Polypeptides for treating nonalcoholic fatty liver disease

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a polypeptide for treating non-alcoholic fatty liver disease. The present application provides a novel octapeptide P3-1 (SEQ ID NO: 1) derived from macadamia nut, which can significantly improve liver lipid metabolism disorder, restore liver function, reduce inflammatory response and increase probiotic abundance in non-alcoholic fatty liver disease mice induced by high-fat diet. The polypeptide has small molecular weight, safe source and easy absorption, has good potential for pharmaceutical development, and provides a safe and effective new way for targeted intervention of non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polypeptide for treating non-alcoholic fatty liver disease. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease, with its global prevalence increasing from 25% before 2005 to 38% in recent years, becoming a public health problem affecting more than one-third of adults worldwide. The pathological progression of NAFLD ranges from simple steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and ultimately cirrhosis and even malignant transformation into hepatocellular carcinoma. In the complex pathogenesis of NAFLD, the imbalance between energy intake and expenditure leading to excessive accumulation of lipids in the liver, resulting in fatty acid metabolism disorders, oxidative stress, and inflammatory responses, is the core pathological mechanism of NAFLD. Long-term HFD leads to hepatic free fatty acid overload, subsequently causing lipid metabolism disorders, activating the switch of the fatty acid synthase (FASN)-mediated lipid synthesis pathway, and further affecting the expression of key genes in fatty acid synthesis and transport, such as stearoyl-CoA desaturase-1 (SCD-1) and fatty acid-binding protein 1 (FABP1). In addition, the large accumulation of lipid droplets in hepatocytes leads to lipid metabolism disorders, resulting in increased lipid peroxidation levels, which further induces inflammation and causes liver damage, causing NAFLD to gradually develop into NASH.

[0003] The liver and gastrointestinal tract are closely linked physiologically and pathologically. The liver forms a unique "hepatic-enterocirculatory" system with the intestines via the portal vein. Cholesterol is converted into bile acids in the liver and secreted into the intestines. Most of these bile acids are reabsorbed in the distal small intestine and then transported back to the liver. Gut microbiota also play a role in metabolic diseases (including NAFLD) through the hepatic-enterocirculatory axis. Dysbiosis can disrupt the intestinal mucosal barrier, increasing intestinal permeability and allowing harmful gut bacteria and harmful metabolites such as lipopolysaccharide (LPS) to enter the liver via the portal vein. This induces endoplasmic reticulum stress, oxidative stress, and other intracellular reactions, activating signaling pathways such as nuclear factor-κB (NF-κB) to produce pro-inflammatory factors, thus exacerbating hepatocellular damage and the progression of fatty liver disease.

[0004] Currently, drugs used to treat obesity, such as orlistat and metformin, face risks of insufficient specificity and side effects. Most drugs under development, such as glucose-dependent insulinotropic peptide (GIP) receptor agonists, BMP4 inhibitors, and FGF21 analogs, focus on regulating abnormal lipid accumulation or insulin resistance via a single receptor, resulting in limited target targeting and significant gastrointestinal side effects. Therefore, finding safe and effective drugs for the prevention and treatment of NAFLD is of great practical significance. Numerous studies have shown that plant protein peptides possess various physiological activities, including antioxidant, anticancer, blood pressure-lowering, lipid-regulating, and immune-modulating effects. Furthermore, the small molecular weight of protein peptides makes them easier for the human body to absorb, making nut protein peptides a current research hotspot.

[0005] Nuts, as a healthy food, contain protein peptides that can improve the intestinal barrier and regulate gut microbiota homeostasis. Macadamia nuts, in particular, are rich in plant protein and are an excellent source for preparing peptides. Previous studies by our research group have found that macadamia nut peptides can alleviate the pathological response of acetaminophen-induced liver damage in mice by inhibiting the Toll-like receptor 4 (TLR-4) / NF-κB inflammatory signaling pathway and downregulating the levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Summary of the Invention

[0006] This invention covers the following technical solutions:

[0007] One aspect of the present invention relates to a polypeptide having the amino acid sequence shown in SEQ ID NO: 1.

