Marine-derived polypeptide for alleviating gouty kidney injury and preparation method thereof

Abstract

This invention relates to the field of alleviating gouty kidney injury. To address the problem that existing xanthine oxidase inhibitory peptides cannot alleviate kidney cell damage caused by uric acid, this invention provides a marine-derived polypeptide for alleviating gouty kidney injury and its preparation method. The marine-derived polypeptide is a glycosylated skipjack tuna xanthine oxidase inhibitory peptide, obtained from skipjack tuna xanthine oxidase inhibitory peptide via a Maillard reaction. This glycosylated skipjack tuna xanthine oxidase inhibitory peptide can effectively inhibit xanthine oxidase activity, thereby reducing uric acid production, and has a alleviating effect on kidney cell damage caused by uric acid.

Description

Technical Field

[0001] This invention relates to the field of relieving gouty kidney injury, and more particularly to a marine-derived polypeptide for relieving gouty kidney injury and its preparation method. Background Technology

[0002] Gout is a group of metabolic diseases caused by abnormal purine metabolism in the human body. Its biochemical basis is hyperuricemia. Uric acid is the final product of purine metabolism in the human body. It is slightly soluble in the blood environment. When its metabolism is disordered, that is, when the production is much greater than the excretion, the uric acid content in the blood continues to rise. The sodium urate crystals formed are deposited in tissues such as joint synovium, cartilage and kidneys, causing repeated attacks of inflammation, which eventually leads to the occurrence of gout.

[0003] Studies have found that xanthine oxidase (XOD) is one of the key enzymes regulating the metabolism of purines into uric acid. Certain amino acids in food protein-derived uric acid-lowering active peptides can competitively / non-competitively bind to amino acid residues in XOD through hydrophobic interactions, hydrogen bonds, electrostatic interactions, and van der Waals forces, thereby altering its spatial structure and inhibiting XOD activity, thus reducing uric acid production. Furthermore, food protein-derived uric acid-lowering active peptides can reduce the mRNA expression level of XOD, thereby reducing the intracellular XOD content. For example, invention publication CN115715796A discloses "a composition that can lower uric acid and its application," which includes a xanthine oxidase inhibitory peptide, having the effect of reducing uric acid production, but without significant effect on uric acid-induced kidney cell damage. Summary of the Invention

[0004] To overcome the problem that xanthine oxidase inhibitory peptides in the prior art cannot alleviate kidney cell damage caused by uric acid, this invention provides a marine-derived polypeptide that alleviates gouty kidney injury. This marine-derived polypeptide can effectively inhibit xanthine oxidase activity, thereby reducing uric acid production, and also has a alleviating effect on kidney cell damage caused by uric acid. This invention also provides a method for preparing the marine-derived polypeptide that alleviates gouty kidney injury. The marine-derived polypeptide prepared by this method has a good alleviating effect on kidney injury caused by uric acid.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A marine-derived polypeptide for alleviating gouty kidney injury, wherein the marine-derived polypeptide is a glycosylated skipjack tuna xanthine oxidase inhibitor peptide.

[0007] Skipjack tuna XOD inhibitory peptide has strong inhibitory activity against xanthine oxidase. However, cell experiments showed that skipjack tuna XOD inhibitory peptide has no effect on uric acid-induced kidney cell damage. The inventors found that after glycosylation modification, it can alleviate uric acid-induced kidney cell damage.

[0008] Preferably, the glycosyl donor of the glycosylated skipjack tuna xanthine oxidase inhibitory peptide is one or more of glucose, fructose, xylose and galactose.

[0009] Modifying skipjack tuna xanthine oxidase inhibitory peptides using glucose, fructose, xylose, and galactose as glycosyl donors can enhance their protective effect against uric acid-induced renal cell damage. The glycosylated skipjack tuna xanthine oxidase inhibitory peptides obtained by glycosylation modification using xylose showed the best efficacy.

[0010] Preferably, the skipjack tuna xanthine oxidase inhibitory peptide is obtained by enzymatic hydrolysis of skipjack tuna using trypsin or aquatic protease.

[0011] Preferably, the molecular weight of the glycosylated bonito xanthine oxidase inhibitory peptide is not higher than 1000 Da.

[0012] Molecular weight is an important parameter for evaluating protein hydrolysates, and it may affect their antioxidant activity and other functional properties. When the molecular weight of glycosylated skipjack tuna XOD inhibitory peptide is 1000 Da or below, it exhibits good absorption and strong antioxidant activity.

