method for treating ulcerative colitis, comprising the step of administering a safe and effective amount of the pharmaceutical composition as described above to a subject.

[0058] The phrase "safe and effective amount" means, as used herein, a compound or composition in a reasonable pharmaceutically modifiable amount sufficient to significantly and effectively relieve the symptoms or condition being treated, but small enough to avoid serious side effects (with a reasonable benefit / risk ratio). The safe and effective amount of the active ingredient in the pharmaceutical composition used in the methods of this invention varies depending on the specific symptoms being treated, the age and physical condition of the treated subject, the severity of the disease, the duration of treatment, concurrent treatments, the specific active ingredient used, the specific pharmaceutically acceptable excipients used, and factors including the knowledge and skills of the physicians involved in the treatment.

[0059] The pharmaceutical compositions of the present invention can be administered via any route, as will be known to those skilled in the art. In some embodiments, the delivery system / pharmaceutical compositions of the present invention are administered orally (PO), intravenously (IV), intramuscularly (IM), intra-arterially, intramedullaryly, intrathecally, subcutaneously (SQ), intravenously, percutaneously, intradermally, intradermally, transrectally (PR), transvaginally, intraperitoneally (IP), intragastrically (IG), topically (e.g., using powders, ointments, creams, gels, lotions and / or drops), mucous membranes, intranasally, intrabuccally, transintestinally, vitreously, sublingually; via tracheal instillation, bronchial instillation and / or inhalation; as an oral spray, nasal spray and / or aerosol and / or via a portal vein catheter.

[0060] The term "subject" as used in this invention can refer to a patient or other animal receiving the drug described in this invention to treat, prevent, alleviate, and / or relieve the disease, condition, or symptom described in this invention. Subjects include warm-blooded animals, such as mammals like pandas, elephants, primates (chimpanzees, orangutans, gibbons, macaques, marmosets), and preferably humans. Non-human primates are also considered individuals. The term "individual" includes domesticated animals such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, goats), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs).

[0061] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0062] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0063] Example

[0064] 1. Materials and Methods

[0065] 1.1 MPAF Preparation

[0066] Macadamia nut protein powder was hydrolyzed with neutral protease (enzyme dosage 200 U / g, pH adjusted to 7.0, temperature 55℃, time 3 h), and ultrafiltration was used to obtain peptides smaller than 1000 Da. The peptides were then separated and purified sequentially using macroporous resin and cross-linked dextran gel, and their sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The obtained peptide sequences were used for bioactivity prediction. The heptapeptide Ala-Cys-Asn-Pro-Phe-Gly-Trp (ACNPFGW, MPAF), with a PeptideRanker score exceeding 0.9, was selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for chemical synthesis as part of subsequent experimental studies. MPAF was scored using the PeptideRanker database, toxicity was predicted using ToxinPred, purity was determined by high-performance liquid chromatography (HPLC), and molecular weight was determined by LC-MS.

[0067] 1.2 Animal grouping and administration

[0068] Forty healthy male SPF-grade C57BL / 6 mice, weighing (26±2) g, were used. The animals were housed in a clean-grade animal facility at a temperature of 25±2℃, humidity of 55%±8%, with 12-hour light exposure and free access to food and water. The mice were randomly divided into four groups (n=10 per group): control (CON), model (DSS), low-dose MPAF (MPAF-L), and high-dose MPAF (MPAF-H). Starting on day 1, mice in the CON group had free access to distilled water, while the other three groups had free access to 3% DSS solution to induce the ulcerative colitis (UC) model, for 7 consecutive days. Starting on day 8, the low-dose MPAF (MPAF-L, 50 mg / kg) and high-dose MPAF (MPAF-H, 100 mg / kg) groups were administered the two doses of MPAF by gavage once daily for 28 consecutive days. Twelve hours after the last administration, the mice were sacrificed and serum, liver, and colon tissue were collected for subsequent experiments.

