Abalone peptide and application thereof

Abstract

The invention discloses an abalone peptide. The amino acid sequence of the abalone peptide is SDAPWWQTPV. The invention further discloses application of the abalone peptide in preparation of functional food or medicine. The functional food or medicine is used for (a) treating or preventing hyperuricemia; and / or (b) reducing kidney cell damage caused by hyperuricemia. The abalone peptide disclosed by the invention is a safe and effective food-borne active peptide with a natural uric acid reducing function by taking a by-product viscera of haliotis discus hannai processing as a raw material, has the advantages of being easy to absorb, low in off-target toxicity, high in activity and strong in stability, and is suitable for being applied to functional foods or medicines for preventing and treating hyperuricemia.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to an abalone peptide and its application in the preparation of pharmaceuticals. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disease characterized by abnormal purine metabolism or impaired uric acid excretion. In recent years, with changes in dietary structure due to socioeconomic development, the prevalence of HUA has shown a significant upward trend. Xanthine oxidase (XOD) is a key rate-limiting enzyme in uric acid biosynthesis, thus becoming an important target for the clinical treatment of HUA. Although traditional XOD inhibitors (such as allopurinol) have been widely used in uric acid-lowering therapy, their metabolite, oxypurine, may induce adverse reactions such as renal impairment, which greatly limits its long-term safe clinical application.

[0003] Compared with traditional small molecule drugs, marine-derived bioactive peptides have advantages such as strong structural designability, high bioavailability, strong targeting and fewer adverse reactions, showing good application potential in the field of XOD inhibition.

[0004] my country has a high abalone production, but the discarded viscera (accounting for 15%-25% of body weight) during processing is both a waste of resources and pollutes the environment. Studies have found that abalone viscera are rich in protein and can be used in functional food development; however, current research mainly focuses on antioxidant and anti-inflammatory effects, and their uric acid-lowering activity has not been systematically explored. Developing uric acid-lowering peptides from abalone viscera could not only enhance the value of this byproduct but also provide new raw materials for uric acid-lowering products. Summary of the Invention

[0005] The purpose of this invention is to provide an abalone peptide for the development of functional foods or drugs with uric acid-lowering function.

[0006] To achieve the above objectives, this invention discloses an abalone peptide with the amino acid sequence: SDAPWWQTPV.

[0007] Furthermore, the abalone peptide is derived from the viscera of the wrinkled abalone.

[0008] The present invention also discloses the application of the above-mentioned abalone peptide in the preparation of functional foods or drugs, wherein the functional foods or drugs are used for: (a) treating or preventing hyperuricemia; and / or (b) reducing renal cell damage caused by hyperuricemia.

[0009] The concentration of the abalone peptide is less than 5 mM.

[0010] Preferably, the concentration of the abalone peptide is 2.5 mM.

[0011] Furthermore, the treatment or prevention is achieved through one or more of the following mechanisms:

[0012] (1) Inhibits the activity of xanthine oxidase and / or adenosine deaminase; (2) Downregulate the gene expression level of GLUT9, a uric acid reabsorption transporter; (3) Upregulate the gene expression level of uric acid excretion transporter OAT1; (4) Clear reactive oxygen species from kidney cells.

[0013] Based on the above scheme, the present invention has the following beneficial effects: The abalone peptide disclosed in the present invention uses the viscera, a by-product of the processing of wrinkled abalone, as raw material. It is a safe and effective natural food-derived active peptide with the function of lowering uric acid. It has the advantages of easy absorption, low off-target toxicity, high activity and strong stability. It can be used in functional foods or drugs for the prevention and treatment of hyperuricemia. Attached Figure Description

[0014] Figure 1 This is the HPLC chromatogram of the abalone peptide of the present invention.

[0015] Figure 2 This is the mass spectrum of the abalone peptide of this invention.

[0016] Figure 3a This is a 3D schematic diagram of the docking results between the abalone peptide and the XOD active pocket molecule of the present invention.

[0017] Figure 3b This is a 2D schematic diagram of the docking results between the abalone peptide and the XOD active pocket molecule of the present invention.

[0018] Figure 4a This is a 3D schematic diagram of the docking results between the abalone peptide and the XOD whole protein molecule of the present invention.

[0019] Figure 4b This is a 2D schematic diagram of the docking results between the abalone peptide and the XOD whole protein molecule of the present invention.

[0020] Figure 5 This is a schematic diagram of the molecular dynamics simulation results of abalone peptide and XOD in this invention.

[0021] Figure 6 This is the fitting curve of the MST determination of abalone peptide and XOD molecule in this invention.

[0022] Figure 7 This invention relates to abalone peptide and XOD IC. 50 Measurement results.

[0023] Figure 8 The effects of different concentrations of SDAPWWQTPV on zebrafish with high uric acid (Note: # indicates a significant difference from the normal group (p < 0.05). This indicates a significant difference from the model group (p < 0.05), and the same applies below. Figure 9a This invention relates to the effect of abalone peptide on the activity of XOD enzyme in zebrafish.

[0024] Figure 9b This invention relates to the effect of abalone peptide on the activity of ADA enzyme in zebrafish.