[0008] Another aspect of the present invention relates to a nucleic acid capable of expressing the polypeptide as described above.

[0009] Another aspect of the present invention relates to a carrier containing the nucleic acid described above.

[0010] Another aspect of the present invention relates to a host cell containing the nucleic acid as described above or the vector as described above.

[0011] Another aspect of the present invention relates to a pharmaceutical composition comprising the polypeptide as described above and a pharmaceutically acceptable excipient.

[0012] Another aspect of the present invention relates to the use of the polypeptide described above in the preparation of a medicament for treating non-alcoholic fatty liver disease.

[0013] This invention provides a novel octapeptide P3-1 (SEQ ID NO: 1) derived from macadamia nuts, which significantly improves hepatic lipid metabolism disorders, restores liver function, reduces inflammatory response, and increases probiotic abundance in mice with high-fat diet-induced non-alcoholic fatty liver disease. This polypeptide has a small molecular weight, safe origin, and is easily absorbed, possessing good potential for pharmaceutical development and providing a safe and effective new approach for targeted intervention in non-alcoholic fatty liver disease. Attached Figure Description

[0014] 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.

[0015] Figure 1 Isolation, identification, and chemical synthesis of P3-1. A: Amino acid sequence of P3-1; B: LC-MS / MS chromatogram of P3-1 isolation and identification; C: HPLC chromatogram of P3-1 chemical synthesis purity; D: LC-MS chromatogram of P3-1 molecular weight.

[0016] Figure 2 Effects of P3-1 on HFD-induced NAFLD. A: Animal experimental design; B: Mouse liver weight; C: Body weight; D: Liver weight ratio; E: Mouse body weight change trend graph; F: HE staining images of liver sections from the control group and P3-1 group; G: HE staining images of liver sections from the 5 treatment groups.

[0017] Note: Compared with the control group, P<0.05, P<0.01, P<0.001; compared with the HFD group, #P<0.05, ##P<0.01, ###P<0.001, and the same applies below.

[0018] Figure 3 P3-1 improves lipid metabolism disorders caused by NAFLD. A: Representative anatomical image of the liver; B: Liver section HE staining image; C: Liver section Oil Red O staining image; D: Liver section Masson staining image; E: NAFLD histological score and Masson collagen fiber region; F: Blood AST, ALP, ALT and TG levels; G: Liver SOD, MDA, CAT and GSH-Px levels.

[0019] Figure 4P3-1 alleviates intestinal barrier damage caused by NAFLD. A: HE staining of jejunal sections; B: PAS staining; C: Immunohistochemical detection of ZO-1 protein; D: Goblet cell count; E: ZO-1 positive area; F: ZO-1 mRNA transcription level.

[0020] Figure 5 P3-1 improves gut microbiota dysbiosis caused by NAFLD. A: Venn diagram for each group; B: Bar chart of species abundance at the phylum level; C: Heatmap analysis at the genus level.

[0021] Figure 6 P3-1 improves NAFLD by regulating the gut microbiota. A: Liver section HE staining image; B: Liver section Oil Red O staining image; C: Blood ALP, ALT, AST and TG levels; D: Liver TNF-α, IL-1β and IL-6 levels.

[0022] Figure 7 KEGG enrichment pathway analysis and molecular docking. A: Analysis of the top 10 enriched pathways in NAFLD based on the KEGG database; B: Volcano plot generated using fold change values ​​and P-adjustment values, with red dots indicating genes with significant fold change values ​​and P-values, annotating differentially expressed genes in NAFLD; C: P3-1 docking with PPARα; D: P3-1 docking with RXRα; E: P3-1 docking with NF-κB.

[0023] Figure 8 PPARα inhibitors suppress the PPARα / RXRα pathway, exacerbating the pathological response of NAFLD. A: Blood levels of AST, ALP, TG, and ALT; B: MRNA transcription levels of PPARα, APOA1, FABP1, and SCD-1; C, D: Western blot analysis of liver PPARα, APOA1, FABP1, and SCD-1 protein levels; E: Liver levels of TNF-α, IL-6, and IL-1β.

