A tetrapeptide LILP with uric acid lowering activity and its application

Abstract

The present invention discloses a tetrapeptide LILP with uric acid lowering activity and its application, belonging to the field of biomedical technology. The amino acid sequence of the tetrapeptide LILP is Leu‑Ile‑Leu‑Pro. The present invention discloses for the first time that the tetrapeptide LILP has uric acid lowering activity, which can reduce uric acid production by reducing xanthine oxidase activity in the liver; it can also increase uric acid excretion by reducing uric acid reabsorption in the kidneys. The present invention provides a multi-target uric acid-lowering active substance, which can be used to prepare drugs for preventing or treating hyperuricemia, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a tetrapeptide LILP having uric acid-lowering activity and an application thereof. Background Art

[0002] Uric acid is the final product of purine metabolism and is mainly produced in the liver. In order to maintain the balance of uric acid in the human body, about 75% of uric acid is excreted through the kidneys and the remaining 25% is excreted through the intestines. Excessive production and insufficient excretion of uric acid will cause high blood uric acid, which will lead to hyperuricemia in the long run. When the human body's blood uric acid content is oversaturated, urate (mainly MSU crystals) will precipitate in the blood. The deposition of MSU crystals in joints and other body tissues (such as bursae and tendons) will cause a spontaneous immune response in the human body, leading to local inflammation. This arthritis accompanied by severe but intermittent pain is gout.

[0003] At present, clinical uric acid-lowering drugs are mainly divided into three categories: drugs that reduce uric acid production, such as allopurinol and febuxostat; drugs that promote uric acid excretion, such as probenecid and benzbromarone; drugs that accelerate uric acid decomposition, such as polyethylene glycol uricase. However, long-term use of the above three types of drugs in clinical practice will have obvious toxic side effects, such as gastrointestinal discomfort (nausea, vomiting, diarrhea), skin allergies, intermittent headaches, muscle pain, and liver and kidney toxicity. Therefore, it is urgent to develop natural, highly effective and low-toxic drugs.

[0004] Food-derived bioactive peptides are a class of small molecule peptide compounds with beneficial physiological functions that are prepared from food raw materials by enzymatic hydrolysis, fermentation, separation and purification. At present, there are many studies on bioactive peptides from animals, especially many peptides prepared by enzymatic hydrolysis of marine animals have shown excellent uric acid-lowering activity. For example, the study by WAN H et al. showed that the hydrolysates of two sea cucumbers could inhibit uric acid biosynthesis and promote uric acid excretion in a diet-induced hyperuricemia mouse model, thereby alleviating the hyperuricemia phenotype (Comparisons of protective effects between two sea cucumber hydrolysates against diet induced hyperuricemia and renal inflammation in mice. Food Funct, 2020, 11(1): 1074-1086). The study by HE W et al. showed that tuna protein hydrolysate prepared by alkaline protease hydrolysis showed uric acid-lowering effect in a hyperuricemia rat model. The dipeptide FH obtained by further separation and purification showed XOD inhibitory activity IC 50is 25.7 mmol / L (In vivo anti-hyperuricemic and xanthine oxidase inhibitory properties of tuna proteinhydrolysates and its isolated fractions. Food Chem, 2019, 272: 453-461.).

[0005] Small molecule bioactive peptides from natural sources have good bioavailability, are easily metabolized and eliminated by the human body, have low toxic side effects, and have the potential to be developed into natural, safe and efficient new uric acid-lowering products. Therefore, discovering more efficient small molecule bioactive peptides is a technical problem that technicians in this field need to solve. Summary of the invention

[0006] The purpose of the present invention is to provide a natural small molecule biological peptide with uric acid lowering activity, which is used to develop a safe and efficient new uric acid lowering product.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention simulates gastrointestinal digestion of tuna peptides in vitro and identifies peptides that are stable before and after digestion by LC-MS / MS. Molecular docking technology is used to explore the interaction between peptides and xanthine oxidase (XOD) protein, from which a candidate peptide LILP is screened out, whose amino acid sequence is Leu-Ile-Leu-Pro. Further artificial synthesis of tetrapeptide LILP was found to show efficient uric acid-lowering activity in a hyperuricemia cell model.

[0009] Therefore, the present invention provides a tetrapeptide LILP having uric acid-lowering activity, wherein the amino acid sequence of the tetrapeptide LILP is Leu-Ile-Leu-Pro.

[0010] The present invention also provides a method for preparing the tetrapeptide LILP. The tetrapeptide LILP can be prepared by solid phase synthesis. The specific method includes: adopting an Fmoc solid phase synthesis strategy, using Fmoc-protected amino acids as raw materials, selecting Wang resin as a solid phase carrier, sequentially synthesizing proline, leucine, isoleucine, and leucine residues to extend the peptide chain from the C-terminus to the N-terminus, and solid phase synthesizing the tetrapeptide LILP.

