A lipid-lowering peptide, its screening method and application

Abstract

This application provides a lipid-lowering peptide, its screening method, and its application, belonging to the peptide field. The lipid-lowering peptide provided in this application includes at least one of 116 peptides with amino acid sequences such as FFPFF, VFFFPF, PFFFF, AFFFPFF, and LFFFPFY. The lipid-lowering peptide provided in this application can effectively reduce blood lipids without significant toxic side effects. In in vitro enzyme activity tests, it shows good inhibitory activity against sterol esterases and lipases; in vivo animal experiments also show effective reduction of blood lipids; and toxicity prediction and in vivo animal models both show no significant toxic side effects.

Description

Technical Field

[0001] This application relates to the field of peptides, specifically a lipid-lowering peptide, its screening method, and its application. Background Technology

[0002] Hyperlipidemia is a common disease worldwide, leading to a range of related illnesses including cardiovascular disease. It is projected that by 2030, the number of deaths from cardiovascular disease globally will reach 22.3 million, highlighting the significant importance of preventing and treating hyperlipidemia.

[0003] There are many types of lipid-lowering drugs. Although a large amount of evidence-based medicine suggests that statins are the cornerstone of the prevention and treatment of atherosclerotic cardiovascular disease, clinical data shows that about 30% of patients still do not achieve ideal blood lipid levels even after long-term statin use. On the one hand, some patients cannot take statins at the required dose due to side effects (such as myalgia and rhabdomyolysis); on the other hand, high-dose statin therapy increases the incidence of new-onset diabetes, further limiting the use of maximum-dose statins.

[0004] Peptide drugs, due to their high biological activity and specificity, exhibit unique advantages and clinical application value in drug development. Furthermore, the degradation products of peptides are amino acids, which generally do not accumulate in specific organs or tissues and are easily cleared from the body through the liver and kidneys, thus having almost no side effects. Compared to macromolecular biologics, peptides have lower immunogenicity, relatively higher stability, and lower commercial costs.

[0005] Therefore, it is essential to develop peptide drugs that effectively inhibit lipid-lowering effects and have fewer toxic side effects. Summary of the Invention

[0006] In view of this, this application provides a lipid-lowering peptide, a screening method thereon, and its application, with the aim of finding peptide drugs that effectively inhibit lipid-lowering and have fewer toxic side effects.

[0007] The embodiments of this application are implemented as follows: A lipid-lowering polypeptide is provided, comprising one or more of the following 116 polypeptides with the following amino acid sequences:

[0008] FPFFF, FFPFF, FFFFFP, PFFFF, FFFPFF, FFFPFW, FFFPFL, FFFPFG, FFFPFY, FFFPFI, FFFPFA, FFFPFV, FFFFFPF, WFFFPF, LFFFPF, GFFFPF, YFFFPF, IFFFPF, AFFFPF, VFFFPF, FFFFFPFF, FFFFFPFW, FFFFFPFL, FFFFFPFG, FFFFFPFY, FFFFFPFI, FFFFFPFA, FFFFFPFV, FFFFFFPF, FWFFFPF, FLFFFPF, FGFFFPF, FYFFFPF, FIFFFPF, FAFFFPF, FVFFFPF, FFFPFFF, FFFPFWF, FFFPFLF, FFFPFGF, FFFPFYF, FFFPFIF, FFFPFAF, FFFPFVF, FFFFFPFF, WFFFPFF, LFFFPFF, GFFFPFF, YFFFPFF, IFFFPFF, AFFFPFF, VFFFPFF, FFFPFFW, FFFPFWW, FFFPFLW, FFFPFGW, FFFPFYW, FFFPFIW, FFFPFAW, FFFPFVW, FFFFFPFW, WFFFPFW, LFFFPFW, GFFFPFW, YFFFPFW, IFFFPFW, AFFFPFW, VFFFPFW, WFFFPFF, WFFFPFW, WFFFPFL, WFFFPFG, WFFFPFY, WFFFPFI, WFFFPFA, WFFFPFV, WFFFFPF, WWFFFPF, WLFFFPF, WGFFFPF, WYFFFPF, WIFFFPF, WAFFFPF, WVFFFPF, WFFFPFL, FFFPFFL, FFFPFWL, FFFFFPFL, LWFFFPF, LFFFPFF, LFFFPFW, LFFFFPF, GFFFPFL, AFFFPFL, FFFPFL, YFFFPFL, LGFFFPF, LFFFPFL, LFFFPFL, LYFFFPF, FFFPFL, FFFPFYL, IFFFPFL, LAFFFPF, FFFPFIL, VFFFPFL, LIFFFPF, LFFFPFG, LLFFFPF, LFFFPFY, FFFPFL, FFFPFVL, LFFFPFI, LVFFFPF, LFFFPFA, LFFFPFV.