[0069] 1.3 Disease Activity Index

[0070] Twelve hours after the last DSS treatment, the body weight, fecal characteristics, and presence of blood in the feces of mice in each group were recorded and scored from 0 to 4: weight loss (0: 0-1%; 1: 1-5%; 2: 5-10%; 3: 10-15%; 4: >15%, percentage refers to mass fraction); fecal viscosity (0: normal; 2: loose stool; 4: diarrhea); and fecal bleeding (0: normal; 2: bloody; 4: total bleeding). The Disease Activity Index (DAI) score was the mean of the three scores, calculated as: DAI = (weight loss score + fecal viscosity score + fecal bleeding score) / 3.

[0071] 1.4 Colon length measurement

[0072] Mice were euthanized by cervical dislocation, and the colonic and rectal segment from the anus to the end of the cecum was removed. The segment was rinsed with phosphate-buffered saline (PBS) to ensure that the surface was clean and free of impurities. The length of the treated colon was measured using a ruler.

[0073] 1.5 Hematoxylin-eosin and periodic acid-Schiff staining

[0074] Mouse liver, kidney, and colon tissues were fixed in 4% paraformaldehyde for 48 h, dehydrated, embedded in paraffin, and then cut into 6 µm thick sections. Hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining kits were used for staining and mounting. Images were observed and captured using an Olympus CX43 microscope (Japan). Changes in colonic morphology were analyzed, and the number of goblet cells per unit field of view among mucosal epithelial cells was calculated.

[0075] 1.6 Immunohistochemistry

[0076] Paraffin sections were routinely dewaxed and rehydrated. The sections were placed in a container containing pH 6.0 citrate buffer and boiled over medium heat for 2-3 minutes. After natural cooling, they were washed with PBS. They were then incubated with 3% hydrogen peroxide for 30 minutes and washed with PBS. Three antibodies (ZO-1, NF-κB, and TNF-α, 1:300) were added separately and incubated overnight at 4°C. The sections were washed with PBS. Enzyme-labeled secondary antibody was added and incubated at 37°C for 30 minutes. The sections were washed with PBS. DAB staining was performed for a few seconds, followed by hematoxylin counterstaining for 5 minutes. The sections were dehydrated using a routine gradient of ethanol, cleared with xylene, mounted with neutral resin, observed under a microscope, and images were acquired and analyzed using ImageJ software.

[0077] 1.7 Detection of liver transaminases and colonic antioxidant markers

[0078] 0.3 g of liver and colon tissue from each group were weighed and homogenized in glass homogenizing tubes with 2.7 mL of physiological saline to obtain a 10% tissue homogenate. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AKP) in the liver were measured using a kit from Nanjing Jiancheng Bioengineering Institute to evaluate liver function. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-pX), and catalase (CAT) in the colon homogenate were measured according to the kit instructions from Nanjing Jiancheng Bioengineering Institute to evaluate the antioxidant capacity of the colon.

[0079] 1.8 Molecular docking

[0080] NF-κB and NLRP3 were designated as the core targets, and MPAF was designated as the ligand. The protein receptor and small molecule ligand were optimized using PyMOL 4.0 software. Docking simulations were performed using Autodock Vina, running 20 times, and ligand complexes with binding energies less than -5 kcal / mol were selected. The binding modes of the ligand and receptor in the two-dimensional structure were visualized using Discovery Studio software.

[0081] 1.9 Western blot analysis

[0082] Western blotting (WB) was used to detect the expression of NLRP3 and NF-κB p65 proteins in mouse colon tissue. Protein concentrations were determined using a BCA assay kit. After sample loading, electrophoresis, and transfer to a membrane, the tissue was incubated with NLRP3 antibody, NF-κB p65 antibody, and GAPDH antibody, respectively, followed by incubation with secondary antibodies. Images were then acquired using a gel imaging system.