[0011] The tetrapeptide LILP can also be obtained by enzymatic hydrolysis of tuna meat. Further, tuna dorsal and ventral meat is used as raw material, and enzymatic hydrolysis is performed using neutral protease, the enzymatic hydrolysis temperature is 30-60°C, the pH of the reaction system is controlled at 7-8, and the enzymatic hydrolysis time is 4-6 hours. Furthermore, the enzymatic hydrolysis conditions are neutral protease (500 U / g, pH=7) at 50°C for 5 hours to obtain a polypeptide enzymatic hydrolyzate, and the polypeptide enzymatic hydrolyzate contains the tetrapeptide LILP.

[0012] The present invention provides the use of the tetrapeptide LILP in the preparation of a uric acid-lowering drug. The tetrapeptide LILP provided by the present invention can significantly reduce the uric acid content in a hyperuricemia cell model, and can be used to develop drugs for treating hyperuricemia and / or gout.

[0013] Hyperuricemia is a metabolic syndrome caused by purine metabolism disorder, usually referring to fasting blood uric acid levels that are long-term higher than the critical value (adult females>6.0mg / dL, adult males>7.0mg / dL, adolescents>5.5mg / dL). Gout is a complex arthritis characterized by intermittent attacks of pain, which is caused by the body's natural immune response to deposited monosodium uric acid crystals.

[0014] Furthermore, the effects of the drug include: inhibiting the activity of xanthine oxidase and reducing the mRNA expression of uric acid reabsorption transporter.

[0015] Mechanism studies have shown that the tetrapeptide LILP provided by the present invention has a highly effective xanthine oxidase inhibitory activity, and the in vitro XOD inhibitory activity IC 50 The value is 10.18±1.35 mmol / L, which can reduce the liver's metabolism of purine to produce uric acid by inhibiting the activity of xanthine oxidase, thereby reducing the production of uric acid in the body; at the same time, the tetrapeptide LILP can significantly reduce the mRNA expression of uric acid reabsorption transporters, and increase uric acid excretion by reducing renal uric acid reabsorption. The tetrapeptide LILP provided by the present invention can reduce the deposition of uric acid in the body through multi-target effects, thereby achieving the purpose of alleviating hyperuricemia and / or gout.

[0016] Furthermore, the uric acid reabsorption transporter includes glucose transporter 9 and uric acid transporter 1 in kidney cells. Glucose transporter 9 and uric acid transporter 1 in renal proximal tubular epithelial cells are responsible for uric acid reabsorption, and the tetrapeptide LILP provided by the present invention can significantly reduce the expression of glucose transporter 9 and uric acid transporter 1, thereby reducing the renal reabsorption of uric acid.

[0017] Furthermore, the drug is a drug for alleviating hyperuricemia and / or gout in mammals. The mammals may be, but are not limited to, humans.

[0018] Specifically, the present invention provides a pharmaceutical composition for treating hyperuricemia and / or gout, comprising an effective dose of a tetrapeptide LILP having an amino acid sequence of Leu-Ile-Leu-Pro and a pharmaceutically acceptable carrier.

[0019] The pharmaceutical composition is prepared by adding a pharmaceutically acceptable carrier with the tetrapeptide LILP as the main active ingredient, and can be prepared according to the preparation method recorded in pharmacy. The tetrapeptide LILP in the pharmaceutical composition provided by the present invention can be used as the only active ingredient that exerts the effect of lowering uric acid, and can also be compounded with other active ingredients that have the effect of regulating uric acid content, such as plant extracts (eucommia, sunflower, etc.) reported to have uric acid lowering activity, vitamins (such as vitamin B), polyphenols, protein peptides, etc.

[0020] The pharmaceutically acceptable carrier includes one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant.

[0021] Furthermore, the pharmaceutical composition may be in the form of an oral preparation but is not limited thereto. The tetrapeptide LILP provided by the present invention is an enzymatic hydrolysis product of tuna meat and can still exist stably after being digested by the gastrointestinal tract.

[0022] The pharmaceutical composition can be prepared as, but not limited to, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion. The specific dosage is adjusted according to the type of disease, disease severity, age and purpose of administration.

[0023] In the hyperuricemia cell model, the effective dose of tetrapeptide LILP was 1 mmol / L. CCK-8 experiments showed that tetrapeptide LILP showed no cytotoxicity in the concentration range of 0.1-3.2 mmol / L.

[0024] Terminology Note:

[0025] The term "comprising" is an open expression, that is, it includes the contents specified in the present invention but does not exclude other contents.

[0026] The term "treating" refers in some embodiments to ameliorating a disease or condition (i.e., slowing or arresting or alleviating the development of the disease or at least one clinical symptom thereof). In other embodiments, it refers to alleviating or improving at least one physical parameter, including physical parameters that may not be perceived by the subject. In other embodiments, it refers to regulating a disease or condition physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a physical parameter), or both.