[0024] Figure 9 P3-1 activation of the PPARα / RXRα signaling pathway alleviates lipid metabolism disorders in NAFLD. A: mRNA transcription levels of PPARα, RXRα, APOA1, FASN, FABP1, and SCD-1; B, D: Immunofluorescence staining and expression statistics of PPARα and RXRα; C, E: Immunohistochemical staining and expression statistics of APOA1, FASN, FABP1, and SCD-1; F, G: Western blot analysis of protein expression levels of PPARα, RXRα, APOA1, FASN, FABP1, and SCD-1 in the liver.

[0025] Figure 10P3-1 inhibits the inflammatory response of NAFLD. A: Immunofluorescence staining and expression statistics of NF-κB; B: Immunohistochemical staining and expression statistics of NF-κB; C: Expression levels of TNF-α, IL-1β and IL-6 in the liver. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] The concentration values ​​involved in this invention include fluctuations within a certain range. For example, they can fluctuate within a corresponding precision range.

[0033] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0034] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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).

[0040] This invention relates to polypeptides, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0041] This invention provides a short peptide molecule, named P3-1, which has a significant effect on improving non-alcoholic fatty liver disease. This polypeptide is a small molecule peptide obtained from macadamia nut protein through neutral protease hydrolysis. After separation, purification, and identification by liquid chromatography-tandem mass spectrometry (LC-MS / MS), its amino acid sequence was determined to be Gly-Phe-Cys-Asp-Arg-Met-Pro-Leu, as listed in SEQ ID NO:1. The polypeptide has a linear L-type structure and a molecular weight of approximately 938.23 Da. This polypeptide can be obtained through chemical synthesis, recombinant expression, or enzymatic hydrolysis purification. The polypeptide is stable under in vivo and in vitro conditions and can be prepared and delivered using conventional drug carriers.

[0042] 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.

[0043] Experimental results showed that the peptide P3-1 significantly reduced hepatic fat deposition, improved liver function, and reduced histological damage in a high-fat diet-induced NAFLD mouse model. Its mechanism of action includes:

[0044] 1) The P3-1 peptide has a high binding affinity to peroxisome proliferator-activated receptor α (PPARα), retinoid X receptor α (RXRα), and NF-κB protein, effectively interfering with the expression of pathway genes.

[0045] 2) Activate the PPARα / RXRα signaling pathway, improve hepatic lipid peroxidation, and reduce lipid accumulation.

[0046] 3) Inhibit the NF-κB signaling pathway, suppress the levels of pro-inflammatory factors such as TNF-α, IL-1β and IL-6, and reduce the inflammatory response.

[0047] 4) Repairs the intestinal barrier and regulates the microbiota structure, restores tight junction protein (ZO-1) expression, increases probiotics, and reduces pathogenic bacteria. 。

[0048] In summary, the polypeptides described in this invention regulate the "metabolism-microbiota-inflammation" pathway through the liver-gut axis, improving the pathological process of NAFLD from multiple targets. They exhibit good biosafety and pharmacological activity, providing new candidate peptide molecules for the prevention and treatment of NAFLD. According to one aspect of this invention, a nucleic acid capable of expressing the polypeptides described above is also involved.

[0049] According to one aspect of the invention, a vector containing nucleic acids as described above is also involved.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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).

[0054] The present invention also relates to host cells containing the nucleic acids described above or the vectors described above.

[0055] The present invention also relates to pharmaceutical compositions comprising the polypeptides described above and pharmaceutically acceptable excipients.

[0056] 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.

[0057] In some embodiments, the pharmaceutical composition further comprises a peptide stabilizer or a nucleic acid stabilizer.

[0058] In some embodiments, the pharmaceutical composition is an oral formulation, a rectal formulation, or an enteric-coated formulation for targeted release into the colon.

[0059] 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.

[0060] 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 NAFLD.