[0083] 1.10 Real-time quantitative PCR detection

[0084] Total RNA was extracted from mouse colon tissue using a total RNA extraction kit, and the RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions. Primer sequences for target genes ZO-1, mucin 2 (MUC2), NF-κB, NLRP3, apoptosis-associated speckled protein (ASC), Caspase-1, IL-1β, IL-18, and the internal control β-actin were designed using the NCBI website and synthesized at Sangon Biotech (Shanghai) Co., Ltd. Amplification was performed using the SYBR Green method under the following conditions: 95 °C pre-denaturation for 10 min, 95 °C denaturation for 30 s, and Tms annealing for 30 s, for a total of 32 cycles. The relative mRNA expression levels of each gene were calculated using the 2-ΔΔCt method with GAPDH as the internal control.

[0085] The primer sequences are shown in the table below.

[0086]

[0087] 1.11 Bioinformatics Analysis

[0088] Differential expression analysis was performed between the control group and the colitis group in the dataset using the Gene Expression Comprehensive Database (GEO) and the Limma package in R software (version 4.5.1). P-values ​​were adjusted to minimize potential false positives, and thresholds of P < 0.05 and Log2 (fold change) > 1 or < 1 were adjusted to identify significantly differentially expressed genes. Pathway enrichment analysis of differentially expressed genes was performed using the KOBAS-i database.

[0089] 1.12 Molecular docking

[0090] NF-κB and NLRP3, the core targets, and MPAF, the core components, were used as the receptor and ligand, respectively. Structures of the core components were retrieved from the PubChem and TCMSP databases. The structural file (PDB format) of the target protein was downloaded from the RCSB protein structure database (https: / / www.rcsb.org). Molecular docking was performed using AutoDock software. Models with good binding affinity were mapped, and the molecular docking results were analyzed visually.

[0091] 1.13 Enzyme-linked immunosorbent assay (ELISA)

[0092] Take 10% tissue homogenate from each group of colon tissue and perform the TNF-α and IL-6 enzyme-linked immunosorbent assay (ELISA) according to the instructions of the kit (Wuhan GeneMei Technology Co., Ltd.). Measure the absorbance at 450 nm using an ELISA reader (K3 TOUCH, Thermo Fisher Scientific) and calculate the concentration of the sample protein.

[0093] 1.14 Statistical Analysis

[0094] Data analysis was performed using SPSS 26.0 statistical software, and t-tests were used to compare differences between groups. A p-value > 0.05 was considered statistically insignificant; a p-value < 0.05 indicated a significant difference between groups; and a p-value < 0.01 indicated a highly significant difference.

[0095] 2 Results

[0096] 2.1 MPAF Isolation and Biosynthesis

[0097] A seven-peptide was isolated from macadamia nuts, with the amino acid sequence Ala-Cys-Asn-Pro-Phe-Gly-Trp (ACNPFGW, MPAF). Figure 1 (A), Chinese name: Alanine-cysteine-aspartic acid-proline-phenylalanine-glycine-tryptophan. MPAF identification LC-MS / MS chromatogram as shown... Figure 1 MPAF was chemically synthesized by Sangon Biotech (Shanghai) Co., Ltd. The purity of MPAF was determined by HPLC to be 99.499% (…). Figure 1 The molecular weight of MPAF (C) was determined by LC-MS to be 793.20 Da, meeting the requirements for subsequent functional experiments. Figure 1 (Medium D). The PeptideRanker score is 0.98, close to 1, indicating strong biological activity. ToxinPred predicts that MPAF is non-toxic.

[0098] 2.2 Effects of MPAF on the liver of UC mice

[0099] The effects of MPAF on liver biochemical parameters in mice were investigated. The results showed that, compared with the CON group, DSS treatment significantly increased the levels of ALT, AST, and AKP in the liver (P<0.01). Figure 2The presence of AC (internal inflammatory markers) indicates liver damage in DSS-induced UC mice. Low and high doses of MPAF alleviated these changes (P<0.01 or P<0.01). HE staining revealed no pathological damage in the livers of the CON group mice. In the DSS group mice, hepatocytes were disorganized, with enlarged nuclei, vacuolar degeneration, and some degree of inflammatory cell infiltration. The pathological damage in the liver tissue of the MPAF-L and MPAF-H groups was effectively reduced (…). Figure 2 (D). This indicates that MPAF can improve secondary liver injury induced by DSS in UC mice in a dose-dependent manner.