[0013] Preferably, the molecular weight of the glycosylated bonito xanthine oxidase inhibitory peptide is 200–1000 Da.

[0014] A method for preparing the above-mentioned marine-derived polypeptide for alleviating gouty kidney injury includes the following steps:

[0015] S1 adjusts the pH of the solution containing bonito xanthine oxidase inhibitory peptide to neutral or alkaline, and adds a glycosyl donor;

[0016] S2 involves heating the solution obtained from S1 to carry out a Maillard reaction, followed by cooling to obtain a glycosylated skipjack tuna xanthine oxidase inhibitory peptide.

[0017] This invention modifies the skipjack tuna XOD inhibitory peptide by Maillard reaction through glycosylation.

[0018] Preferably, in step S1, the pH of the solution containing bonito xanthine oxidase inhibitory peptide is adjusted to 7.0–11.0.

[0019] The Maillard reaction rate is faster when the pH of the reaction system is between 7.0 and 11.0.

[0020] Preferably, the total mass of the solution in S2 is 90-120 mg / mL, and the mass ratio of the glycosyl donor to the bonito xanthine oxidase inhibitory peptide is (1-3):(1-3).

[0021] Preferably, the reaction temperature of the Mérald reaction in S2 is 60–120°C.

[0022] Preferably, the reaction time of the Mérald reaction in S2 is 30 to 180 min.

[0023] Preferably, edible alkali is used to adjust the pH in S1.

[0024] Therefore, the present invention has the following beneficial effects: (1) Glycosylation of skipjack tuna xanthine oxidase inhibitor peptides makes them not only inhibit XOD enzyme activity but also relieve gouty kidney damage; (2) The present invention also optimizes the temperature, heating time, pH value and glycopeptide ratio of Maillard reaction so that the prepared glycosylated skipjack tuna xanthine oxidase inhibitor peptides have good DPPH clearing and proliferation promoting effects on uric acid-damaged renal cells. Attached Figure Description

[0025] Figure 1 This is a liquid phase diagram of the skipjack tuna xanthine oxidase inhibitory peptide and the glycosylated skipjack tuna xanthine oxidase inhibitory peptide obtained in Example 1.

[0026] Figure 2 This is a survival curve of HK-2 cells after treatment with different concentrations of STH, STH-M, and STH-M 200-1000.

[0027] Figure 3 This is a bar chart showing the survival rate of uric acid-induced HK-2 cells after treatment with different concentrations of STH-M and STH-M 200-1000.

[0028] Figure 4 These are flow cytometry images of apoptosis in the control group, uric acid model group, STH-M repair experimental group, and STH-M 200-1000 repair experimental group.

[0029] Figure 5 The bar chart shows the apoptosis rate of the control group, the uric acid model group, the STH-M repair experimental group, and the STH-M 200-1000 repair experimental group.

[0030] Figure 6 The images show cell cycle bar charts for the control group, uric acid model group, STH-M repair experimental group, and STH-M 200-1000 repair experimental group.

[0031] Figure 7The figures show the ROS bar charts for the control group, the uric acid model group, the STH-M repair experimental group, and the STH-M 200-1000 repair experimental group.

[0032] Figure 8 This is a bar chart showing the DPPH removal rates of Fru30, Gal30, and Xyl-0.5h-30 in Example 3.

[0033] Figure 9 This is a bar chart showing the proliferation rate of HK-2 cells in Example 4.

[0034] Figure 10 This is a line graph showing the DPPH scavenging rate, browning degree, and fluorescence value of Example 5.

[0035] Figure 11 This is a line graph showing the DPPH scavenging rate, browning degree, and fluorescence value of Example 6.

[0036] Figure 12 This is a line graph showing the DPPH scavenging rate, browning degree, and fluorescence value of Example 7. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific implementation methods.

[0038] The skipjack tuna xanthine oxidase inhibitory peptide in the following specific implementation method is prepared by the following steps:

[0039] (1) Take the bonito processing by-product and water to mix and homogenize to obtain a slurry with a concentration of 25 g / mL. First, add animal protease (purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.) to the slurry. The amount added is 2% of the mass of the bonito processing by-product. After adjusting the pH to 8.0, heat to 55℃ for 4 hours for enzymatic hydrolysis.