[0025] Figure 10a This invention relates to the effect of abalone peptide on the expression level of the HPRT1 gene in a zebrafish model with high uric acid.

[0026] Figure 10b This invention relates to the effect of abalone peptide on the expression level of the GLUT9 gene in a zebrafish model with high uric acid.

[0027] Figure 10c This invention relates to the effect of abalone peptide on the expression level of the OAT1 gene in a zebrafish model with high uric acid.

[0028] Figure 11 These are images of mouse liver pathological examination. (a)-(f) show liver tissue sections (H&E staining, scale bar 50 μm) from the normal group, model group, positive control group, high-dose group, medium-dose group, and low-dose group of the abalone peptide of this invention, respectively. Black arrow: irregular dilation of hepatic sinusoids; green arrow: hepatocyte enlargement; yellow arrow: microvesicle-like fatty degeneration; blue arrow: hepatocyte death.

[0029] Figure 12 These are images of mouse kidney pathological examination. (a)-(f) show kidney tissue sections (H&E staining, scale bar 50 μm) from the normal group, model group, positive control group, high-dose group, medium-dose group, and low-dose group of the abalone peptide of this invention, respectively. Black arrows: inflammatory cell infiltration; green arrows: renal tubular cell edema; red arrows: glomerular lesions; yellow arrows: red-stained material; blue arrows: renal tubular epithelial cell necrosis; circles: irregular dilation of renal tubules.

[0030] Figure 13 This relates to the effect of uric acid on the viability of HK-2 cells.

[0031] Figure 14a The effect of different doses of SDAPWWQTPV on the viability of normal HK-2 cells.

[0032] Figure 14b The effect of different doses of SDAPWWQTPV on the viability of high uric acid HK-2 cells.

[0033] Figure 15 The average fluorescence intensity of HK-2 cells in each group of the SDAPWWQTPV model of uric acid-induced HK-2 cell damage is shown.

[0034] Figure 16 Fluorescence images of HK-2 cells in different groups of the SDAPWWQTPV model of uric acid-induced HK-2 cell damage. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0036] Explanation of the relevant definitions in this invention: Hyperuricemia (HUA) is a metabolic disease characterized by abnormal purine metabolism or impaired uric acid excretion.

[0037] Xanthine oxidase (XOD): hereinafter referred to as XOD, is a key rate-limiting enzyme in uric acid biosynthesis, and therefore an important target for clinical treatment of uric acid hyperacidity (HUA).

[0038] Adenosine deaminase (ADA): ADA participates in the salvage synthesis pathway of uric acid, converting adenosine and deoxyadenosine into inosine and deoxyinosine, while releasing ammonia. Inosine is further converted into hypoxanthine, which is ultimately catalyzed by XOD to produce uric acid. ADA can serve as an effective target for screening uric acid-lowering active ingredients.

[0039] The wrinkled abalone (Haliotis discus hannai) is a mollusk belonging to the genus Haliotis in the family Haliotisidae. Its shell is oval and thick, with a dark greenish-brown surface and irregular wrinkles. There are 4-5 excretory channels at the end of the shell, and the inner surface is silvery-white with a bluish-green pearly luster.

[0040] Allopurinol is an organic compound with the chemical formula C5H4N4O. It is a competitive XOD inhibitor that inhibits XOD activity by binding to reduced xanthine oxidase to form an irreversible complex.

[0041] HPRT1 (Hypoxanthine Phosphoribosyltransferase 1): Hypoxanthine phosphoribosyltransferase is a key enzyme that catalyzes the conversion of hypoxanthine and guanine into mononucleotides (IMP and GMP). It maintains the intracellular purine nucleotide balance through the purine salvage synthesis pathway, is a key enzyme in uric acid synthesis, and is a key molecular target for assessing uric acid metabolism.

[0042] GLUT9 (Glucose Transporter 9): A uric acid reabsorption transporter protein, also known as glucose transporter 9, is encoded by the SLC2A9 gene and belongs to the glucose transporter family. However, GLUT9 has higher uric acid transport activity than glucose. On the basement membrane of renal tubular epithelial cells, GLUT9 can transport uric acid from cells into the blood, increasing the uric acid content in the blood. GLUT9, along with urate anion transporter 1 (URAT1), mediates renal uric acid reabsorption, and its expression level can reflect the uric acid excretion status.

[0043] OAT1 (Organic Anion Transporter 1) is a uric acid excretion transporter, belonging to the organic anion transporters (OATs) and the solute carrier 22 (SLC22) superfamily. It is a transmembrane protein expressed in various tissues and participates in the physiological clearance of endogenous and exogenous substances. OAT1 and OAT3 (Organic Anion Transporter 3), as key uric acid transporters in the kidney, directly affect uric acid metabolism and excretion. OAT1 is mainly expressed in the proximal tubular basement membrane, mediating the entry of uric acid from the blood into the renal tubular epithelial cells. OAT3, also located in the basement membrane, works synergistically with OAT1 to promote the transport of uric acid from the blood into the renal tubules. When the expression of both genes is inhibited, uric acid excretion capacity is significantly reduced, leading to elevated serum uric acid levels. This makes OAT1 and OAT3 ideal indicators for evaluating the efficacy of uric acid-lowering drugs.