[0100] 2.3 Effects of MPAF on the pathology of colitis in UC mice

[0101] First, the body weight, DAI score, and colon length of mice in each group were measured. The results showed that compared with the DSS group, the MPAF-L and MPAF-H groups had lower body weight, lower DAI scores, and shorter colon lengths (P < 0.01). Figure 3 (AD). HE staining revealed no obvious pathological changes in the colon in the CON group. In the DSS group, the inherent structure of the colonic ulcer lesions was destroyed, the mucosal layer was thinned, and there was extensive inflammatory cell infiltration. After MPAF intervention, the degree of damage to the colonic mucosal epithelium in mice was reduced, the crypt structure was more orderly arranged, and the inflammatory cell infiltration was significantly reduced. Figure 3 (E). This indicates that MPAF can improve pathological damage in the colon of UC mice.

[0102] 2.4 Effects of MPAF on the intestinal barrier in UC mice

[0103] The number of goblet cells in the mouse colon was observed by PAS staining. Figure 4 In the AB group, abundant goblet cells were found in the colonic epithelial crypts of the CON group, with mucus tissue evenly covering the crypt openings. Mice in the DSS group exhibited typical crypt developmental abnormalities, characterized by goblet cell atrophy, thinning or absence of the mucus layer at the crypt openings, and a significant reduction in the number of goblet cells (P<0.01). Compared with the DSS group, the MPAF-L and MPAF-H groups showed reduced goblet cell atrophy, some recovery of the mucus layer thickness at the crypt openings, and a significant increase in the number of goblet cells (P<0.05 or P<0.01).

[0104] To investigate the effect of MPAF on intestinal barrier function, immunohistochemistry was used to analyze ZO-1 protein expression, and qPCR was used to detect the mRNA transcription levels of MUC2 and ZO-1. The results showed that compared with the CON group, the expression of ZO-1 and MUC2 in the DSS group was significantly downregulated (P<0.01), indicating that DSS damage impaired the intestinal mucosal barrier in mice. After MPAF intervention, the ZO-1 protein level in the colon of UC mice decreased (…). Figure 4 The mRNA expression of CD1, ZO-1, and MUC2 was significantly reduced (P<0.01). Figure 4 (In the middle EF). Especially in the MPAF-H group, the changes in ZO-1 expression were significantly reversed. This indicates that NF-κB activation can downregulate the expression of ZO-1 and MUC2, increase intestinal epithelial permeability, and MPAF can repair the degree of damage to the intestinal barrier caused by DSS.

[0105] 2.5 Effects of MPAF on colonic antioxidant markers in UC mice

[0106] Compared with the CON group, the activities of SOD, GSH-Px, and CAT in the colon tissue of the DSS group were significantly decreased (P < 0.01). Figure 5 (AB). Compared with the DSS group, the activities of the three indicators in the MPAF-L group and MPAF-H group were significantly increased (P<0.01). This indicates that MPAF can increase the activities of SOD, GSH-Px and CAT in the colonic tissue of UC mice and improve the antioxidant level of the colon.

[0107] 2.6 MPAF docking analysis with target protein molecules

[0108] Based on the KEGG database, colonic data from mice with ulcerative colitis (UC) were reanalyzed. KEGG enrichment pathway analysis revealed that the NF-κB pathway is involved in the development of UC. Figure 6 (A), and differential gene analysis revealed that NF-κB-related TNF was upregulated ( Figure 6 (Middle B). NF-κB / NLRP3 plays an important role in cell activation and the production of inflammatory factors. Using AutoDockVina software, molecular docking was performed on MPAF with key target proteins NF-κB and NLRP3 to determine the binding ability of the ligand to the protein. The lower the binding energy, the more stable the binding between the molecule and the target. It was found that MPAF can bind to both proteins, and the binding energy of NF-κB to MPAF is -28.4512 kJ / mol. Figure 6 The binding energy between NLRP3 and MPAF is -31.38 kJ / mol (C). Figure 6 (D). This indicates that MPAF has a regulatory effect on the core targets of NF-κB and NLRP3.