[0009] In some embodiments, the hypolipidemic polypeptide comprises at least one polypeptide having an amino acid sequence of FFPFF, VFFFPF, PFFFF, AFFFPFF, or LFFFPFY.

[0010] In some embodiments, the amino acid sequence of the lipid-lowering polypeptide is FFPFF, VFFFPF, PFFFF, AFFFPFF, LFFFPFY.

[0011] In some embodiments, the lipid-lowering polypeptide comprises PFFFF; or, the amino acid sequence of the lipid-lowering polypeptide is PFFFF.

[0012] This application also provides a method for screening lipid-lowering peptides, including:

[0013] Provide peptides to be screened, and use the Peptide Ranker database to predict the probability of bioactivity, and screen out peptides with a bioactivity prediction score > 0.8.

[0014] Peptides with a predicted bioactivity score > 0.8 were screened using molecular docking to identify peptides that inhibit lipid activity, thus obtaining lipid-lowering peptides.

[0015] In some embodiments, the peptide to be screened is a polypeptide with 5-7 amino acids; and / or,

[0016] The proportion of hydrophobic amino acids in the peptides to be screened is ≥80%.

[0017] In some embodiments, the screening of peptides with a bioactivity prediction score > 0.8 using molecular docking includes: screening peptides with a bioactivity prediction score > 0.8 by semi-flexible molecular docking with cholesterol esterase.

[0018] In some embodiments, the crystal structure information of the cholesterol esterase is obtained from the RCSB database, and the PDB number of the cholesterol esterase is 1F6W; and / or,

[0019] The molecular docking was performed using AutoDock 4.2.6 software; and / or,

[0020] Prior to the molecular docking, the PDB file of the cholesterol esterase is preprocessed, including the removal of water molecules and ligands and the addition of hydrogen atoms.

[0021] In some embodiments, the lipid-lowering peptides further undergo one or more of the following screening processes:

[0022] In in vitro activity tests, the lipid-lowering peptide showed an inhibition rate of ≥30% against cholesterol esterase activity.

[0023] In in vitro activity tests, the lipid-lowering peptide showed an inhibition rate of 30% or greater against lipase.

[0024] In animal models of hyperlipidemia, the lipid-lowering peptides showed no toxicity.

[0025] In a hyperlipidemic animal model, the lipid-lowering polypeptide drug group showed a greater reduction in cholesterol than the aliximumab positive drug group.

[0026] This application also provides an application of the lipid-lowering polypeptide described above for the treatment of lowering blood lipids.

[0027] The lipid-lowering peptides provided in this application include at least one of 116 peptides with amino acid sequences such as FFPFF, VFFFPF, PFFFF, AFFFPFF, and LFFFPFY. These peptides effectively reduce blood lipids and cholesterol without significant toxic side effects. In vitro enzyme activity tests show good inhibitory activity against sterol esterases and lipases, and in vivo animal experiments also show effective reduction of blood lipids and cholesterol. Toxicity prediction and in vivo animal models both demonstrate no significant toxic side effects. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a schematic flowchart of an embodiment of the lipid-lowering peptide screening method provided in this application;

[0030] Figure 2 Diagram showing the docking site and details of the lipid-lowering peptide FFPFF with cholesterol esterase;