[0044] Example 1 This invention discloses an abalone peptide with the amino acid sequence SDAPWWQTPV, as shown in SEQ NO.1.

[0045] This invention uses the viscera, a byproduct of abalone processing, as raw material. Through screening of active peptides and analysis of their mechanisms of action, it develops food-derived bioactive peptides with uric acid-lowering functions. The preparation and screening process of this invention is detailed below.

[0046] 1. Preparation of crude extract from abalone viscera Fresh wrinkled abalone (purchased from Dashun Aquatic Farm, Chencheng Town, Dongshan County, Zhangzhou City, Fujian Province) were dissected, with the internal organs preserved. The abalone were minced and then dried in a vacuum freeze dryer to remove the moisture from the internal organs. The powder was then crushed and sealed for storage at -20℃.

[0047] Accurately weigh the freeze-dried muscle or viscera powder, add deionized water at a material-to-liquid ratio of 1:50 (W / V), stir well, and preheat in a water bath for 1 hour. Then add alkaline protease for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme in a 100°C water bath for 20 minutes, cool naturally, and centrifuge at 4°C, 8000 rpm / min for 30 minutes. Collect the supernatant, which is the abalone viscera enzymatic hydrolysate.

[0048] The enzymatic hydrolysate of abalone viscera obtained by enzymatic hydrolysis was passed sequentially through a ceramic membrane and an ultrafiltration membrane under low temperature conditions to separate components with a molecular weight of ≤1 kDa.

[0049] 2. Identification of peptide sequences using Nano-HPLC-MS / MS A suitable amount of abalone peptide ≤1 kDa was desalted using a C18 desalting column, and the sample was analyzed by LC-MS / MS equipped with an online nanospray ionization source. The entire system was a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) with EASY-nanoLC 1200 in series. A total of 1 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm). The sample was separated using a gradient at a rate of 60 min, with the column flow rate controlled at 300 nL / min, the column temperature at 40°C, and the electrospray voltage at 2 kV. The gradient started at 2% B phase, increased non-linearly to 35% at 47 min, increased to 100% within 1 min, and held for 12 min. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: Scan range (m / z): 200-2000; Resolution: 70,000; AGC target: 3e6; Maximum injection time: 50 ms; (2) HCD-MS / MS: Resolution: 17,500; AGC target: 1e5; Maximum injection time: 45ms; Collision energy: 28; Dynamic exclusion time: 30s.

[0050] Nano-HPLC-MS / MS analysis identified 743 abalone peptides in the fractions obtained in process 1. A candidate peptide database was constructed by setting screening criteria (-log10P>20 and bioactivity>0.75).

[0051] 3. Virtual Filtering The crystal structure of 3NVY (XOD) was obtained from the RCSB protein database and modified using Discovery Studio 2019 software, including dehydration, hydrogenation, and retention of key residues in the active pocket, while minimizing energy consumption. Using 3NVY as the receptor protein, the identified peptide sequence was used as the ligand, and the active pocket of the target protein (X: 40.749, Y: 20.794, Z: 19.52) was precisely located by determining the amino acid residues. Virtual screening was performed under the defined pocket range and box edge conditions.

[0052] 4. Molecular docking The X-ray crystal structure of 3NVY was obtained from the PBD protein database. The ligands underwent charge optimization under physiological conditions, and their three-dimensional configuration was generated using Open Babel. The receptor-ligand system was preprocessed using AutoDock Tools, the binding domain was defined using AutoGrid, and flexible docking was performed using Autodock Vina (v1.2.0). The lowest conformation of ΔG was selected to resolve the interaction mode, and the final visualization was presented using PyMOL.

[0053] Figure 3a , 3b The results of active pocket docking between SDAPWWQTPV and XOD are presented. Figure 4a , 4b The full-protein docking results of SDAPWWQTPV and XOD are presented. The XOD protein is represented as a dark blue cartoon model, the ligands as cyan stick models, and their binding sites as magenta stick structures. Nonpolar hydrogen atoms are omitted, and hydrogen bonds are depicted as yellow dashed lines. Table 1 shows the main interaction force analysis between SDAPWWQTPV and XOD, where the underlined amino acid binding sites of XOD are highlighted.

[0054] Table 1. Types of molecular docking interactions between SDAPWWQTPV and XOD and their corresponding amino acid analyses.