[0109] 2.7 Effects of MPAF on the colonic NF-κB / NLRP3 pathway in UC mice

[0110] During disease development, NF-κB can activate the formation of NLRP3, ASC, and Caspase-1 inflammasomes, activate inflammatory factors such as IL-1β and IL-18, and promote the occurrence and development of inflammatory responses. Immunohistochemical staining was used to detect the expression of NF-κB protein in colonic tissue, with brown-yellow positive cells. Results showed that compared with the CON group, the NF-κB protein level in the DSS group mice was significantly increased. After intervention with different concentrations of MPAF, the expression of NF-κB protein in the colon was significantly decreased (P<0.01). Figure 7 (AB). Western blotting was used to detect the expression of NLRP3 and NF-κB P65 proteins in colonic tissue. MPAF significantly inhibited the expression of NLRP3 and NF-κB proteins in the colon of UC mice (P<0.01). Figure 7 (CE).

[0111] The effect of MPAF on the mRNA transcription levels of key genes in the NF-κB / NLRP3 pathway in mouse colon tissue was further detected using q-PCR. The results showed that, compared with the CON group, the levels of NF-κB, NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the colon tissue of mice in the DSS group were significantly increased (P<0.01). Compared with the DSS group, the mRNA transcription levels of each gene in the colon tissue of the MPAF-L and MPAF-H groups were decreased (P<0.01). Figure 7 (Middle F). This indicates that MPAF can improve inflammatory damage in the colon by regulating the NF-κB / NLRP3 signaling pathway.

[0112] 2.8 Effects of MPAF on colonic inflammatory factors in UC mice

[0113] Immunohistochemistry was used to detect the expression level of TNF-α in the colonic tissue of mice in each group. Compared with the CON group, the DSS group showed significantly increased TNF-α content and expression in the colon (P<0.01). Compared with the DSS group, the MPAF-L and MPAF-H groups showed significantly decreased TNF-α expression in the colonic tissue of mice (P<0.01). Figure 8 (AB). This suggests that MPAF significantly inhibits TNF-α protein expression in the colonic tissue of IBD mice. The expression levels of pro-inflammatory factors TNF-α and IL-6 in each group were measured by ELISA. The results showed that compared with CON, the expression levels of TNF-α and IL-6 in the colonic tissue of mice in the DSS group were significantly increased (P<0.01), while the expression levels of TNF-α and IL-6 in the colonic tissue of mice in the MPAF-L and MPAF-H groups were significantly decreased (P<0.01). Figure 8(CD). This suggests that MPAF can inhibit the levels of TNF-α and IL-6 inflammatory factors in the colon tissue of UC mice.

[0114] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A polypeptide, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. Nucleic acid, characterized in that, It can express the polypeptide described in claim 1.

3. A carrier, characterized in that, It contains the nucleic acid as described in claim 2.

4. A host cell, characterized in that, It contains the nucleic acid as described in claim 2 or the vector as described in claim 3.

5. A pharmaceutical composition, characterized in that, It comprises the polypeptide of claim 1 and a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is an oral or rectal formulation.

7. The pharmaceutical composition according to claim 6, characterized in that, The oral preparation is selected from tablets, capsules, granules, oral solutions, or oral emulsions.

8. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is an enteric-coated formulation for targeted release into the colon.

9. The pharmaceutical composition according to any one of claims 5-8, characterized in that, The pharmaceutical composition further comprises a delivery carrier selected from liposomes, solid lipid nanoparticles, PLGA nanoparticles, or chitosan nanogels.

10. The use of the polypeptide of claim 1 in the preparation of a medicament for treating ulcerative colitis.

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