[0027] The term "effective dose" refers to an amount of a compound sufficient to be effective for the treatment of a disease when administered to a subject. The effective dose may vary with the severity of the disease, and the physical condition, age, weight, sex, etc. of the subject to be treated.

[0028] The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium that can deliver an effective dose of the active substance of the present invention without interfering with the biological activity of the active substance and having no toxic side effects on the host or subject.

[0029] The present invention has the following beneficial effects:

[0030] The present invention discloses for the first time that the tetrapeptide LILP has uric acid lowering activity. The tetrapeptide LILP not only has efficient xanthine oxidase inhibitory activity, but also reduces uric acid production by reducing xanthine oxidase activity in the liver; at the same time, the tetrapeptide LILP can also significantly reduce the mRNA expression of uric acid reabsorption transporters, and increase uric acid excretion by reducing renal uric acid reabsorption. The tetrapeptide LILP provided by the present invention can reduce the deposition of uric acid in the body through multi-target effects, and the tetrapeptide LILP is a natural peptide segment with high biological safety. Therefore, it can be used to prepare drugs for preventing or treating hyperuricemia. The present invention provides a multi-target uric acid-lowering active substance with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the primary mass spectrum of the tetrapeptide LILP.

[0032] Figure 2 This is the secondary mass spectrum of the tetrapeptide LILP.

[0033] Figure 3 Molecular docking of the ligand peptide LILP and the XOD receptor protein at the MOS site.

[0034] Figure 4 Molecular docking of the ligand peptide LILP and the XOD receptor protein at the FAD site.

[0035] Figure 5 This is a cytotoxicity test of the tetrapeptide LILP.

[0036] Figure 6 This is the detection of uric acid content in the supernatant of the HK-2 hyperuricemia cell model after the action of the tetrapeptide LILP.

[0037] Figure 7 It is the mRNA expression level of uric acid reabsorption protein GLUT9 in the HK-2 hyperuricemia cell model after the action of tetrapeptide LILP.

[0038] Figure 8It is the mRNA expression level of uric acid reabsorption protein URAT1 in the HK-2 hyperuricemia cell model after the action of tetrapeptide LILP. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0041] Pepsin, trypsin, and porcine bile salt were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Adenosine, xanthine, xanthine oxidase, allopurinol, uric acid (UA) content detection kit, xanthine oxidase (XOD) activity detection kit, and CCK-8 kit were purchased from Beijing Solebow Technology Co., Ltd. Human renal cortical proximal tubule epithelial cells (HK-2), MEM medium containing NEAA, fetal bovine serum (FBS), penicillin / streptomycin solution (Penicillin / Streptomycin, P / S), and trypsin were purchased from Dalian Meilun Biotechnology Co., Ltd. RNAsimple total RNA extraction kit (spin column type) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Reverse transcription kit and fluorescence quantitative PCR kit were purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0042] Example 1: Screening of active peptides

[0043] In this example, the stable peptides before and after digestion were identified by LC-MS / MS through in vitro simulated gastrointestinal digestion of tuna peptides, molecular docking technology was used to explore their specific mechanism of action with XOD protein, and highly effective XOD inhibitory peptides were screened out.

[0044] 1. The tuna peptide used in this study was prepared by a single-factor experimental optimization method described in the reference (Zou Lin. Enzymatic preparation and functional activity evaluation of skipjack tuna xanthine oxidase inhibitory peptide [D], 2019.), using tuna dorsal and ventral meat as raw material and obtained by enzymatic hydrolysis with neutral protease (500 U / g, pH=7) at 50°C for 5 h.

[0045] 2. In vitro simulated digestion: Under light-proof and oxygen-free conditions, take 10 mL of tuna peptide sample solution, preheat in a 37 °C water bath, add 9 mg / mL NaCl solution preheated in a 37 °C water bath, adjust the pH to 2 with 1 mol / L HCl, and add 4 mL of gastric juice (0.4 g pepsin dissolved in 0.01 mol / L HCl solution) to the system. Simulate gastric digestion after shaking at 37 °C water bath and 120 r / min for 2 h. Inactivate protease in a boiling water bath, centrifuge at 4 °C, 12000 r / min for 15 min, take the supernatant, obtain the peptide after in vitro simulated gastric digestion, and store at -20 °C in the dark.