[0055] like Figure 3aAs shown in Figure 3b, SDAPWWQTPV forms specific interactions with multiple amino acid residues (such as PHE 649, ASN768, and GLU 802) in the XOD active pocket, with a binding energy of -5.8 kcal / mol. These interactions may not be entirely dependent on the binding energy, but rather stabilize the peptide's position in the active pocket through specific chemical bonds or spatial conformations, thereby effectively hindering the catalytic activity of XOD. Furthermore, tryptophan (Trp) located in the middle of the SDAPWWQTPV structure forms conventional hydrogen bonds and π-π interactions with PHE 649, respectively. The former fixes the peptide chain position, while the latter further restricts the swinging of the PHE 649 side chain, potentially disrupting the dynamic equilibrium of the active pocket through synergistic effects and compensating for the binding energy disadvantage. Figure 4a As shown in Figure 4b, SDAPWWQTPV (ΔG = -8.9 kcal / mol) is anchored to THR 354, LEU 257, THR396, ARG 394, and ILE 353 of the FAD domain via seven hydrogen bonds (interatomic distance 2.8–3.2 Å), with its binding sites directly adjacent to the flavin ring region of the FAD cofactor. This binding mode may hinder the electron transfer function of FAD through steric hindrance or interfere with the conformational coordination between FAD and the Mo-Pt domain through allosteric regulation, thereby inhibiting the catalytic activity of XOD.

[0056] 5. Molecular dynamics simulation This invention constructs a molecular model based on the Desmond / Maestro academic version (2022.1), employs the TIP3P solvent model for hydration of the complex, and achieves charge neutralization by supplementing with 0.15 M NaCl solution. After energy optimization and relaxation pretreatment, the system undergoes a 100 nsec kinetic simulation under the NPT ensemble. Trajectory data are acquired and stored at 10² ps intervals, and conformational evolution analysis is performed using the Simulation Event Analysis module, focusing on the dynamic changes of hydrogen bonds and the stability of the binding pocket.

[0057] like Figure 5Molecular dynamics simulations showed that the overall RMSD of XOD was stable (2.6-3.0 Å), indicating that the protein backbone did not undergo global conformational collapse. However, the RMSF of the ligand increased to 3 Å within 40-60 nsec, suggesting that its binding to the FAD domain induces enhanced local flexibility. SDAPWWQTPV mainly interacts with amino acid residues at positions 200-400 of XOD, indicating that it may induce allosteric regulation by preferentially binding to the FAD domain of XOD (rather than the traditional active pocket). The 40-60 nsec window is a critical allosteric window, characterized by significant expansion of rGyr (7.2 Å), enhanced hydrogen bond network stability (2-6 intraHBs), and synergistic fluctuations in surface properties (MolSA / SASA peak difference >120 Ų). Combining whole-protein docking and in vitro inhibitory activity data, it can be inferred that SDAPWWQTPV mainly triggers long-range conformational changes in XOD through the FAD binding domain, thereby interfering with the function of its catalytic active site and ultimately achieving efficient inhibition.

[0058] 6. Solid-phase synthesis of XOD-inhibiting peptides The results of the virtual screening are ranked by Grid Score. Based on the ranking and interaction, the high-scoring abalone peptides are finally selected, including the abalone peptide of the present invention: SDAPWWQTPV (Ser-Asp-Ala-Pro-Trp-Trp-Gln-Thr-Pro-Val).

[0059] The abalone peptide of this invention was synthesized using a solid-phase method, and its purity and molecular weight were identified using HPLC and LC / MS. Figure 1 As shown, HPLC analysis revealed that the purity of SDAPWWQTPV reached 99.53%. Figure 2 As shown, the relative molecular mass was determined to be 1186.29 by LC / MS. The physicochemical parameters of SDAPWWQTPV were analyzed using ProtParam, as shown in Table 2.

[0060] Table 2. Physicochemical parameters of SDAPWWQTPV

[0061] 7. MST verification of XOD-peptide binding affinity This experiment used microthermophoresis (MST) to determine the binding affinity of SDAPWWQTPV to XOD. The experiment was conducted on a NanoTemper Monolith NT.115 instrument platform, using 1% DMSO PBST as the run buffer at a constant temperature of 25.0°C. Data analysis was performed using MO.Affinity Analysis v2.3 software. The specific procedure is as follows: First, the His-tagged XOD protein was labeled with RED-tris-NTA second-generation dye. 33 nM dye and 66.00 nM protein were mixed in equal volumes (100.00 µL each), incubated at room temperature for 30 minutes, and then centrifuged to collect the supernatant. Subsequently, a ligand concentration gradient (250.00 µM to 7.63 nM) was established in PCR tubes. 10 µL of PBS buffer was added to each tube, and the reporter probe complex was serially diluted from tubes 1 to 16 using a serial dilution method to finally form the ligand concentration gradient system. For detection, a moderate MST power (LED power 30%) was used, and the target peptide concentration was fixed at 200 nM. Interaction detection was performed via capillary loading. A fitting curve was plotted with ligand concentration on the x-axis and normalized fluorescence change percentage on the y-axis.

[0062] Kd reflects the dissociation equilibrium constant of the ligand-protein complex; a smaller value indicates more stable binding and stronger specificity. For example... Figure 6 As shown, the binding curve was directly fitted and the calculated MST binding affinity was 248 nM. This directly corresponds to the preferential binding characteristic of the FAD domain observed in molecular dynamics simulations, indicating that SDAPWWQTPV can form a high-affinity binding by specifically recognizing the stable conformation of this region. The low Kd of SDAPWWQTPV is directly related to its stable hydrogen bond network and long-range conformational transfer effect, indicating that it not only has high static binding strength but can also interfere with the function of the enzyme active site through allosteric effects.