[0040] (2) After heating and inactivation, trypsin (purchased from Henan Yangshao Biochemical Engineering Co., Ltd.) was added at a rate of 2% of the mass of the bonito processing by-product, and enzymatic hydrolysis was continued for 4 hours.

[0041] (3) After centrifuging the enzymatic hydrolysate at 10,000 r / min for 15 min, discard the upper oil film and bottom residue, and spray dry the middle clear liquid to obtain bonito xanthine oxidase inhibitory peptide STH.

[0042] The following specific implementation methods used SPSS 17.0 software and GraphPad Prism 8.0 for statistical processing and data analysis; experimental data are expressed as mean ± standard deviation (mean ± SD), and one-way ANOVA was used to analyze the statistical results; ANOVA with P < 0.05 was considered statistically significant.

[0043] Example 1

[0044] The preparation steps of the glycosylated skipjack tuna xanthine oxidase inhibitory peptide are as follows:

[0045] S1 The pH of the aqueous solution containing bonito xanthine oxidase inhibitory peptide was adjusted to 7.0 with sodium hydroxide, and xylose was added and fully dissolved. The total concentration of bonito xanthine oxidase inhibitory peptide and xylose was 100 mg / mL, and the glycopeptide ratio was 1:2.

[0046] S2 The solution obtained in S1 was heated to 120℃ and maintained for 90 min, and then cooled to obtain the glycosylated bonito xanthine oxidase inhibitory peptide STH-M.

[0047] The inhibition rates of xanthine oxidase activity of Example 1, STH, and febuxostat (Feb) were determined by ultraviolet spectrophotometry.

[0048] Weigh 4.56 g of dipotassium hydrogen phosphate and 2.71 g of potassium dihydrogen phosphate, and dilute to 100 mL of distilled water respectively. Mix at an 80:20 ratio to obtain a 0.2 M, pH 7.5 phosphate buffer. Prepare: Weigh 0.0228 g of xanthine powder, dissolve in a trace amount of 4 mol / L NaOH, and dilute to 100 mL with buffer to obtain a 1.5 mM xanthine solution. Take 400 μL of 5 U stock solution and dilute to 50 mL with buffer to obtain a 0.0125 U / mL XOD solution. Add 40 μL of 0.0125 U / mL XOD solution and 50 μL of STH or STH-M to the reaction tube as the experimental group. Add 40 μL of 0.0125 U / mL PBS solution and 50 μL of STH-M to the reaction tube as the experimental group. STH (1.67 mg / mL), STH-M (1.67 mg / mL), or Feb (4.64 ng / mL) served as control groups. 40 μL of 0.0125 U / mL XOD solution and 50 μL of PBS solution were added to the reaction tubes as blank groups. 90 μL of PBS solution was added to the reaction tubes as blank control groups. After shaking each tube thoroughly, the mixture was incubated at 25°C for 10 min. Then, 50 μL of 1.5 mM xanthine solution was added, and the mixture was shaken thoroughly and incubated at 25°C for 30 min. The absorbance (OD) at 290 nm was measured, and the XOD inhibition rate was calculated using the following formula.

[0049] The results are shown in Table 1.

[0050] Table 1 XOD Inhibition Rate

[0051] project XOD inhibition rate / % Standard deviation Significance STH 85.05 2.20 a STH-M 81.15 3.22 a Feb 56.96 9.44 b

[0052] The data in Table 1 show that the glycosylated skipjack tuna xanthine oxidase inhibitory peptide still has the effect of inhibiting XOD activity, and the inhibitory effect is better than febuxostat, a commonly used drug for the treatment of hyperuricemia in gout patients.

[0053] Based on Appendix A of QB / T 2879-2007 Marine Fish Oligopeptide Powder, modifications were made to the acetylamino acid-acetylamino acid chromatographic conditions: column: TSKgel G2500 SWXL 300nm×7.8mm gel column; mobile phase: acetonitrile:water:trifluoroacetic acid, 45:55:0.1 (volume ratio); detection wavelength: UV 220nm; flow rate: 0.5mL / min; column temperature: 30℃; injection volume: 10μL. The molecular weights of the skipjack tuna xanthine oxidase inhibitory peptide and the glycosylated skipjack tuna xanthine oxidase inhibitory peptide obtained in Example 1 were determined. The detection results are as follows: Figure 1 As shown, the molecular weight of the skipjack tuna xanthine oxidase inhibitory peptide and its glycosylated products is below 1000 Da.