[0046] Under light-proof and oxygen-free conditions, the pH value of the sample after gastric digestion was pre-adjusted to 6.5. 2 mL of the sample after gastric digestion was drawn into a centrifuge tube, and 4 mL of intestinal juice was added to each tube (0.02 g trypsin and 0.12 g pig bile salt were dissolved in 10 mL of 0.1 mol / L NaHCO3 solution, respectively, to obtain 2 mg / mL trypsin solution and 12 mg / mL bile solution, and pancreatic juice and bile were mixed 1:1), and the pH was adjusted to 7.0. The intestinal digestion simulation was completed after shaking at 120 r / min for 4 h in a 37°C water bath. The protease was inactivated in a boiling water bath, and the sample was centrifuged at 12000 r / min for 15 min at 4°C. The supernatant was taken to obtain peptides after in vitro simulated gastric and intestinal digestion, which were stored at -20°C in the dark.

[0047] 3. LC-MS / MS identification of active peptides of tuna peptides before and after digestion

[0048] Sample pretreatment: Dissolve the sample with 50 mmol / L NH4HCO3. Take an appropriate amount of the dissolved sample, add dithiothreitol solution to the sample to make the final concentration of 10 mmol / L, and reduce it in a 56℃ water bath for 1 h. Add iodoacetamide solution to make the final concentration of 50 mmol / L, and react for 40 min in the dark. Desalt using a self-filled desalting column and evaporate the solvent in a 45℃ vacuum centrifugal concentrator.

[0049] Liquid chromatography conditions: analytical column: 150 μm id×150 mm, packed with Acclaim Pep MapRPLC C18, 3 μm, 100 Å; mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% ACN; flow rate: 600 nL / min; analysis time for each component: 66 min. Gradient elution time is shown in Table 1.

[0050] Table 1. Gradient elution time

[0051] time Phase A (%) Phase B (%) 0 96 4 2 92 8 45 72 28 55 60 40 56 5 95 66 5 95

[0052] The mass spectrometry conditions are shown in Table 2. The mass spectrometry raw files were retrieved using Byonic software to retrieve the target protein data.

[0053] Table 2. Primary and secondary mass spectrometry parameters

[0054] parameter Primary mass spectrometry Secondary mass spectrometry Resolution 70000 17500 AGCtarget 3e6 1e5 MaximumIT (ms) 100 50 Scanrange (m / z) 300-1800 / TopN / 20 NCE / steppedNCE / 28

[0055] 4. Molecular docking to screen active peptides

[0056] Xanthine oxidase (XOD) can catalyze hypoxanthine to generate xanthine in the human body, and can also directly catalyze the metabolism of xanthine into uric acid, playing a key role in the purine metabolic pathway. Therefore, this study selected peptides that existed stably before and after digestion for homology modeling, and used them as ligand small molecules to perform molecular docking with the XOD receptor protein at the two main binding active sites, molybdenum dioxide ion (MOS) and flavin adenine dinucleotide (FAD), and used the ligand existing in XOD itself as a positive control.

[0057] Ligand peptide preparation: Use ChemBio3D Ultra software to input the peptide sequence for 3D modeling and generate the peptide structure.

[0058] Receptor pretreatment: The X-ray crystal structure of bovine XOD protein (PDBID: 3NVY) was downloaded from the PDB database (https: / / www.rcsb.org / ), the existing ligand quercetin was removed using PyMol software, and the software MGLTools was used to add charge and correct chemical bonds.

[0059] Molecular docking: AutodockVina software was used for molecular docking, the docking pockets covered MOS and FAD sites respectively, and the docking score was calculated.

[0060] Analysis of docking results: PyMol software was used to analyze the docking results and record the number and sites of hydrogen bonds and π-π interactions.

[0061] 5. Results Analysis

[0062] Tuna peptides (tuna hydrolysate) obtained by enzymatic hydrolysis of tuna with neutral protease were opened with Xcalibur to generate raw files after LC-MS / MS data acquisition. The Byonic database was used for search and matching based on the results of LC-MS / MS. Tuna peptides before digestion could match 1,330 different peptide sequences, while only 151 different peptide sequences could be detected after digestion. This indicates that tuna peptides have extremely high peptide diversity before digestion. During the digestion process, tuna peptides are enzymatically hydrolyzed by gastric and intestinal proteases, and some peptides are cut into smaller fragments, or some peptides are completely degraded, resulting in the number of different peptide sequences detected being reduced to 151.

[0063] Considering the digestion stability of peptides, this study selected peptides that can exist stably before and after digestion for research. Figure 1 and Figure 2 They represent the primary mass spectrum and secondary mass spectrum of one of the four peptides respectively. The fragment ions of peptides include: N-terminal fragment ions (a, b, c types) and C-terminal fragment ions (x, y, z types). The side chains of a, y, and z type ions break to form d, v, and w type ions respectively. In addition, there are internal ions formed by the breakage of both ends. The b and y series ions are the most common. The primary structure of the peptide can be analyzed and inferred based on the b or y series fragment ions of the peptide. Figure 1 m / z 455.32 is the [M+H]+ ion signal of the tetrapeptide, which is basically consistent with the molecular weight of the tetrapeptide of 455.40. The tetrapeptide was further analyzed by secondary mass spectrometry using in-source collision-induced dissociation technology, and the primary structure of the tetrapeptide was determined to be Leu-Ile-Leu-Pro ( Figure 2 ).