[0063] 8. IC50 determination The abalone peptide of this invention was prepared into solutions of different concentration gradients. After co-incubation with XOD, xanthine was added to initiate the reaction. The specific operation method is as follows: Using a 96-well UV microplate, 40.00 μL of the sample to be tested or buffer and 40.00 μL of XOD solution were added to each well sequentially. The plate was preheated at 25 ℃ for 5 min, and then 40.00 μL of xanthine solution was added to start the reaction. Parallel blank control wells were set up for each reaction well. The total reaction time was 30 min, and the absorbance at 290 nm was recorded every 3 min. The XOD inhibition rate was calculated as follows: "y" represents the net absorbance of the reaction system at 290 nm. "x" represents the net absorbance of the reaction system at 290 nm after the sample is added.

[0064]

[0065] The XOD inhibition rate of abalone peptide at different concentrations was calculated. GraphPad Prism 7 software was used to perform nonlinear regression analysis with the logarithm of drug concentration on the x-axis and the inhibition rate on the y-axis. The dose-response curve was fitted and the half maximum inhibitory concentration (IC50) was automatically calculated.

[0066] like Figure 7 As shown, the IC50 of the abalone peptide of the present invention against XOD is 0.58 ± 0.02 mM, indicating that the abalone peptide of the present invention has strong XOD inhibitory activity.

[0067] Example 2 Based on Example 1, this embodiment further discloses the application of the abalone peptide of the present invention in the preparation of functional foods or drugs, which are used to treat or prevent hyperuricemia.

[0068] The specific experimental procedure is detailed below.

[0069] 1. Verification of uric acid-lowering efficacy 1620 normally developed 5-day-fever zebrafish were cultured in 6-well cell culture plates with n=3 (30 zebrafish / well) and 4 mL of culture medium / well. The modeling drugs for hyperuricemic zebrafish were potassium oxonate (OXO) and xanthine sodium salt (XSS). Except for the normal group, all other groups were immersed in the modeling drugs (600 μM OXO and 30 μM XSS, OXO-XSS) for 24 h during the experimental period. The experimental groups were as follows: the normal group was immersed in zebrafish culture medium; the model group was immersed in the drug; the abalone peptide treatment group of this invention was immersed in a safe dose of peptide SDAPWWQTPV and OXO-XSS; the positive control group was immersed in 0.1 mM allopurinol and OXO-XSS. After 24 h, the uric acid content of the zebrafish homogenate supernatant in each group was detected by HPLC.

[0070] The HPLC method for detecting uric acid content in zebrafish was as follows: Zebrafish samples were transferred from six-well plates to centrifuge tubes. After washing away residual drugs on the body surface with DBPS buffer, the samples were transferred to 1.5 mL centrifuge tubes. Residual liquid was thoroughly aspirated using a sterile syringe, and 250.00 μL of pre-chilled DBPS buffer was added for tissue homogenization. The samples were then centrifuged at 8000 rpm / min for 15 min at 4 °C. The supernatant was filtered through a 0.22 μm microporous membrane before analysis.

[0071] (1) Chromatographic conditions Chromatographic column: C18 analytical column (4.6 × 250 mM, 5 μm); mobile phase: 0.2% acetic acid-methanol (94:6, v / v); flow rate: 0.8 mL / min; column temperature: 30°C; detection wavelength: 288 nm; injection volume: 10 μL; analysis time: 30 min (2) Establishment of regression equation With the concentration of uric acid standard as the abscissa (X) and the peak area as the ordinate (Y), a standard curve regression equation was established as Y = 6777 X + 52622, with a correlation coefficient R² = 0.999.

[0072] A zebrafish hyperuricemia model was successfully established using potassium oxonate and sodium xanthine. The uric acid level in the model group was significantly higher than that in the control group (model group: 43.98±1.99 μM; control group: 17.61±0.54 μM). Figure 8 As shown, based on this model, the uric acid-lowering activity of SDAPWWQTPV was systematically evaluated. The effect of SDAPWWQTPV on the uric acid level of zebrafish showed a concentration-dependent effect. At concentrations of 0.63, 1.25 and 2.5 mM, the uric acid level was reduced by 64.71%, 69.96% and 80.81%, respectively.

[0073] It is evident that the abalone peptide SDAPWWQTPV of this invention can significantly reduce the uric acid level in zebrafish, with a uric acid clearance rate of 80.81% at 2.5 mM. Moreover, it is non-toxic at high doses, demonstrating a superior balance between overall efficacy and safety.

[0074] 2. Determination of XOD and ADA enzyme activities in zebrafish The total protein concentration, XOD, and adenosine deaminase (ADA) activity in zebrafish tissue were determined using a kit.