[0061] The present invention also relates to the use of the polypeptides described above in the preparation of medicaments for treating non-alcoholic fatty liver disease.

[0062] The present invention also relates to a method for treating non-alcoholic fatty liver disease, comprising the step of administering a safe and effective amount of the pharmaceutical composition as described above to a subject.

[0063] 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.

[0064] 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.

[0065] 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).

[0066] 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.

[0067] 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.

[0068] Example

[0069] 1. Materials and Methods

[0070] 1.1 Preparation of P3-1

[0071] Macadamia nut kernels were extracted using hydraulic oil extraction to obtain meal (protein content 26.40%). This meal was then subjected to alkali dissolution (pH 10.0), acid precipitation (pH 5.0), washing, horizontal centrifugation, and spray drying to obtain macadamia nut protein powder (protein content 64.37%). The macadamia nut protein powder was hydrolyzed with neutral protease (enzyme dosage 200 U / g, pH 7.0, temperature 55℃, time 3 h), followed by ultrafiltration (<1000 Da) to obtain small molecule peptides. The small molecular weight peptides were separated and purified using macroporous resin and cross-linked dextran gel, and their sequences were identified by LC-MS / MS. The obtained peptide sequences were subjected to PeptideRanker bioactivity prediction (https: / / www.pepdraw.com / ), and the octapeptide Gly-Phe-Cys-Asp-Arg-Met-Pro-Leu (GFCDRMPL, P3-1) with a PeptideRanker score exceeding 0.9 was selected. P3-1 was chemically synthesized by Sangon Biotech (Shanghai) Co., Ltd. The purity of P3-1 was detected by HPLC, and the molecular weight of P3-1 was detected by LC-MS.

[0072] 1.2 Animal grouping and administration

[0073] One hundred and ten six-week-old male SPF mice (C57BL / 6, 23±2 g) were housed in a clean-grade animal room at a temperature of 25±2°C and a humidity of 55%±10%, with a light / dark cycle of 12 h, and free access to food and water.

[0074] Experiment 1: Sixty mice were divided into six groups (n=10): the control group was fed a normal diet, while the P3-1 group was fed a normal diet but administered P3-1 (90 mg / kg) by gavage for toxicological experiments. Mice on a high-fat diet (HFD) for 8 weeks were randomly assigned to the HFD group, the low-dose HFD+P3-1 group, the medium-dose HFD+P3-1 group, and the high-dose HFD+P3-1 group. In addition to HFD, the P3-1 intervention group received P3-1 by gavage daily at doses of 30 mg / kg (low dose), 60 mg / kg (medium dose), and 90 mg / kg (high dose) for 4 weeks. Mice in the HFD group received the same dose of PBS.

[0075] Experiment 2: Thirty SPF-grade mice were randomly divided into three groups (n=10) after 8 weeks of fecal microbiome transplantation (FMT) – Control group (normal diet control group), FMT-HFD group (HFD group as donor), and FMT-HFD+P3-1 group (HFD-P3-1 group as donor). Each group was administered 200 μL of microbial suspension from its respective donor group by gavage for 4 weeks.

[0076] Experiment 3: Twenty SPF-grade mice were subjected to HFD for 8 weeks and then randomly divided into two groups (n=10). The HFD+GW6471 group was treated with a PPARα inhibitor and administered GW6471 by gavage at a dose of 10 mg / kg every other day for 4 weeks. Mice in the HFD group were given the same dose of PBS.

[0077] Mice in each group were euthanized by cervical dislocation on day 84 (12 hours after the last administration).

[0078] Fresh mouse blood was collected and centrifuged at 3000 g for 15 min to separate serum. Liver and jejunum tissues were collected to prepare sections, while other relevant organs were immediately immersed in liquid nitrogen and stored at -80°C for further analysis.

[0079] 1.3 Histopathological examination

[0080] After fixing the right lobe liver tissue and proximal jejunum segment of mice with 4% paraformaldehyde, paraffin sections with a thickness of 6 μm were prepared. The liver sections were stained with hematoxylin-eosin (HE), periodic acid-Schiff (PAS), and Oil Red O staining of frozen liver sections using a kit. The pathological changes of the liver were analyzed under an optical microscope to assess fat accumulation and intestinal barrier function.

