A novel polypeptide with lipid-lowering effect

Abstract

The application belongs to the technical field of biological medicine, and discloses a novel polypeptide with lipid-lowering effect. In view of the problem that polypeptides are prone to be enzymolyzed in the gastrointestinal tract and difficult to play obvious effect in the body, a novel polypeptide is designed, and the amino acid sequence of the polypeptide is GQAIWMY. The polypeptide GQAIWMY has lipid-lowering capacity, is not prone to be degraded by gastrointestinal enzymes and is more stable, and can be used for preparing drugs for preventing or treating cardiovascular diseases (such as hyperlipidemia, atherosclerosis, coronary heart disease) and fatty liver diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a novel lipid-lowering polypeptide and its application in the preparation of drugs for the prevention or treatment of cardiovascular diseases. Background Technology

[0002] Cardiovascular diseases, including hyperlipidemia, hypertension, atherosclerosis, coronary heart disease, and stroke, are among the leading causes of death and disability worldwide. According to the World Health Organization (WHO), cardiovascular diseases cause approximately 18 million deaths annually, accounting for 31% of all deaths globally. With an aging population and changes in unhealthy diets and lifestyles, the incidence of cardiovascular diseases continues to rise, placing a significant burden on healthcare systems and the socioeconomic system. Cholesterol homeostasis is crucial for maintaining good health; the body needs it to participate in cell formation, vitamin production, and the generation of other hormones. However, excessive cholesterol can lead to cardiovascular disease. Bile acids are a class of structurally similar molecules, consisting of a hydrophobic steroid moiety and a polar alkyl side chain (forming the flexible hydrophilic portion of the bile acid structure). In mammals, bile acid synthesis is the primary pathway (95%) for cholesterol catabolism. The human liver synthesizes 200-600 mg of bile acids daily, and the same level is excreted in feces to maintain adequate bile acid levels in the body. Bile acid synthesis occurs via two pathways: a neutral pathway and an acidic pathway, forming primary bile acids such as cholic acid (CA) and chenodeoxycholic acid (CDCA). In the classical bile acid metabolism pathway, primary bile acids account for approximately 75% of all bile acids, with CA and CDCA at comparable levels. Before bile acids are transported through the tubules, CA and CDCA undergo N-acylation modification with taurine and glycine to form bile salts, such as taurocholic acid (TCA) and glycocholic acid (GCA). Most primary bile acids are reabsorbed in the terminal ileum, while some unabsorbed ones enter the colon, where they are converted into secondary bile acids by the gut microbiota. CA forms deoxycholic acid (DCA), and CDCA forms lithocholic acid (LCA). The main function of bile acid micelles is to promote the dissolution and absorption of lipids in the intestine. Bile acids, as surfactants, can encapsulate hydrophobic molecules, making them stable in an aqueous environment and allowing them to be absorbed into the bloodstream through the intestinal mucosa. In lipid metabolism, the formation of bile acid micelles is a key step in lipid absorption. If bile acid micelles are disrupted, lipid absorption efficiency decreases, thereby reducing blood lipid levels. On the other hand, bile acids can improve insulin sensitivity and alleviate the development of type 2 diabetes by activating Takeda G protein-coupled receptor 5 (TGR5). Therefore, regulating the solubility of bile acid micelles is an important strategy for developing cardiovascular disease drugs or functional foods.

[0003] Bile acid sequestrants are positively charged, non-absorbable macromolecules that bind to negatively charged bile acids in the intestine, affecting the formation of bile acid micelles, reducing the body's absorption of lipids, and lowering lipid levels. A dose of approximately 16-32g of bile acid sequestrant can remove some bile acids from the enterohepatic circulation, thereby consuming about 40% of the body's bile acids, promoting the conversion of cholesterol into bile acids, and increasing the excretion of fecal sterols. For example, the first-line drug cholestyramine intervenes in cholesterol metabolism and activates TGR5 through this mechanism to reduce the risk of diseases such as atherosclerosis, coronary heart disease, and type 2 diabetes. However, bile acid sequestrants can cause varying degrees of side effects, such as constipation and flatulence, leading to low patient adherence.