[0075] like Figure 9a As shown in Figure 9b, the activities of XOD and ADA enzymes in the liver of zebrafish in the model group were significantly increased by 46.98% and 10.52% respectively compared with the normal group, indicating abnormal activation of the uric acid synthesis pathway. The XOD enzyme activity in the 0.10 mM allopurinol intervention group decreased by 90.09% compared with the model group, confirming that the sensitivity of the model to XOD inhibitors met the experimental design requirements. After low, medium, and high doses of SDAPWWQTPV intervention, XOD enzyme activity decreased by 33.5%, 46.67%, and 48.72% respectively compared with the model group, indicating that SDAPWWQTPV reduces uric acid production by targeting and inhibiting XOD. Furthermore, medium and high doses of SDAPWWQTPV simultaneously downregulated ADA enzyme activity by 20.49% and 38.44%, synergistically blocking uric acid synthesis.

[0076] 3. Real-time quantitative PCR detection of uric acid synthesis and transport gene expression levels in zebrafish Total RNA was extracted from zebrafish according to the RNA extraction kit instructions, and the isolated RNA was quantified. β-actin was used as an internal reference gene, and the primer design results are shown in Table 3. The reverse transcription reaction solution was prepared according to the components in Table 4, and the extracted RNA was reverse transcribed into cDNA in a PCR instrument after sequential loading. The cDNA was stored at -20 ℃. The reaction system was prepared according to the components in Table 5, and the reaction was performed according to the program. Finally, the 2-ΔΔCT value was calculated.

[0077] Table 3. Primer sequences for mRNA

[0078] Table 4. Reagent components and dosage for cDNA synthesis

[0079] Table 5. qPCR amplification reaction system configuration

[0080] The key enzymes in the uric acid synthesis pathway, HPRT1, the uric acid reabsorption protein GLUT9, and the secretory protein OAT1, are three genes that regulate the synthesis, reabsorption, and secretion of uric acid in zebrafish, respectively. These three genes work synergistically to maintain uric acid homeostasis and are key molecular targets for assessing uric acid metabolism. Figure 10a As shown in Figures 10b and 10c, SDAPWWQTPV significantly regulates the gene expression of uric acid reabsorption protein GLUT9 and secretion protein OAT1: low, medium, and high doses of SDAPWWQTPV downregulated GLUT9 expression by 55.45%, 79.21%, and 82.18%, respectively, while 2.5 mM SDAPWWQTPV simultaneously upregulated OAT1 expression by 70.33%, indicating that this concentration can bidirectionally regulate the inhibition of uric acid reabsorption and the promotion of secretion, effectively accelerating uric acid excretion. However, SDAPWWQTPV has limited regulatory effect on the gene expression of HPRT1, a key enzyme in the uric acid synthesis pathway: high uric acid led to a 79.81% downregulation of HPRT1 expression compared to the normal group. Although intervention with 2.5 mM SV10 restored its expression to 45.28% of the normal level, there was no significant difference compared to the model group.

[0081] In summary, the present invention SDAPWWQTPV reduces uric acid levels by decreasing the activity of XOD and ADA enzymes, downregulating the classical pathway of uric acid reabsorption transporter GLUT9 and upregulating excretion transporter OAT1, which is consistent with the traditional drug mechanism: reducing uric acid synthesis and promoting uric acid metabolism.

[0082] Example 3 Based on Example 1 or Example 2, this embodiment further discloses the application of the abalone peptide of the present invention in the preparation of functional foods or drugs. The functional foods or drugs can be used to reduce kidney cell damage caused by hyperuricemia.

[0083] The specific experimental procedure is detailed below.

[0084] 1. Establishment of an animal model of hyperuricemia Thirty-six SPF-grade male KM mice, 5 weeks old and weighing (20.00±1.50) g, were purchased from Shanghai Silex Laboratory Animal Co., Ltd. All animals were housed in the animal facility of the Fujian Provincial Fisheries Research Institute in strict accordance with the requirements of the "Regulations on the Management of Laboratory Animals". Experiments began after 7 days of housing under the following conditions (ambient temperature 20-25℃, air humidity 40%-60%, daily supplementation of pure water and standard feed, and a 12-hour light-dark cycle).

[0085] Thirty-six SPF-grade male KM mice were randomly divided into six groups (n=6): normal control group (NC), model control group (MC), low-dose group (L-AP), medium-dose group (M-AP), high-dose group (H-AP) of the abalone peptide of this invention, and positive control group (PC). Except for the normal control group, the other groups were continuously administered the modeling drug by gavage: 150 mg / kg potassium oxonate + 125 mg / kg adenine (OXO-Ade), dissolved in 0.5% sodium carboxymethyl cellulose. The experiment lasted for 2 weeks, and the daily gavage volume of the drug was calculated as 0.01 mL / g of mouse body weight. Intervention regimens: ① NC group: daily gavage administration of 0.5% sodium carboxymethyl cellulose; ② MC group: gavage administration of OXO-Ade for 1 hour followed by an equal volume of solvent; ③ L-AP, M-AP, and H-AP groups: gavage administration of OXO-Ade for 1 hour followed by administration of 100, 200, and 400 mg / kg of the abalone peptide solution of this invention, respectively; ④ PC group: gavage administration of OXO-Ade for 1 hour followed by administration of 5 mg / kg allopurinol solution. Patients were fasted for 12 hours prior to the last administration (free access to water). Whole blood samples were collected via the orbital venous plexus 1 hour after administration, followed by euthanasia via cervical dislocation. The liver and kidneys were immediately dissected for removal, and cecal contents were collected as fecal samples.