[0064] The molecular docking results of the tetrapeptide LILP are shown in Figure 3 , Figure 4 As shown in the figure, the XOD protein used in this experiment comes from bovine milk, and there is a quercetin ligand at the MOS site, with a binding energy of -8.7. Molecular docking revealed the binding mode of quercetin to the receptor, forming hydrogen bonds with the amino acid residues of XOD at the GLU802, ARG880 and THR1010 sites, and forming π-π interactions with amino acids PHE914 and PHE1009. The binding energy of the tetrapeptide LILP is -6.8, forming two hydrogen bonds with the amino acid residue LYS771, and one hydrogen bond each with SER876 and LEU648.

[0065] FAD is a ligand structure existing in xanthine oxidase itself, and is involved in the electron transfer process of xanthine and hypoxanthine oxidation reactions. Molecular docking showed that FAD and XOD formed a stable complex structure with a docking score of -12.4, and hydrogen bonds were formed at 9 amino acid residue binding sites: VAL259, ASN261, THR262, GLU263, ILE264, ASP360, ASN351, SER347 and LEU404. The binding energy of tetrapeptide LILP at the FAD site was -8.4, forming a total of 7 hydrogen bonds, 3 hydrogen bonds with THR354, 2 hydrogen bonds with GLY350, and 1 hydrogen bond with ASN351 and ILE264 at the same site as the FAD ligand itself.

[0066] The above results indicate that the tetrapeptide LILP is a XOD inhibitor with good performance at the molecular docking level and has the potential to be developed into a multi-target uric acid-lowering active substance.

[0067] Example 2: Synthesis of active peptide LILP

[0068] In this example, the peptide LILP was artificially synthesized. Specifically, Jier Biochemical (Shanghai) Co., Ltd. was commissioned to synthesize the peptide LILP with a purity of ≥98%, and the method was as follows:

[0069] A solid phase synthetic resin based on Wang resin with FMOC protective group and amino acid residues was used. The glass reaction column was swollen with DCM for 30 min, and DCM was removed under reduced pressure. The resin was washed with DMF 3 times, and a 20% piperidine / DMF solution was added to react for 20 min to remove the protective group fmoc, and the solution was removed under reduced pressure, and washed with DMF 6 times. Subsequent amino acids containing FMOC protective group were added to the resin in sequence, dissolved in DMF and DIEA was added; reacted for 30 min, the resin was taken for color test reaction, and the solution color test and resin color were observed. The solution was bright yellow and the resin was yellow, indicating that the reaction was complete. The solvent was removed under reduced pressure. The cleavage solution was added to remove the resin and amino acid side chain protective groups, and the sand core was filtered. Ether was added to the filtrate to precipitate, centrifuged, washed the solid 3 times, dried, and detected by mass spectrometry. Purification method: The main peak time of the crude peptide with a concentration of 5%-95% was analyzed by HPLC, dissolved in acetonitrile and water, clarified, filtered, and separated by HPLC-MS to obtain the pure product and determine the molecular weight of the peptide.

[0070] Example 3: In vitro XOD inhibitory activity assay of tetrapeptide LILP

[0071] 1. Determination of XOD inhibition rate

[0072] In a 96-well plate, 50 μL of PBS buffer was added to each well of the blank control group, 50 μL of allopurinol (0.1 mg / mL) was added to each well of the positive control group, and 50 μL of the sample peptide LILP solution (20 mg / mL) synthesized in Example 2 was added to each well of the experimental group. 50 μL of XOD solution (0.02 U / mL) was added to each well of each group, oscillated for 30 s, and incubated at 37 °C for 5 min. After that, 150 μL of xanthine solution (0.48 mmol / L) was added to start the reaction, oscillated for 30 s, and incubated at 37 °C for 25 min. Finally, 80 μL of HCl (1 mol / L) was added to terminate the reaction, and the reaction system was diluted 5 times with ultrapure water, filtered through a 0.22 μm filter membrane, and then subjected to high performance liquid chromatography injection analysis to determine the uric acid content.

[0073] HPLC conditions: C18 column (4.6 mm × 250 mm, 5 μm), injection volume 20 μL, column temperature 25 °C, mobile phase A was 0.1% (v / v) formic acid in water, mobile phase B was methanol, flow rate 1 mL / min, gradient elution (0-3 min was 90% A, 3-6 min was 90%-0% A, 6-8 min was 0%-90% A, 8-10 min was 90% A), detection wavelength was 290 nm.