[0054] Example 2

[0055] The glycosylated skipjack tuna xanthine oxidase inhibitory peptide is a glycosylated skipjack tuna xanthine oxidase inhibitory peptide with a molecular weight between 200 and 1000, separated from the glycosylated skipjack tuna xanthine oxidase inhibitory peptide STH-M obtained in Example 1 by spiral wound membranes with molecular weights of 1000 Da and 200 Da, and is denoted as STH-M 200-1000.

[0056] The efficacy of Examples 1-2 in alleviating gouty kidney injury was tested, and the testing process is as follows:

[0057] (1) Detection of the toxicity of skipjack tuna xanthine oxidase-inhibited peptide glycosylation products on HK-2 cells

[0058] HK-2 cells with a cell density of 80% were collected and processed at a rate of 6 × 10⁻⁶ cells / year. 4 100 μL of cell suspension was evenly seeded into each well of a 96-well cell culture plate. After approximately 48 hours of seeding, when the cell density reached about 70%, the complete culture medium in each well was aspirated. After washing once with PBS, HK-2 cells were incubated for 24 hours with different concentrations of STH, STH-M, and STH-M 200-1000 prepared in RPMI 1640 (1:1) basal medium. After adding CCK8 solution, the cells were incubated for another 1 hour in a 37°C, 5% CO2 incubator. The absorbance at 450 nm was measured using a microplate reader, and a cell viability curve was plotted.

[0059] The survival rate of HK-2 cells after treatment with STH at concentrations ranging from 0.1 to 6.4 mg / mL, STH-M at 0.025 to 1.6 mg / mL, and STH-M200-1000 at 0.1 to 5 mg / mL for 24 hours was as follows: Figure 2As shown in the figure, treatment with 0.1-0.2 mg / mL STH, 0.025-0.2 mg / mL STH-M, and 0.1-2.0 mg / mL STH-M 200-1000 all increased cell viability compared to the control group. The highest cell viability (108.39%) was observed at an STH concentration of 0.1 mg / mL; the highest cell viability (17.14%) was observed at an STH-M concentration of 0.1 mg / mL; and the highest cell viability (144.69%) was observed at an STH-M 200-1000 concentration of 0.2 mg / mL. This indicates that skipjack tuna XOD inhibited peptide glycosylation and enhanced the ability to promote HK-2 cell growth.

[0060] (2) Establishing a uric acid-induced kidney injury HK-2 cell model

[0061] HK-2 cells with a cell density of 80% were collected and processed at a rate of 6 × 10⁻⁶ cells / year. 4 One cell per well was evenly seeded into a 96-well cell culture plate, with a cell suspension volume of 100 μL per well. After about 48 hours of seeding, when the cell density reached approximately 70%, the complete culture medium in each well was aspirated, and the cells were washed once with PBS. The samples were then diluted to different concentrations with RPMI 1640 (1:1) complete culture medium for grouping and treatment, with 6 replicates per group. The cell culture plates were placed in a 37°C, 5% CO2 cell culture incubator for 24 hours. After that, the original culture medium was discarded, and the cells were washed once with PBS. HK-2 cells were then stimulated with 0.672 mg / mL (4.0 μmol / mL) uric acid in RPMI 1640 (1:1) basal medium for 24 hours to establish an HK-2 cell renal injury model.

[0062] The IC50 value of uric acid for HK-2 cells is 0.71 mg / mL. When the concentration is higher than this value, the uric acid solution is unstable and easily precipitates. At a concentration of 0.672 mg / mL, the survival rate of HK-2 cells after 24 hours of modeling is 57.8%. HK-2 cells themselves have a certain recovery ability, so 24 hours was chosen as the modeling time to stimulate cells to reach a cell death rate of 50%.