[0086] 2. Determination of XOD and ADA activities in mouse liver tissue Weigh an appropriate amount of liver tissue sample and add pre-chilled DBPS buffer at a ratio of 1:9 (w / v). Homogenize mechanically using a high-speed homogenizer under ice bath conditions for 60 s. After centrifuging the homogenate at 3000 rpm / min for 15 min, collect the supernatant as the liver tissue homogenate sample. Use a kit to determine the total protein concentration, XOD, and ADA enzyme activity of the sample.

[0087] Detecting liver XOD and ADA activity can clarify whether abalone XOD inhibitory peptides improve kidney function by targeting and inhibiting key enzymes in uric acid production.

[0088] like Figure 11 As shown in a and 11b, the liver XOD enzyme activity in the MC group (23.40±0.85 U / gprot) was significantly higher than that in the NC group (10.14±0.63 U / gprot) by 130.77%, suggesting that the modeling drug induces uric acid metabolism disorder by upregulating XOD activity. The abalone peptide intervention of this invention can dose-dependently inhibit XOD activity: the enzyme activities in the L-AP, M-AP, and H-AP groups were 18.48±1.01 U / gprot, 17.40±0.56 U / gprot, and 14.45±0.64 U / gprot, respectively, which were 21.03%, 25.64%, and 38.25% lower than those in the MC group. These results indicate that the abalone peptide of this invention affects uric acid synthesis by regulating XOD activity. The ADA enzyme activity in the MC group mice (32.11±1.56 U / L) was significantly increased by 99.94% compared to the NC group (16.06±0.66 U / L). However, the ADA activity decreased in a dose-dependent manner after intervention with the abalone peptide of this invention, with values ​​of 28.41±0.77 U / L, 26.19±1.44 U / L, and 23.09±0.96 U / L in the L-AP, M-AP, and H-AP groups, respectively. This suggests that the abalone peptide of this invention has dual inhibitory properties against XOD and ADA. The positive control group (allopurinol) significantly reduced XOD activity in the MC group to 11.92±0.74 U / gprot (a decrease of 49.06% compared to the MC group), but had no significant inhibitory effect on ADA activity (MC group 32.11±1.56 U / L, PC group 31.85±1.23 U / L).

[0089] 3. Histopathological observation of mouse liver and kidney tissues Histopathological evaluation was performed using hematoxylin-eosin (HE) staining. The specific procedure was as follows: Mouse kidney and liver tissues were dissected and isolated, rinsed repeatedly with pre-cooled physiological saline, and then fixed in 4% paraformaldehyde fixative for 24-48 h. After fixation, the tissues were sequentially dehydrated with graded ethanol (75%-95%), with each grade of ethanol treatment lasting 1 h. Subsequently, the tissues were cleared with xylene for 5-10 min, embedded in paraffin, and sectioned to a thickness of 4 μm. After dewaxing with xylene, the sections were stained according to the standard HE staining procedure, and the morphological changes were observed under an optical microscope.

[0090] Pathological changes in mouse kidneys were observed using H&E staining, such as... Figure 12 As shown. NC group ( Figure 12 a) The glomeruli were of regular shape, and the renal tubules were tightly arranged, with no pathological damage observed. MC group ( Figure 12b) Presented typical characteristics of hyperuricemic nephropathy: thickening of the glomerular basement membrane with inflammatory exudation and atrophy, dilation of renal tubules, swelling and apoptosis of epithelial cells, and deposition of homogeneous red-stained material within the tubular lumen. PC group ( Figure 12 c) The degree of renal tubular dilation was reduced compared to the MC group, but compensatory glomerular hypertrophy and persistent interstitial inflammation were observed, suggesting that allopurinol may exacerbate the metabolic burden on the kidneys. The abalone peptide intervention of this invention significantly improved kidney injury in a dose-dependent manner: H-AP ( Figure 12 d) Glomerular morphology was basically restored, renal tubular dilation was significantly relieved, with only a small amount of residual edema and tubular deposition; M-AP group ( Figure 12 e) Thyroid-like casts and protein deposits are visible in the renal tubules; L-AP ( Figure 12 f) Glomerular basement membrane thickening and luminal deposits remained significant. Furthermore, all abalone peptide groups of this invention showed reduced interstitial inflammatory cell infiltration compared to the MC group, but the L-AP group exhibited significantly more lesions than the M-AP and H-AP groups.

[0091] As can be seen, H&E staining showed that the liver in the model group exhibited typical sinusoidal cavitation and focal hepatocyte necrosis, while the kidney tissue showed vacuolization of renal tubular epithelial cells and inflammatory infiltration of glomeruli. After intervention with the abalone peptide of this invention, the tightness of hepatocyte arrangement was significantly improved in all dose groups, and the kidney tissue structure tended to be more intact, with the high-dose group showing the most significant effect.