[0081] 1.4 Biochemical Measurements

[0082] Fresh mouse blood was collected and centrifuged at 3000 g for 15 min to obtain the supernatant serum. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and triglycerides (TG) in the blood were measured using a kit.

[0083] 1.5 Detection of Antioxidant Index

[0084] Left lobe liver tissue was collected from mice in each group and prepared into a 10% (w / w) tissue homogenate using physiological saline. The levels of superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), and malondialdehyde (MDA) in the liver tissue were detected using a kit.

[0085] 1.6 Immunohistochemical assay

[0086] Immunohistochemical staining of liver paraffin sections was performed using a kit. Antibodies APOA1 (1:500), FASN (1:1000), SCD-1 (1:500), FABP1 (1:500), ZO-1 (1:100), and NF-κB (1:500) were added and incubated overnight at 4°C. Enzyme-labeled secondary antibody was added, and the sections were incubated at 37°C for 30 min, followed by DAB staining for a few seconds, hematoxylin counterstaining for 5 min, xylene clearing, and mounting with neutral resin. Images were observed under a microscope and analyzed using Image ProPlus 6.0 software.

[0087] 1.7 Real-time quantitative PCR detection

[0088] Total RNA was extracted from mouse liver tissue using a tissue RNA extraction kit. The RNA was then reverse transcribed into cDNA using a reverse transcription kit. PCR amplification was performed using the SYBR Green method with GAPDH as an internal control, and the relative mRNA expression levels of each gene were calculated using the 2-ΔΔCt method. Primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0089] The primer sequences are shown in the table below.

[0090]

[0091] 1.8 Sequencing and Analysis of 16S rRNA Gene Amplicon

[0092] 16S rRNA amplicon sequencing and analysis: 16S rRNA amplicon sequencing DNA was extracted from ex vivo human fecal cultures or mouse feces, and high-throughput sequencing of the 16S rRNA gene was performed using the Illumina NovaSeq PE250 platform. The V3-V4 region of the 16S rRNA gene was amplified using primers F341 (SEQ ID NO: 18) and R806 (SEQ ID NO: 19). The data quality obtained from Illumina NovaSeq sequencing was assessed using FastQC, and analysis was performed using the DADA2 software package. The DADA2 output sequence listing was converted to biological format using bioformat software; this data was used to assess sequence variation abundance and generate counts for each sample. The complete analysis workflow was performed at (https: / / www.microbiomeanalyst.ca / ).

[0093] 1.9 Fecal microbiome transplantation

[0094] Fresh fecal samples were collected from donor mice in each group. Approximately 200 mg of the collected fecal sample was vortexed vigorously in 2 mL of sterile saline for 1 min until the fecal particles were completely dispersed. Then, the sample was centrifuged at 800 g for 3 min. The supernatant was collected and administered orally via gavage within 10 min to minimize changes in microbial content.

[0095] 1.10 Immunofluorescence assay

[0096] Immunofluorescence detection was performed on liver paraffin sections using a kit. Antibodies PPARα (1:500), RXRα (1:500), and NF-κB P65 (1:500) were added and incubated at 4°C for 12 h. Then, fluorescent secondary antibody IgG (H+L) (1:1000) was added and incubated at 37°C in the dark for 30 min. DAPI staining was then added for 5 min. After washing with PBS, anti-quenching agent was added, and the sections were mounted. Images were acquired using a fluorescence microscope and analyzed using Image ProPlus 6.0 software.

[0097] 1.11 Western blot analysis

[0098] Total protein was extracted from mouse liver tissue and transferred to a PVDF membrane via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membrane was then sealed with 5% skim milk powder. Antibodies PPARα (1:3000), RXRα (1:5000), APOA1 (1:2000), FASN (1:10000), SCD-1 (1:12000), FABP1 (1:5000), and GAPDH (1:5000) were added and incubated overnight at 4°C. Secondary antibody was added, and the membrane was incubated at 37°C in the dark for 30 min. Images were then acquired using a gel imaging system.