[0031] Figure 3 A diagram showing the docking site and details of the lipid-lowering peptide VFFFPF with cholesterol esterase;

[0032] Figure 4 Diagram showing the docking site and details of the lipid-lowering peptide PFFFF with cholesterol esterase;

[0033] Figure 5 A diagram showing the docking site and details of the lipid-lowering peptide AFFFPFF with cholesterol esterase;

[0034] Figure 6 Diagram showing the docking site and details of the lipid-lowering peptide PFFFFY with cholesterol esterase;

[0035] Figure 7 The graph shows the inhibition rate of lipid-lowering peptide cholesterol esterase activity.

[0036] Figure 8This is a graph showing the inhibition rate of lipid-lowering peptide lipase activity. Detailed Implementation

[0037] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. In addition, it should be understood that the specific embodiments described herein are only for illustration and explanation of this application and are not intended to limit this application. In this application, unless otherwise stated, directional terms such as "upper" and "lower" specifically refer to the drawing directions in the accompanying drawings. In addition, in the description of this application, the term "including" means "including but not limited to".

[0038] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0039] In this application, "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0040] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c," or "at least one of a, b, and c," can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0041] This application provides a lipid-lowering polypeptide with lipid-lowering activity, wherein the lipid-lowering polypeptide includes at least one polypeptide with the amino acid sequence FFPFF, VFFFPF, PFFFF, AFFFPFF, or LFFFPFY.

[0042] In one embodiment, the lipid-lowering polypeptide includes PFFFF.

[0043] In one embodiment, the amino acid sequence of the lipid-lowering polypeptide is FFPFF, VFFFPF, PFFFF, AFFFPFF, LFFFPFY. Preferably, the amino acid sequence of the lipid-lowering polypeptide is PFFFF.

[0044] It is understood that any modification and processing of the lipid-lowering peptide, such as bi-terminal blocking, intermediate residue modification, cyclization modification, etc., are within the protection scope of this patent.

[0045] The lipid-lowering peptides provided in this application can effectively reduce blood lipids without significant toxic side effects. In in vitro enzyme activity tests, they showed good inhibitory activity against sterol esterase and lipase. In animal in vivo experiments, they can also effectively reduce blood lipids and cholesterol. In toxicity prediction and in animal models, no significant toxic side effects were observed.

[0046] This application also provides a method for screening lipid-lowering peptides, see reference. Figure 1 , Figure 1 This is a schematic flowchart of an embodiment of the lipid-lowering peptide screening method provided in this application, including the following steps:

[0047] Step S1: Provide peptides to be screened, predict the bioactivity probability using the Peptide Ranker database, and screen out peptides with a bioactivity prediction score > 0.8.

[0048] Step S2: Using molecular docking, peptides with a predicted bioactivity score > 0.8 are screened to identify peptides that inhibit lipid activity, thus obtaining lipid-lowering peptides.

[0049] In this embodiment, by performing bioactivity screening and molecular docking screening, lipid-lowering peptides with good lipid-lowering activity can be quickly screened out.

[0050] In step S1:

[0051] The peptides to be screened are designed based on the principle of high proportion of hydrophobic amino acids and small molecular weight. Specifically, the peptides to be screened are polypeptides with 5-7 amino acids, and the proportion of hydrophobic amino acids is ≥80%.

[0052] The hydrophobic amino acids mentioned above are also known as nonpolar amino acids. The side chains of these amino acids are mainly composed of carbon and hydrogen, and they do not easily form hydrogen bonds with water molecules, thus exhibiting hydrophobicity. Hydrophobic amino acids include: glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), and tryptophan (Trp, W).

[0053] In this embodiment, the presence of hydrophobic amino acids in peptides, especially small peptides, has a positive impact on the lipid-lowering effect of active peptides, significantly increasing the lipid-lowering effect of active peptides.

[0054] In step S2:

[0055] The molecular docking screening of peptides with a bioactivity prediction score > 0.8 can be specifically performed by screening through semi-flexible molecular docking with cholesterol esterase.