[0092] 4. In vitro culture of HK-2 cells and establishment of HK-2 cell injury model HK-2 cells were supplemented with DMEM medium containing 15% FBS and 1% antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin) and cultured in an incubator at 37°C with 5% CO2. Cells adhered and grew, with the culture medium changed every 2 days. Cells were passaged when confluence reached 80%. To screen for effective concentrations of uric acid that induce HK-2 cell damage, cells were stimulated with gradients of different concentrations of uric acid, and cell viability was measured and IC50 was calculated. Figure 13 As shown, the half-maximal inhibitory concentration (WMC) of uric acid on HK-2 cell viability was 1.48 ± 0.12 mM, at which a significant proliferation arrest phenomenon was observed. Microscopic morphological analysis revealed that with increasing uric acid concentration, the cell phenotype gradually evolved from an initial regular cobblestone-like structure to a pathologically characteristic spindle-shaped aberration. This morphological transformation confirms that a 1.48 mM uric acid concentration can effectively simulate the pathological process of renal arrest induced by hyperuricemia. Based on these experimental results, this critical concentration was subsequently selected to establish an in vitro injury model for intervention and evaluation.

[0093] 5. Cell viability and reactive oxygen species level analysis HK-2 cells in logarithmic growth phase were digested with 0.25% trypsin, and the digestion was terminated by adding an equal volume of complete culture medium. The cells were centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. The cell density was adjusted to 1.5 × 10⁶ cells / min with complete culture medium. 5 Cells were seeded at a density of 100 cells / mL into 96-well cell culture plates and incubated for 24 h. Experimental groups were as follows: the normal control group was cultured in basal medium for 24 h; the model group was cultured in basal medium containing 1.48 mM uric acid for 24 h; the positive control group was co-cultured in medium containing 1.48 mM uric acid and 0.1 mM allopurinol for 24 h; and the XOD inhibitory peptide treatment groups were treated in medium containing 1.48 mM uric acid and 2.5 mM and 1.25 mM peptides SDAPWWQTPV, respectively, for 24 h. After treatment, cell viability was assessed using a CCK-8 assay kit. Superoxide dismutase levels were detected using a ROS assay kit, and fluorescence images were observed and recorded using a fluorescence microscope.

[0094] like Figure 14a and 14b As shown, cells were incubated with 0.15625-5 mM SDAPWWQTPV for 24 h. Results indicated no cytotoxicity within this concentration range. Further investigation of the nephrotoxicity-protective effect of the peptide was conducted on hyperuricemic HK-2 cells using the same SDAPWWQTPV concentration as in the zebrafish experiment. 1.48 mM uric acid significantly reduced HK-2 cell survival. After neutralization and high-dose intervention, HK-2 cell survival significantly recovered compared to the model group; however, it did not improve the cellular damage. The survival rate of cells in the SDAPWWQTPV group was still significantly different from the blank control group. These results suggest that SDAPWWQTPV may alleviate hyperuricemia-induced HK-2 cell damage and improve the survival rate of damaged cells to some extent by promoting cell proliferation or exerting antioxidant effects.

[0095] ROS is an important marker of cellular redox imbalance, and hyperuricemia may lead to excessive accumulation of ROS in renal tubular cells. Figure 15 and Figure 16 As shown, in the uric acid-induced HK-2 cell damage model, fluorescence images revealed large areas of cell damage with significantly higher ROS fluorescence intensity than the normal group. This difference indicates that high concentrations of uric acid can exacerbate oxidative stress in HK-2 cells. Observation of ROS clearance in HK-2 cells after intervention with 0.63 mM, 1.25 mM, and 2.5 mM SDAPWWQTPV revealed that ROS levels in HK-2 cells treated with SDAPWWQTPV gradually decreased in a concentration-dependent manner.

[0096] It is evident that a high uric acid environment significantly increases the level of reactive oxygen species (ROS) in HK-2 cells, while SDAPWWQTPV effectively scavenges ROS in a concentration-dependent manner. The fluorescence intensity corresponding to the low, medium and high dose groups decreased sequentially, confirming that its antioxidant capacity is positively correlated with the dose, providing direct molecular evidence for its protective effect.

[0097] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. An abalone peptide, characterized in that, Its amino acid sequence is: SDAPWWQTPV.

2. The abalone peptide as described in claim 1, characterized in that: The abalone peptide is derived from the viscera of the wrinkled abalone.

3. The use of the abalone peptide according to claim 1 in the preparation of functional foods or pharmaceuticals, wherein the functional foods or pharmaceuticals are used for: (a) Treatment or prevention of hyperuricemia; and / or (b) Reduce kidney cell damage caused by hyperuricemia.

4. The application as described in claim 3, characterized in that: The concentration of the abalone peptide is less than 5 mM.

5. The application as described in claim 4, characterized in that: The concentration of the abalone peptide is 2.5 mM.

6. The application as described in claim 3, characterized in that: The treatment or prevention is achieved through one or more of the following mechanisms: (1) Inhibits the activity of xanthine oxidase and / or adenosine deaminase; (2) Downregulate the gene expression level of GLUT9, a uric acid reabsorption transporter; (3) Upregulate the gene expression level of uric acid excretion transporter OAT1; (4) Clear reactive oxygen species from kidney cells.

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