[0004] Based on the characteristics of amino acids, this invention designs a polypeptide GQAIWMY (G-glycine, Q-glutamine, A-alanine, I-isoleucine, W-tryptophan, M-methionine, Y-tyrosine). Compared with other polypeptide drugs, GQAIWMY does not have the enzyme cleavage sites of active enzymes in the intestine, thus better maintaining its lipid-lowering ability in the body and making it better suited for the development of drugs to prevent and treat cardiovascular diseases. Summary of the Invention

[0005] This invention designs and synthesizes a polypeptide GQAIWMY (G-glycine, Q-glutamine, A-alanine, I-isoleucine, W-tryptophan, M-methionine, Y-tyrosine). This polypeptide has good bile acid micelle disruption ability and is not easily degraded by gastrointestinal enzymes. In addition, it has a significant in vivo lipid-lowering effect, providing a new biological resource for the treatment of cardiovascular diseases.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A polypeptide having the amino acid sequence GQAIWMY (SEQ ID NO.1).

[0007] The polypeptide GQAIWMY has the following uses: (1) Prepare lipid-lowering products, including products that lower cholesterol, triglycerides or phospholipids; (2) To prepare drugs for the prevention or treatment of fatty liver disease, including hyperlipidemia, atherosclerosis or coronary heart disease, etc. Preferably, the polypeptide is an oral preparation.

[0008] Compared with the prior art, the present invention has the following beneficial effects: At the molecular level, the polypeptide GQAIWMY has a certain ability to disrupt different bile acid micelles. Figure 4At the animal level, the peptide GQAIWMY can significantly inhibit the absorption of fluorescent cholesterol in the animal intestine, thereby reducing the level of fluorescent cholesterol in the animal serum and achieving a lipid-lowering effect. Furthermore, its effect in intestinal tissue is comparable to that of the positive control drug cholestyramine. Figure 5 ). Attached Figure Description

[0009] Figure 1 This is the high-performance liquid chromatogram of the polypeptide GQAIWMY.

[0010] Figure 2 This is the mass spectrum of the polypeptide GQAIWMY.

[0011] Figure 3 The predicted cleavage sites of pepsin, trypsin, and chymotrypsin in the polypeptide GQAIWMY are shown.

[0012] Figure 4 The effect of the polypeptide GQAIWMY on the solubility of different bile acid micelles.

[0013] Figure 5 The effect of the peptide GQAIWMY on the absorption of fluorescent cholesterol in hamsters. Detailed Implementation

[0014] Example 1: Synthesis of polypeptide GQAIWMY (1) Swelling resin: Weigh 600 mg of dichloro resin and add it to the cleaned and dried fully automatic peptide synthesizer. First, add an appropriate amount of dichloromethane (DCM) to soak for 5 min to allow the resin to swell fully. Then, add 7 mL of N,N-dimethylformamide (DMF) to wash the resin 3 times.

[0015] (2) Adding the first amino acid: Dissolve Fmoc-protected tyrosine Y in DMF at a resin:amino acid equivalent ratio of 1:0.6 and add it to the resin. Add N,N-diisopropylethylamine (DIEA) at 3-5 times the amino acid equivalent, mix gently, and then add to the reactor. React at room temperature for 1 h. Add methanol to end unreacted dichloro sites, react for 30 min, and wash the resin 3 times with DMF after the reaction. Dry the resin under vacuum.

[0016] (3) Adding the second amino acid: Peptide synthesis proceeds from the C-terminus to the N-terminus. Weigh 600 mg of methionine M, add 2 mL each of condensing agent DIEA and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), and microwave at 45°C for 300 s to carry out the condensation reaction. Drain the liquid, add 7 mL of DMF, and wash three times with the system, each time for 25 s. Drain the washing liquid. Add 7 mL of deprotecting agent piperidine, and microwave at 45°C for 300 s to carry out the condensation reaction. Drain the liquid, add 7 mL of DMF, and wash three times with the system, each time for 25 s. Drain the washing liquid.

[0017] (4) Subsequent amino acid addition: The method of addition is the same as the process of adding the second amino acid, until all amino acids are added.