[0099] 1.12 Bioinformatics Analysis

[0100] The dataset (GSE208735) was downloaded from the Gene Expression Omnibus (GEO) database in MINiML format. Differential expression analysis between the HFD group and the control group was performed using the Limma package in R software. P-values ​​were adjusted to minimize potential false positives, and thresholds of P < 0.05 and Log2 (fold change) > 1 or Log2 (fold change) < 1 were adjusted to identify significantly differentially expressed genes. This gene set was uploaded to the KOBAS-i database (KOBAS) for KEGG enrichment pathway analysis.

[0101] 1.13 Molecular docking

[0102] The core targets of PPARα and RXRα, and the core components of P3-1 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.

[0103] 1.14 Enzyme-linked immunosorbent assay (ELISA)

[0104] Fresh liver tissue from each group of mice was prepared into a 10% tissue homogenate. The levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in mice were detected using an enzyme-linked immunosorbent assay (ELISA) kit. The absorbance was measured at a wavelength of 450 nm, and the sample concentration was calculated.

[0105] 1.15 Statistical Analysis of Data

[0106] SPSS statistical software was used for data analysis. All data are expressed as mean + standard deviation (x±s). The LSD-t method was used to compare the means between two groups, and one-way ANOVA was used to compare the means among multiple groups.

[0107] 2 Results

[0108] 2.1 Isolation and Chemical Synthesis of P3-1

[0109] A novel 8-peptide was isolated from macadamia nuts using LC-MS / MS. The amino acid sequence is Gly-Phe-Cys-Asp-Arg-Met-Pro-Leu (GFCDRMPL). Figure 1 (AB). The P3-1 score was 0.94 using PeptideRanker, close to 1, indicating good biological activity. ToxinPred was used to predict the non-toxicity of P3-1. P3-1 was chemically synthesized by Sangon Biotech (Shanghai) Co., Ltd., and its purity was determined by HPLC. Figure 1 The C content was 99.08%, meeting the requirements for subsequent functional experiments. The molecular weight of P3-1 was determined using LC-MS. Figure 1 The value of (D) is 938.23 Da.

[0110] 2.2 P3-1 reduces obesity and hepatic steatosis in NAFLD mice

[0111] Mice were fed HFD for 8 weeks to induce obesity and hepatic steatosis. The effects of low, medium, and high doses of P3-1 on NAFLD were investigated by gavage administration to mice. Figure 2 (A). The body weight and liver index of mice in the HFD group were significantly higher than those in the control group. After P3-1 intervention, the obesity trend in mice was slowed down. Figure 2 (Medium BE). No significant pathological differences were observed in the liver tissues of the control group and the P3-1 group alone. Figure 2 (Medium F). The livers of mice in the HFD group were enlarged and yellowish in color, while the livers in the P3-1 dose groups were bright red. Furthermore, histopathological observation revealed that the liver tissue structure of the control group and the P3-1-only group was intact, with no obvious pathological changes in the liver lobules. In the HFD group, the hepatocyte cords were disordered, the hepatocytes were enlarged, and a large number of lipid droplets were visible in the cytoplasm. In contrast, the degree of hepatocyte steatosis was reduced in the three HFD+P3-1 dose groups, with the medium dose group showing the lowest degree of steatosis, suggesting that this concentration had the most significant intervention effect. Figure 2 (G). Therefore, the concentration of P3-1 used in subsequent experiments was 60 mg / kg.