[0056] Furthermore, in one specific embodiment, peptides with a binding energy value < -1.22 kcal that dock with cholesterol esterase molecules were screened to obtain five polypeptides with amino acid sequences of FFPFF, PFFFF, VFFFPF, AFFFPFF, and LFFFPFY.

[0057] The crystal structure information of the cholesterol esterase can be obtained from the RCSB database, and the PDB number of the cholesterol esterase is 1F6W.

[0058] The molecular docking was performed using AutoDock 4.2.6 software. The cholesterol esterase was used as the acceptor, and the original PDB file underwent pretreatment before molecular docking, including deleting water molecules, adding hydrogen atoms, and removing ligands carried by the original acceptor.

[0059] Cholesterol esterase, also known as cholesterol ester hydrolase, mainly participates in the hydrolysis of cholesterol esters, breaking them down into cholesterol and free fatty acids. Cholesterol esterase plays a crucial role in cholesterol metabolism, exerting important physiological functions in maintaining the balance between cholesterol and fatty acids and regulating cholesterol metabolism in the body.

[0060] In one embodiment, the lipid-lowering polypeptide is further predicted to have the following physicochemical properties:

[0061] (1) Toxicity and steric hindrance were predicted using the ToxinPred website (ToxinPred(iiitd.edu.in));

[0062] (2) Hydrophilicity was predicted using the ExPasy website (Expasy-ProtParam);

[0063] (3) Predict human intestinal absorption (HIA) using the admetSAR website (admetSAR@LMMD(ecust.edu.cn));

[0064] (4) Predict the isoelectric point using the PepD website (PepD http: / / www.pepdraw.com / ).

[0065] The above-mentioned physicochemical property predictions provide valuable reference for subsequent drug formulation development. The predicted physicochemical properties indicate that these five lipid-lowering peptides are non-toxic, have an isoelectric point less than 7, are acidic, and have a steric hindrance of 0.63-0.64, suggesting good binding to cholesterol esterase. The overall average hydrophilicity is positive, indicating strong hydrophobicity. Furthermore, FFPFF and PFFFF peptides exhibit good intestinal absorption, while VFFFPF, AFFFPFF, and LFFFPFY peptides show poor intestinal absorption. This provides support for the selection of drug formulations for these lipid-lowering peptides, such as oral or injectable formulations.

[0066] In one embodiment, the lipid-lowering peptide further undergoes one or more of the following screening processes:

[0067] (1) In the in vitro activity test, the lipid-lowering peptide inhibits cholesterol esterase activity by ≥30%, such as 32%, 46%, 48%, 51%, 63%, etc.

[0068] (2) In the in vitro activity test, the lipid-lowering peptide inhibits lipase by 30% or more, such as 37%, 43%, 48%, 51%, 69%, etc.

[0069] (3) In animal models of hyperlipidemia, the lipid-lowering peptides showed no toxicity;

[0070] (4) In the hyperlipidemia animal model, the lipid-lowering peptide drug group showed a significant lipid-lowering effect, and the cholesterol reduction was greater than that of the aliximab positive drug group.

[0071] In this embodiment, peptides with good inhibitory performance were screened by testing the inhibitory performance of cholesterol esterase and lipase in vitro; peptides with no obvious toxic side effects and that can significantly reduce blood lipid levels in animals were screened by testing in an in vivo animal model with hyperlipidemia, and the cholesterol-lowering effect was better than that of commercially available aliximumab.

[0072] As described above, the lipid-lowering peptide screening method provided in this application involves designing multiple small molecule peptides, predicting their biological activity, screening the peptides through molecular docking and in vitro lipid-lowering experiments, and then validating their functions. This provides technical support for the development of safe and effective lipid-lowering drugs.

[0073] This application also provides the use of a lipid-lowering peptide in lipid-lowering drugs for the treatment of lipid-lowering diseases.

[0074] The technical solutions and effects of this application will be described in detail below through specific embodiments, comparative examples, and experimental examples. The following embodiments are only some embodiments of this application and are not intended to limit this application in any specific way. It should be understood that unless otherwise specified, the reagents used in the various embodiments, comparative examples, and experimental examples are all commercially available reagents.