[0018] (5) Resin lysis and drying: First, wash the resin with 6 mL of methanol and vacuum dry for 2 min. Add 8 mL of lysis buffer (containing 90% TFA, 2.5% phenol, 2.5% EDT, 2.5% benzyl mercaptan, and 2.5% diethyl ether), and lyse at 30℃ for 3 h. Add 40 mL of ice-cold diethyl ether to precipitate, shake well, and centrifuge for 2 min. Remove the supernatant, resuspend in ice-cold diethyl ether, and centrifuge once more to obtain crude peptide.

[0019] (6) Crude product purification: The peptide was purified using a Shimadzu LC-20AP preparative high-performance liquid chromatograph. The peptide was weighed, dissolved in a measured amount of 70% acetonitrile aqueous solution by sonication, filtered through a 0.45 μm filter membrane, and then loaded onto the sample. A quantitative loop was inserted into the sample injector, and the crude peptide solution was loaded at a flow rate of 12 ml / min. Gradient elution was performed according to the following procedure: Column: 20 mm × 250 mm 10 μM C18 reverse-flow silica column Mobile phase A: 0.1% TFA aqueous solution Mobile phase B: 0.1% TFA acetonitrile solution Gradient elution program: 0-25 min 5-65% mobile phase B; 25-30 min 95% mobile phase B; 30-35 min 5% mobile phase B; detection wavelength: 220 nm.

[0020] (7) Purity identification: After lyophilization, the purified peptides were subjected to purity identification using a Shimadzu LC-20AB analytical high-performance liquid chromatography system under the following conditions: Column: Inertsil ODS-3 4.6×250 mm Mobile phase A: 0.065% TFA aqueous solution Mobile phase B: 0.05% TFA acetonitrile solution Gradient elution program: 0-25 min 5-65% mobile phase B; 25-27 min 95% mobile phase B; 27-35 min 5% mobile phase B; detection wavelength: 220 nm.

[0021] The chromatogram of the peptide detected by HPLC is as follows: Figure 1 As shown, a major absorption peak is observed at a retention time of 9.808 min at a wavelength of 220 nm, indicating a purity of 95.87%. The secondary spectrum of the peptide, identified by a Shimadzu LCMS2020 mass spectrometer, is shown below. Figure 2 As shown, the strongest ion signal is present at a mass-to-charge ratio of 868.4, which can be identified as the main detected component. The predicted cleavage sites of pepsin, trypsin, and chymotrypsin in the polypeptide GQAIWMY are shown below. Figure 3 As shown, the polypeptide does not contain pepsin or trypsin cleavage sites, and the chymotrypsin cleavage site does not affect the sequence of the polypeptide itself.

[0022] Example 2: Peptide GQAIWMY reduces bile acid micelle solubility Solutions of 2 mM cholesterol, 4 mM oleic acid, 2.4 mM lecithin, and 2 mM monoacylglycerol were prepared using methanol. 250 μL of each solution was thoroughly mixed and concentrated by rotary freezing until complete evaporation. Then, 1 mL of PBS-prepared 4 mM taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), or glycochenodeoxycholic acid (GCDCA) solutions were added. The mixture was sonicated for 20 min and incubated at 37°C for 24 h to prepare TCA micelles, GCA micelles, TCDCA micelles, and GCDCA micelles. 5 mM peptides and cholestyramine were prepared using PBS. Bile acid micelles and peptide solutions or cholestyramine were mixed at a 1:1 volume ratio and reacted at 37°C and 250 rpm for 1 h. After centrifugation at 15000×g for 30 min, the cholesterol concentration in the supernatant was measured, and the solubility of bile acid micelles after peptide or cholestyramine treatment was calculated. The control group used PBS solution without micelles, while the model group was tested for micelle cholesterol levels without peptides or cholestyramine.

[0023] like Figure 4 As shown, for TCA and GCA micelles, the peptide group GQAIWMY significantly reduced the solubility of TCA and GCA micelles compared to the model group, and its effect was stronger than that of the first-line drug cholestyramine. For TCDCA and GCDCA micelles, the peptide group GQAIWMY significantly reduced the solubility of TCDCA and GCDCA micelles compared to the model group, and its effect was comparable to that of the first-line drug cholestyramine.