[0074] The above experiment was repeated 3 times in parallel, and the XOD inhibition rate was calculated as follows:

[0075] XOD inhibition rate (%) = (1-uric acid peak area of ​​experimental group / uric acid peak area of ​​blank group) × 100%.

[0076] 2. IC of XOD inhibitory peptide 50 Calculation of values

[0077] A gradient concentration (1, 2, 4, 8, 16, 32 mg / mL) was set, and 50 μL of the sample peptide solution with gradient concentration was added to the 96-well plate as the experimental group, and 50 μL of PBS buffer was added to the blank group. 50 μL of XOD solution (0.02U / mL) was added to each well, oscillated for 30 s, and incubated at 37°C for 5 min. Then, 150 μL of xanthine solution (0.48 mmol / L) was added to start the reaction, oscillated for 30 s, and incubated at 37°C for 25 min. Finally, 80 μL of HCl (1 mol / L) was added to terminate the reaction, and the reaction system was diluted 5 times with ultrapure water, filtered through a 0.22 μm filter membrane, and then analyzed by high-performance liquid chromatography to determine the uric acid content. The high-performance liquid chromatography conditions and the calculation method of XOD inhibition rate were the same as step 1. The above experiment was repeated 3 times in parallel, and a scatter plot was made with the logarithm of the peptide concentration and the XOD inhibition rate as the horizontal and vertical coordinates, respectively. The XOD inhibition IC was calculated after fitting the trend line. 50 value.

[0078] 3. Results Analysis

[0079] As shown in Table 3, the in vitro XOD inhibitory activity IC 50 =10.18±1.35 mmol / L, which also verified the results of molecular docking. The efficient XOD inhibitory activity of the peptide may be related to its ability to form a stable binding conformation with XOD at both MOS and FAD active sites.

[0080] Table 3. In vitro XOD inhibitory activity of active peptides

[0081] Peptide sequence <![CDATA[XOD inhibitory IC 50 Value (mmol / L)]]> LILP 10.18±1.35

[0082] The above results indicate that the tetrapeptide LILP has the effect of inhibiting XOD activity and thus reducing the production of uric acid in the body.

[0083] Example 4: Cytotoxicity analysis of tetrapeptide LILP (CCK-8)

[0084] 1. Cell culture: HK-2 human kidney cortex proximal tubule epithelial cells (Human Kidney Cortex Proximal Tubule Epithelial Cell Line, HK-2), growth morphology is adherent growth. Culture system: MEM medium + 10% FBS + 1% P / S. Subculture cycle 2-3 days. Subculture ratio 1:3-1:4, medium change frequency 2-3 days. Culture environment: 95% air + 5% carbon dioxide, temperature 37°C.

[0085] 2. Cytotoxicity experiment: Take HK-2 cells in the logarithmic growth phase, discard the cell culture medium, add an appropriate amount of PBS to rinse the cells and discard, add trypsin to digest for 3 minutes, then add culture medium to terminate digestion, resuspend the cells after centrifugation, blow and evenly use a pipette, count the number of cells with a hemocytometer and dilute, and divide the number of cells into 10 5 100 μL cell suspension were inoculated into 96-well plates, and blank, 0 drug group (cells only) and drug group (gradient concentration peptide treatment) were set up, with 3 replicates in each group. The culture plate was placed in an incubator for pre-culture, and the cell culture medium was aspirated after the cells attached to the wall, and PBS was rinsed three times. The blank group and 0 drug group were added with culture medium, and the drug group was added with peptide-containing culture medium containing concentrations of 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mmol / L, respectively. After 24 hours of intervention, the culture medium was aspirated, and 10 μL of CCK-8 solution was added to each well. The culture plate was incubated in the incubator for 1-4 hours, and the absorbance at 450 nm was measured with an enzyme marker to calculate the cell survival rate.

[0086] Cell viability (%) = [A (drug addition) - A (blank)] / [A (0 drug addition) - A (blank)] × 100;

[0087] In the formula, A(drug added): absorbance of wells with cells, CCK-8 solution and drug solution; A(blank): absorbance of wells with culture medium and CCK-8 solution but no cells; A(0 drug added): absorbance of wells with cells, CCK-8 solution but no drug solution; cell viability: cell proliferation viability or cytotoxicity viability.

[0088] 3. Data processing and analysis: Graphpad Prism 9 software was used for statistical difference analysis and drawing. All data were expressed as mean ± standard deviation (Mean ± SD). Each group of data was tested for normal distribution and homogeneity of variance. One-way ANOVA was used for inter-group comparison, and P < 0.05 was considered significant.

[0089] 4. Results Analysis

[0090] The results are as follows Figure 5 As shown in the results, when HK-2 cells were cultured for 24 hours, the tetrapeptide LILP showed no cytotoxicity in the concentration range of 0.1-3.2 mmol / L. The CCK8 experiment showed that the cell survival rate in each experimental group was between 90% and 110%, with no significant difference from the control group. Therefore, the peptides in the concentration range of 0.1-3.2 mmol / L can be used to intervene in HK-2 cells for 24 hours in subsequent experiments.