[0075] Example 1: Short peptide design, activity evaluation, and screening of lipid-lowering peptides based on molecular docking technology

[0076] Following the principle of high proportion of hydrophobic amino acids and small molecular weight, 116 peptides with 5-7 amino acids were designed. These 116 peptides can be synthesized or extracted, and their sequences were identified by LCMS / MS. The amino acid sequences of each peptide are shown in Table 1. Peptide Ranker performed preliminary screening on the above 116 peptides, identifying peptides with a bioactivity prediction score >0.8.

[0077] The crystal structure of cholesterol esterase (PDB number: 1F6W) was obtained from the RCSB database (http: / / www.rcsb.org / ). Semi-flexible molecular docking was performed between the above-mentioned peptides with bioactivity prediction scores higher than 0.80 and the cholesterol esterase using AutoDock 4.2.6 software. Table 1 shows the active peptide sequences, activity scores, and molecular docking scores.

[0078] Table 1. Results of active peptides, activity scores, and molecular docking scores.

[0079]

[0080]

[0081]

[0082]

[0083] Of the 116 designed peptides, all achieved bioactivity scores exceeding 0.8, consistent with the characteristic of high bioactivity in small peptides. Semi-flexible docking of these 116 peptides with cholesterol esterase was performed, and five peptides with relatively stable docking conformations with cholesterol esterase were selected, with sequences FFPFF, VFFFPF, PFFFF, AFFFPFF, and LFFFPFY.

[0084] The polypeptide FFPFF undergoes a semi-flexible docking with cholesterol esterase. The key amino acid residues involved in its interaction with cholesterol esterase are ASP-72 and LYS-243, and the interaction force is a hydrogen bond. (See [link to intermolecular interactions]). Figure 2 .

[0085] The polypeptide VFFFPF undergoes a semi-flexible docking with cholesterol esterase. Other key amino acid residues for cholesterol esterase activity include ASP-72 and LYS-271, and the interaction force is hydrogen bonding. (See [link to intermolecular interactions]). Figure 3 .

[0086] The polypeptide PFFFF undergoes a semi-flexible docking with cholesterol esterase. The key amino acid residues involved in its interaction with cholesterol esterase are LYS-271 and LEU-274, and the interaction force is a hydrogen bond. Intermolecular interactions are detailed below. Figure 4 .

[0087] The polypeptide AFFFPFF undergoes a semi-flexible docking with cholesterol esterase. The key amino acid residues for its interaction with cholesterol esterase are GLY-24, SER-26, ASP-72, GLY-90, and LYS-271, and the interaction force is van der Waals force. Intermolecular interactions are described in [reference needed]. Figure 5 .

[0088] The polypeptide LFFFPFY undergoes a semi-flexible docking with cholesterol esterase. The key amino acid residues for cholesterol esterase activity are ASP-72, GLN-71, and TYR-270, and the interaction force is a hydrogen bond. Intermolecular interactions are detailed below. Figure 6 .

[0089] Example 2: Prediction of the physicochemical properties of lipid-lowering peptides

[0090] Based on the five potential lipid-lowering peptides screened in Example 1, their physicochemical properties were predicted using relevant software and websites. The websites used for toxicity and steric hindrance prediction were ToxinPred (ToxinPred(iiitd.edu.in)), total average hydrophilicity prediction was ExPasy (Expasy-ProtParam), human intestinal absorption prediction was admetSAR (admetSAR@LMMD(ecust.edu.cn)), and isoelectric point prediction was pepdraw (PepDhttp: / / www.pepdraw.com / ). As shown in Table 2, all five potential lipid-lowering peptides were non-toxic, had isoelectric points less than 7 (acidic), and steric hindrance of 0.63-0.64, which is favorable for binding to cholesterol esterases. All five peptides had positive total average hydrophilicity values, indicating strong hydrophobicity. FFPFF and PFFFF peptides showed good intestinal absorption, while VFFFPF, AFFFPFF, and LFFFPFY peptides showed poor intestinal absorption. The prediction of these physicochemical properties is of reference value for subsequent drug formulation development.