[0112] 2.3 P3-1 reduces liver damage and lipid accumulation in NAFLD mice

[0113] Through liver morphology ( Figure 3 (A) Section HE staining ( Figure 3 Medium B), Oil Red O staining ( Figure 3 (C) Masson staining ( Figure 3 Pathological changes observed in the HFD group showed enlarged liver volume, a yellowish color, and lipid droplets of varying sizes in the cytoplasm, indicating severe lipid accumulation in the liver. Numerous irregular reticular fibrosis areas were also observed, indicating a severe degree of liver fibrosis. After P3-1 intervention, the NAFLD liver pathological score decreased, the number of lipid droplets decreased, lipid accumulation significantly decreased, and the area of ​​collagen fibrosis significantly reduced. Figure 3 In the HFD group, the activities of ALT, AST, TG, and ALP were significantly increased, indicating impaired liver function in mice. After P3-1 intervention, liver function indicators showed some recovery. Figure 3 (Middle F). In the HFD group, the activities of SOD, CAT, and GSH-Px in the liver of mice were significantly decreased, while the content of MDA was significantly increased. After P3-1 intervention, the antioxidant capacity was enhanced. Figure 3 The results (G) further confirmed that P3-1 has a protective effect on NAFLD liver.

[0114] 2.4 P3-1 reduces intestinal barrier damage in NAFLD mice

[0115] Intestinal HE ( Figure 4 (A) and PAS staining ( Figure 4Observations in the HFD group revealed that the intestinal barrier was damaged, jejunal epithelial cells in the mucosal layer sloughed into the intestinal tract, and the number of goblet cells decreased. After P3-1 intervention, the degree of jejunal mucosal damage decreased, and the number of goblet cells increased. Figure 4 In the middle D), intestinal barrier function was improved. This was confirmed by immunohistochemistry. Figure 4 C, E) and real-time quantitative PCR ( Figure 4 Further examination of the expression of intestinal tight junction protein (ZO-1) revealed that the expression levels of ZO-1 mRNA and protein were decreased in the HFD group mice, but increased after P3-1 intervention. This indicates that P3-1 can alleviate intestinal mucosal inflammation in NAFLD mice and maintain the integrity of the intestinal barrier.

[0116] 2.5 P3-1 restores the structure and abundance of gut microbiota in NAFLD mice

[0117] The effect of P3-1 on the gut microbiota of NAFLD mice was investigated using 16S rRNA amplicon sequencing. ASV analysis revealed that the abundance of gut microbiota was downregulated in the HFD group mice, and increased after P3-1 treatment. Figure 5 (A). At the phylum level, the expression of Firmicutes / Bacteroidetes in the HFD group mice was upregulated by 74.96%, and downregulated by 34.49% after P3-1 intervention, approaching the control group. The increase in Firmicutes / Bacteroidetes is an important marker reflecting changes in the NAFLD gut microbiota. Figure 5 (B). At the genus level, P3-1 increases Akkermansia ( Akkermansia ), Bacteroides ( Bacteroides Beneficial bacteria such as ) reduce the number of Isprevotella spp. ( Alloprevotella ), genus Tize ( Tyzzerella Streptococcus spp. Streptococcus Harmful bacteria such as ) improve the gut microbiota composition of NAFLD mice. Figure 5 (C)

[0118] 2.6 P3-1 alleviates liver pathology in NAFLD mice by regulating gut microbiota

[0119] HE staining revealed sparse villi arrangement in the jejunum of mice in the FMT-HFD group, with some shedding of small intestinal epithelial cells. In contrast, the villi in the FMT-HFD+P3-1 group were more neatly and densely arranged, and the degree of small intestinal epithelial cell shedding was improved. This suggests that the intestinal structure of NAFLD mice was improved after microbial transplantation. Figure 6(A). Oil Red O staining revealed that the FMT-HFD+P3-1 group showed significantly reduced hepatocyte steatosis and significantly lower lipid deposition in liver tissue compared to the FMT-HFD group. Figure 6 In the FMT-HFD+P3-1 group of mice, serum biochemical levels of ALT, AST, ALP, and TG were significantly decreased, indicating that liver damage was alleviated and lipid metabolism disorder was improved. Figure 6 (C). The levels of key pro-inflammatory factors TNF-α, IL-6, and IL-1β in the FMT-HFD+P3-1 group were significantly lower than those in the FMT-HFD group. Figure 6 (Middle D). These results suggest that the beneficial gut microbiota after P3-1 intervention plays a regulatory role in the gut of NAFLD mice, improving liver lipid metabolism disorders and inflammatory status in NAFLD mice by repairing intestinal structure.