[0091] Example 5: Effect of tetrapeptide LILP on uric acid content in HK-2 hyperuricemia cell model

[0092] 1. HK-2 cells in logarithmic growth phase were cultured at 2×10 5 The cells were inoculated into 6-well plates and cultured with complete medium for 24 h. After the cells adhered to the wall, the culture medium was aspirated and the cells were washed three times with PBS. The following groups were divided and administered: (1) blank group; (2) modeling group; (3) positive control group; (4) tetrapeptide group, with 3 replicate wells in each group.

[0093] Blank group: The cells were cultured in complete medium for 24 h, the culture medium was aspirated and washed three times with PBS, and then cultured in serum-free medium for another 24 h.

[0094] Modeling group: The cells were cultured in complete medium for 24 h, the culture medium was aspirated and washed three times with PBS, and then cultured in serum-free medium containing 0.5 mmol / L adenosine for 24 h.

[0095] Positive control group: cultured in complete medium containing 0.1 mmol / L febuxostat for 24 h, the culture medium was aspirated and washed three times with PBS, and then cultured in serum-free medium containing 0.5 mmol / L adenosine for 24 h.

[0096] Tetrapeptide group: The cells were cultured in complete medium containing 1 mmol / L tetrapeptide LILP for 24 h, the culture medium was aspirated and washed three times with PBS, and then cultured in serum-free medium containing 0.5 mmol / L adenosine for 24 h.

[0097] After the above grouping treatments were completed, 0.005 U / mL of xanthine oxidase (XOD) was added to each group for 1 h, and the cell supernatant was collected to determine the uric acid content according to the kit instructions. If the uric acid content in the cell supernatant of the model group was significantly higher than that of the blank control group, and the final uric acid production in the positive control group was significantly lower than that in the model group, and there was a certain concentration-dose effect, it indicated that the model was basically constructed successfully.

[0098] 2. Data processing and analysis: Graphpad Prism 9 software was used for statistical difference analysis and drawing. All data were expressed as mean ± standard deviation (Mean ± SD). Each group of data was tested for normal distribution and homogeneity of variance. One-way ANOVA was used for inter-group comparison, with P < 0.05 as significant difference (compared with the model group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, model group compared with blank, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001, there was a significant difference, indicating that the model was successfully established).

[0099] 3. Results Analysis

[0100] like Figure 6 As shown in the figure, 1 mmol / L of peptide was used as the intervention dose. The results of uric acid content in the cell supernatant showed that the uric acid in the modeling group was significantly higher than that in the control group ( P <0.0001), indicating that 0.5 mmol / L adenosine and 0.005 U / mL exogenous xanthine oxidase can be used to model the hyperuricemia cell model.

[0101] The uric acid content in the positive control group was significantly lower than that in the modeling group (P<0.0001), indicating that 0.1 mmol / L febuxostat can be used as a positive drug control.

[0102] At a concentration of 1 mmol / L, the tetrapeptide LILP had a significant effect in lowering uric acid.

[0103] Example 6: Effect of tetrapeptide LILP on uric acid reabsorption in HK-2 hyperuricemia cell model

[0104] In order to maintain the balance of uric acid in the human body, about 75% of uric acid is excreted through the kidneys, and the remaining 25% is excreted through the intestines. The transporters responsible for uric acid excretion and reabsorption are mainly distributed on the basolateral membrane and apical membrane of renal proximal tubule cells and intestinal epithelial cells. Studies have shown that glucose transporter 9 (GLUT9) is composed of SLC2A9 Gene encoding, mainly located in the basement membrane and apical membrane of renal proximal tubular cells, responsible for uric acid reabsorption; SLC22A12 The gene-encoded urate transporter 1 (URAT1) is located on the apical membrane of renal proximal tubular epithelial cells and mediates the reabsorption of uric acid through small hydrophilic organic anion exchange.

[0105] In this example, the mRNA expression of uric acid reabsorption proteins URAT1 and GLUT9 after the tetrapeptide LILP acted on the HK-2 hyperuricemia cell model, and the method was as follows:

[0106] 1. Construction of HK-2 hyperuricemia cell model and grouping treatment were the same as in Example 5.

[0107] 2. Add 1 mL of cell lysis buffer to each well, extract RNA according to the kit instructions, and detect the mRNA expression levels of urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9).