[0091] Table 2. Predicted physicochemical properties of five lipid-lowering peptides

[0092]

[0093] Example 3: In vitro lipid-lowering experiment

[0094] Five lipid-lowering peptides were prepared using the Fmoc solid-phase synthesis method, and the peptide purity was determined to be greater than 95% by high performance liquid chromatography and mass spectrometry analysis.

[0095] The specific procedure for the cholesterol esterase activity inhibition experiment is as follows:

[0096] The concentration of all five lipid-lowering peptides was 2 mg / mL. In a 96-well microplate, 50 μL of sample, 50 μL of 25 μg / mL cholesterol esterase (Sigma-Aldrich) solution, and 50 μL of 10 mM p-nitrophenylbutyrate were incubated at 25°C for 5 min in phosphate buffer (pH 7.0, containing 100 mM NaCl and 5.16 mM sodium taurocholate). The absorbance was recorded at 405 nm using a microplate reader. Simvastatin was used as a positive control, and the results were calculated according to formula (1).

[0097]

[0098] In formula (1): A: absorbance of the control; B: absorbance of the control blank; C: absorbance of the sample; D: absorbance of the sample blank.

[0099] The specific procedure for the lipase activity inhibition experiment is as follows:

[0100] Add 4 mL of PBS buffer (pH 7.4), 4 mL of polyvinyl alcohol, and 0.330 g / mL of olive oil to test tubes, respectively. Simultaneously add 2 mL (2 mg / mL) of lipid-lowering peptide solution and mix. Incubate at 37°C for 10 min. Then add 1 mL of 2 mg / mL pancreatic lipase (Sigma) solution. After reacting for 15 min, add 15 mL of 95% ethanol to terminate the enzyme reaction. Add phenolphthalein and titrate with standard sodium hydroxide solution until a slightly reddish tint is achieved. In the blank experiment, add pancreatic lipase solution after stopping the reaction. The experiment was performed in triplicate, and calculations were performed according to formula (2).

[0101]

[0102] See results Figure 7 and Figure 8 , Figure 7 This is a graph showing the inhibition rate of lipid-lowering peptide cholesterol esterase activity. Figure 8 The graph shows the inhibition rate of lipase activity by the lipid-lowering peptide. At a concentration of 2 mg / mL, PFFFF showed a cholesterol esterase activity inhibition rate of 63%, which is higher than that of the other four peptides. At a concentration of 2 mg / mL, PFFFF showed a lipase inhibition rate of 69%. In summary, among the five peptides screened through activity evaluation and molecular docking, PFFFF exhibited the best lipid-lowering activity, effectively inhibiting not only cholesterol esterase but also lipase activity, preventing lipase from hydrolyzing fats into glycerol and free fatty acids.

[0103] Example 4: Lipid-lowering peptide animal experiment

[0104] To establish a hyperlipidemia animal model, 80 SD rats with a weight of 80±10g were used. Ten rats were used as the blank control group and fed with normal feed. The remaining 70 rats were used as the model group and fed with a high-fat diet (commercially available, 60% fat content).

[0105] During the modeling period, the blank control group was fed a normal diet daily, while the modeling group was fed a high-fat diet. All rats had free access to water, and their body weight was measured regularly. After 4 weeks of high-fat diet, rats in both the blank control and modeling groups were decapitated and blood was collected without fasting. Serum was separated as soon as possible after blood collection, and serum TC, TG, LDL-C, and HDL-C levels were measured. The modeling group was randomly divided into 7 groups (high-fat model group, FFPFF polypeptide group, VFFFPF polypeptide group, PFFFF polypeptide group, AFFFPFF polypeptide group, LFFFPFY polypeptide group, and positive drug group). There were no significant differences in TC, TG, LDL-C, and HDL-C among the groups. Compared with the blank control group, the increases in TC, TG, and LDL-C in each modeling group were significant, indicating successful modeling. The administration method was intraperitoneal injection. The solvent for all peptide groups was 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% normal saline. The aliximab positive control group was diluted with normal saline. The injection dosage for each group is shown in Table 3.