[0024] Example 3: Effect of peptide GQAIWMY on fluorescent cholesterol uptake in hamsters Forty male Syrian golden hamsters (5 weeks old, weighing 100-120 grams) were used as animal models and randomly divided into groups. Food was removed from their cheek pouches, and they were acclimatized to gavage using a 12-gauge gavage syringe with an empty gavage tube. After restraint, one ear was exposed, iodine was applied, and an ear tag was placed at the base of the ear using ear-tag pliers. They were kept hungry but allowed free access to water for 18 hours. Gavage treatment: Control group: 200 μL corn oil; Model group: 200 μL corn oil + 0.5 mg NBD-cholesterol (fluorescent cholesterol); Peptide group: 200μL corn oil + 0.5 mg NBD-cholesterol + 50 mg peptide (GQAIWMY); Cholestyramine group: 200 μL corn oil + 0.5 mg NBD-cholesterol + 50 mg cholestyramine.

[0025] Four zirconium beads were added to the gavage sample before administration. The sample was broken up at 6 Hz for 10 seconds, with a 10-second interval, and repeated 10 times to ensure complete mixing. 1.5 h later, the sample was anesthetized with isoflurane, and blood was collected by enucleation. The blood was left at room temperature for at least 30 minutes, then centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. This process was repeated twice. 20 μL of serum was added to 500 μL of cholesterol extraction buffer (isopropanol: n-heptane: 125 mM sulfuric acid, volume ratio = 80:19:16). After centrifugation, 200 μL of the supernatant was added to a black ELISA plate for detection (excitation wavelength 465 nm, detection wavelength 535 nm). A weighed jejunal tissue sample was broken up with 200 μL of lysis buffer, centrifuged at 2000 × g for 5 minutes, and 20 μL of the supernatant was collected. 480 μL of cholesterol extraction buffer was added to the supernatant, and after centrifugation, 200 μL of the supernatant was added to a black ELISA plate for detection (excitation wavelength 465 nm, detection wavelength 535 nm). Centrifuge the remaining sample at 12000×g for 15 minutes, collect the supernatant, and measure the protein level (detection wavelength 562nm). Figure 5 As shown, compared with the model group, the peptide GQAIWMY group can significantly reduce the NBD-cholesterol level in hamster serum and jejunal tissue, and in jejunal tissue, its ability is comparable to that of the first-line drug cholestyramine.

[0026] In summary, the peptide GQAIWMY exhibits significant lipid-lowering capabilities at both the molecular and animal levels. Specifically, GQAIWMY is less susceptible to degradation by intestinal enzymes, making it more stable, and its ability to disrupt the solubility of TCA and GCA micelles is stronger than that of cholestyramine. In the intestine, bile acid micelles promote the dissolution and absorption of lipids (such as cholesterol, triglycerides, and phospholipids). When bile acid micelles are disrupted, the body's absorption of lipids decreases, thus lowering the body's lipid levels. At the animal level, GQAIWMY significantly inhibits lipid absorption, and its effect in intestinal tissue is comparable to that of the first-line drug cholestyramine. This indicates that GQAIWMY has a similar mechanism of action to cholestyramine, with better lipid-lowering potential, and can be used to treat indications related to cholestyramine, including hyperlipidemia, coronary heart disease, fatty liver, and atherosclerosis.

Claims

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

1.

2. The use of the polypeptide of claim 1 in the preparation of lipid-lowering products.

3. The application according to claim 2, characterized in that, Products that lower cholesterol, triglycerides, or phospholipids.

4. The application according to claim 2, characterized in that, The products include food, health products, or medicines.

5. The use of the polypeptide of claim 1 in the preparation of drugs for the prevention or treatment of hyperlipidemia, atherosclerosis or coronary heart disease.

6. The use of the polypeptide of claim 1 in the preparation of a drug for the prevention or treatment of fatty liver disease.

7. The application according to any one of claims 2 to 6, characterized in that, The polypeptide is an oral preparation.

Images