[0108] The method of real-time fluorescence quantitative PCR is as follows:

[0109] (1) Total RNA extraction: Use a total RNA extraction kit and RNase-free centrifuge tubes to extract total RNA from cells in each experimental group using the adsorption column method. Use a NanoDrop micro-spectrophotometer to measure the concentration and purity of the extracted RNA. If the OD 260 / 280 The value is around 2.0, and OD 260 / 230 When the value is between 2.0 and 2.4, the purity of the obtained RNA is considered appropriate;

[0110] (2) Reverse transcription of cDNA: Use a reverse transcription kit to obtain cDNA in a 96-well rapid thermal cycler (reaction program: 37°C, incubation for 15 min, 85°C, incubation for 5 s), and store at -80°C;

[0111] (3) Fluorescence quantitative PCR: Use a qPCR kit and place the sample on ice and avoid light. Add 8.5 µL of solution A and 1.5 µL of solution B to each well of a 384-well plate. Solution A contains 2.5 µL of dd water, 1 µL of cDNA template (diluted 10 times in advance), and 5 µL of mixed reagent (containing deoxynucleoside triphosphates, Mg 2+ , fluorescent dye and DNA polymerase). Solution B contains 1 µL of dilution buffer containing fluorescent dye, 0.25 µL of forward primer and 0.25 µL of reverse primer. After adding the sample, store it on ice and avoid light. After centrifugation, use real-time fluorescence quantitative PCR instrument to perform qPCR reaction.

[0112] The primer sequences are as follows:

[0113] Slc2a9- F: 5'-GCCCACGCTACCTTTCTCTTTG-3';

[0114] Slc2a9- R: 5'-AACCAGATCGCATTGAGTCCA-3';

[0115] Slc22a12- F: 5'-CGTGGGACCTGGTATGTAACT-3';

[0116] Slc22a12- R: 5'-CCAAACCTATCTGAGGCATGG-3';

[0117] Gapdh- F: 5'-TGACCTCAACTACATGGTCTACA-3';

[0118] Gapdh- R: 5'-CTTCCCATTCTCGGCCTTG-3'.

[0119] The qPCR reaction procedure was as follows: (1) pre-denaturation: 95°C for 60 s; (2) amplification cycle: 95°C for 15 s, 60°C for 15 s, 65°C for 30 s, 40 cycles; (3) melting curve: 60°C for 60 s, 95°C for 15 s; (4) annealing: 40°C for 30 s.

[0120] (4) Using Gapdh as the internal reference gene, calculate the number of renal uric acid transporter-related genes Slc2a9 , Slc22a12 The mRNA expression level is as follows:

[0121] ΔCT = CT value of each group of target genes - CT value of each group of internal reference genes;

[0122] ΔΔCT=ΔCT value of experimental group-ΔCT value of control group;

[0123] The relative expression of target genes in each group = 2 -ΔΔCT .

[0124] Data processing and analysis are the same as in step 2 of Example 5.

[0125] 3. Results Analysis

[0126] like Figure 7 and Figure 8 As shown, qPCR results showed that the modeling method of this study can significantly increase the mRNA expression of GLUT9 and URAT1 in HK-2 cells of the modeling group ( P <0.0001), while the tetrapeptide LILP could significantly reduce the mRNA expression of the above-mentioned uric acid reabsorption proteins URAT1 and GLUT9, and has the potential to reduce the renal uric acid reabsorption.

[0127] In summary, the present invention screened out the tetrapeptide LILP from tuna peptides through mass spectrometry identification and molecular docking of peptides. The peptide showed efficient uric acid-lowering activity in a hyperuricemia cell model. The main mechanism of action was to reduce XOD activity to reduce uric acid production and inhibit the mRNA expression of uric acid reabsorption transporters in renal cells to reduce uric acid reabsorption.

[0128] The present invention provides a theoretical basis for the development of new multi-target uric acid-lowering active substances and a possible drug preparation route for preventing and combating hyperuricemia. Specifically, the tetrapeptide LILP can be used alone to prepare uric acid-lowering drugs, or it can be compounded with other active ingredients that have the effect of regulating uric acid content. The active ingredients that have the effect of regulating uric acid content can be plant extracts (eucommia ulmoides, sunflower, etc.), vitamins (such as vitamin B), polyphenols, or other protein peptides. The drug can be prepared into microcapsules to improve the gastrointestinal digestion stability, bioavailability and shelf life of the polypeptide.

Claims

1. The use of the tetrapeptide LILP in the preparation of a uric acid-lowering drug, characterized in that: The amino acid sequence of the tetrapeptide LILP is Leu-Ile-Leu-Pro.

2. The use according to claim 1, characterized in that The medicine is a medicine for alleviating hyperuricemia and / or gout in mammals.

3. The use according to claim 1, characterized in that The drug comprises an effective dose of tetrapeptide LILP with an amino acid sequence of Leu-Ile-Leu-Pro and a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that The pharmaceutically acceptable carrier includes one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant.

5. The use according to claim 3, characterized in that The preparation form of the drug is an oral preparation.

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