[0106] Table 3 Dosage table for each group

[0107]

[0108]

[0109] After a single injection, each group continued feeding for 4 days, then was weighed and their tails were docked for blood collection. The blank control group was fed a normal diet, while the other groups were fed a high-fat diet. Serum was separated as soon as possible after blood collection, and serum TC, TG, LDL-C, and HDL-C levels were measured.

[0110] Table 4. Changes in rat body weight

[0111]

[0112] Table 4 shows the body weight results. Four days after successful modeling and administration, there were no significant differences in body weight among the groups. The weight of the blank control group, fed with normal diet, was slightly lower than that of the other groups fed with high-fat diet. This indicates that the injection of lipid-lowering peptides FFPFF, VFFFPF, PFFFF, AFFFPFF, and LFFFPFY had no significant effect on rat body weight, and the screened lipid-lowering peptides have no obvious toxic side effects as injectable drugs.

[0113] Table 5. Results of serum marker detection in rats after drug injection

[0114]

[0115]

[0116] As shown in the table above, before and after drug administration, the levels of triglycerides, total cholesterol, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in the model groups (high-lipid model group, FFPFF polypeptide group, VFFFPF polypeptide group, PFFFF polypeptide group, AFFFPFF polypeptide group, LFFFPFY polypeptide group, and positive drug group) were significantly different from those in the blank control group, indicating that the hyperlipidemia model was successfully established.

[0117] Elevated low-density lipoprotein (LDL) is the most important risk factor for cardiovascular disease. As shown in the table above, after drug administration, compared with before drug administration, the LDL levels in the FFPFF polypeptide group, VFFFPF polypeptide group, PFFFF polypeptide group, AFFFPFF polypeptide group, LFFFPFY polypeptide group, and positive drug group all showed a decreasing trend. Among them, the LDL levels in the FFPFF polypeptide group, VFFFPF polypeptide group, PFFFF polypeptide group, and positive drug group were significantly reduced (P < 0.05), with reductions of 41.84%, 50.48%, 61.86%, and 50.92%, respectively.

[0118] Compared to pre-drug administration, total cholesterol levels decreased in all groups (FFPFF, VFFFPF, PFFFF, AFFFPFF, LFFFPFY, and the positive control group), with reductions of 30.22%, 30.60%, 33.33%, 27.22%, 27.12%, and 13.91%, respectively. The FFPFF, VFFFPF, and PFFFF groups showed the most significant cholesterol reductions (P < 0.05). The reduction in cholesterol levels in each lipid-lowering peptide group was greater than that in the positive control group.

[0119] Although there were no significant differences in triglycerides and high-density lipoprotein before and after administration, each of the following groups showed corresponding improvements after administration: FFPFF peptide group, VFFFPF peptide group, PFFFF peptide group, AFFFPFF peptide group, LFFFPFY peptide group, and positive drug group.

[0120] According to animal experiments, all five lipid-lowering peptides screened through activity evaluation and molecular docking can improve blood lipids. Among them, PFFFF peptide has the best lipid-lowering effect, and its effect on reducing low-density lipoprotein and total cholesterol is better than that of the positive drug aliximab.

[0121] The lipid-lowering peptides screened in this application can effectively reduce the levels of total cholesterol and low-density lipoprotein in hyperlipidemic rats, and improve the levels of triglycerides and high-density lipoprotein. Furthermore, compared with the marketed positive control drug alimab, the PFFFF peptides have a better lipid-lowering effect.

[0122] The above provides a detailed description of the lipid-lowering polypeptide, its screening method, and its application provided in the embodiments of this application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A lipid-lowering polypeptide, characterized in that, The amino acid sequence of the lipid-lowering polypeptide is LFFFPFY.

2. The use of the lipid-lowering polypeptide as described in claim 1 in the preparation of lipid-lowering drugs.

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