[0120] 2.7 KEGG enrichment pathway and molecular docking

[0121] Based on the KEGG database, NAFLD mouse data were reanalyzed. The study found that the PPAR pathway is closely related to the progression and development of NAFLD, and differential gene analysis revealed upregulation of PPAR-related Pparg, Cyp4a10, and Cyp4a31. Figure 7 (AB). Subsequent molecular docking analysis was used to predict the binding targets of P3-1 to PPARα, RXRα, and NF-κB proteins. The results showed that P3-1 has a high binding affinity to these proteins, effectively interfering with the expression of pathway genes and thus improving the pathological response of NAFLD. Figure 7 (CE).

[0122] 2.8 P3-1 activates the PPARα / RXRα signaling pathway

[0123] To clarify whether P3-1 activates the PPARα / RXRα signaling pathway and exerts its ameliorative effect, the effect of P3-1 on NAFLD was first investigated using the PPARα inhibitor (GW6471). The results showed that GW6471 significantly increased the activities of AST, ALP, TG, and ALT in serum. Figure 8 (A). Real-time quantitative PCR analysis revealed decreased expression levels of PPARα and APOA1 mRNA, while upregulated expression levels of lipid synthesis-related SCD-1 and FABP1 mRNA. Figure 8 (B). Western blot results showed decreased expression levels of PPARα and APOA1 proteins, and increased expression levels of FABP1 and SCD-1 proteins. Figure 8(CD). Simultaneously, serum ELISA analysis revealed that GW6471 intervention increased the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β. Figure 8 (E). The results showed that inhibiting the PPARα / RXRα signaling pathway aggravates liver dysfunction in NAFLD and induces the expression of pro-inflammatory factors.

[0124] 2.9 P3-1 improves lipid metabolism by activating the PPARα / RXRα signaling pathway

[0125] To further understand the impact of PPARα / RXRα signaling pathway activation on NAFLD, qPCR was used to detect the expression of key genes in the PPARα / RXRα signaling pathway in mouse liver. The results showed that the expression levels of PPARα and RXRα mRNA were suppressed in the HFD group mice. After P3-1 administration, PPARα and RXRα were upregulated, APOA1 expression was upregulated, and FASN, SCD-1, and FABP1 expression were downregulated. Figure 9 (A). Further immunofluorescence was used. Figure 9 (B, D) and immunohistochemistry ( Figure 9 The expression levels of PPARα / RXRα proteins in liver tissue were detected by C and E. The results showed that HFD significantly increased the expression of key lipid synthesis factors FABP1, FASN, and SCD-1 in mouse liver, while inhibiting APOA1 expression. Western blotting results showed that P3-1 intervention also increased the expression levels of PPARα and RXRα proteins and regulated their downstream genes APOA1, FABP1, FASN, and SCD-1, thereby improving lipid metabolism in NAFLD. Figure 9 (FG). This indicates that P3-1 improves lipid metabolism in NAFLD by activating the PPARα / RXRα signaling pathway.

[0126] 2.10 P3-1 intervention inhibited the inflammatory response in NAFLD mice.

[0127] The initiation and development of metabolic diseases such as NAFLD are closely related to inflammation; damaged hepatocytes activate NF-κB signaling and upregulate pro-inflammatory factors. Immunofluorescence staining ( Figure 10 (A) and immunohistochemistry ( Figure 10 The results of the B-cell assay showed that the expression level of NF-κB protein in the HFD group mice was significantly upregulated, and P3-1 treatment significantly inhibited the expression level of NF-κB protein. ELISA detection of the expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in liver tissue showed that P3-1 significantly inhibited the expression of the three pro-inflammatory factors in the liver of NAFLD mice. Figure 10 (C). This indicates that P3-1 has a certain inhibitory effect on inflammation caused by NAFLD and can delay the progression of NAFLD cases.

[0128] 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 non-alcoholic fatty liver disease.

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