[0063] (3) Detection of the effect of skipjack tuna xanthine oxidase inhibiting peptide glycosylation products on uric acid-induced HK-2 cell apoptosis (3.1) Detection of HK-2 cell viability by CCK8 assay

[0064] Cells were divided into a control group, a uric acid model group, an STH-M repair experimental group, and an STH-M 200-1000 repair experimental group; cells were processed at a rate of 6 × 10⁻⁶. 4Cells were evenly seeded per well in a 96-well cell culture plate. When the cells reached 80% confluence, 4.0 μmol / mL uric acid was added via medium replacement, and the plate was incubated for 24 hours. After washing the cells twice with PBS, the corresponding concentrations of STH-M (0.1 mg / mL, 0.175 mg / mL) and STH-M 200-1000 (0.1317 mg / mL, 0.2633 mg / mL) were added to each well, and the plate was incubated for 24 hours. The model group was treated with complete culture medium. After washing once with PBS, basal medium containing 10% CCK8 was prepared. 100 μL of CCK8 solution was added to each well, and the plate was incubated at 37°C in a 5% CO2 incubator for 1 hour. The absorbance at 450 nm was measured using a microplate reader, and a cell viability bar chart was plotted.

[0065] like Figure 3 As shown, compared with the uric acid-induced model group, the survival rate of HK-2 cells treated with STH-M and STH-M200-1000 was significantly increased (p<0.05). The low dose of STH-M (0.1 mg / mL) increased the survival rate by 5.04%, while the high dose of STH-M (0.175 mg / mL) increased it by 14.78%. Similarly, the survival rate of STH-M 200-1000 was also dose-dependent, with a cell survival rate of 89.89% at a low dose (0.1317 mg / mL) and 99.23% at a high dose (0.2633 mg / mL). Therefore, STH-M and STH-M 200-1000 have a significant repair effect on uric acid-induced HK-2 cells.

[0066] (3.2) Flow cytometry detection of HK-2 cell apoptosis

[0067] According to 2×10 5 HK-2 cells were evenly seeded into 6-well cell culture plates at a density of 2 mL of cell suspension per well. The plates were then incubated at 37°C with 5% CO2. The modeling and drug administration procedures were the same as in section 3.1. The STH-M concentration was 0.175 mg / mL, and the STH-M 200-1000 concentration was 0.2633 mg / mL. Cell culture medium was collected in 10 mL centrifuge tubes, digested with trypsin (without EDTA), and the digestion was stopped by adding the collected culture medium. The cells were gently pipetted and transferred to centrifuge tubes. After centrifugation at 1000g for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS for cell counting. The cell density was adjusted to 5.0 × 10⁻⁶ cells / well. 5Cells / mL, 1000g, centrifuged for 5 minutes, PBS discarded, 500μL of 1×Binding Buffer added to resuspend cells, then 5μL of Annexin V-FITC and 5μL of PI Staining Solution added, gently mixed, and then incubated in the dark at room temperature for 10-15 minutes. Samples were detected by flow cytometry within 1 hour.

[0068] like Figure 4 and Figure 5 As shown, uric acid has a pro-apoptotic effect on HK-2 cells. After treatment with STH-M and STH-M 200-1000, the cell apoptosis rate decreased significantly.

[0069] (3.3) Flow cytometry detection of HK-2 cell cycle

[0070] Collect cells as described in section 3.2; adjust the cell density to 5.0 × 10⁻⁶. 5 After the cells were counted at 1 / mL, 0.3 mL of PBS was added to resuspend the cells in a single-cell suspension. The suspension was then transferred dropwise to 1.2 mL of anhydrous ethanol at -20°C. After vigorous vortexing, the suspension was placed in a -20°C freezer for 1 hour to fix the cells. After washing, RNase A was added, and the suspension was incubated in a 37°C water bath for 30 minutes. Then, 400 μL of PI was added and thoroughly mixed. The suspension was then incubated in the dark at 4°C for 30 minutes. The red fluorescence at an excitation wavelength of 488 nm was detected by instrumentation.

[0071] like Figure 6 As shown, compared with the normal group, the ratio of G0 / G1 phase and the ratio of G2 / M phase in HK-2 cells after uric acid-induced damage decreased, indicating that uric acid caused HK-2 cells to be arrested in the G2 / M phase. After STH-M treatment, compared with the uric acid model group, the ratio of G0 / G1 phase and the ratio of G2 / M phase decreased, while the ratio of S phase increased. However, after STH-M 200-1000 treatment, there was no significant difference between the two groups.

[0072] (3.4) Detection of ROS levels in HK-2 cells using the fluorescent probe (DCFH-DA) method

[0073] According to 2×10 5 HK-2 cells were evenly seeded into 6-well cell culture plates at a density of 2 mL of cell suspension per well. The plates were then incubated at 37°C in a 5% CO2 incubator. Modeling and drug administration procedures were the same as in section 3.1. STH-M concentrations were 0.025 and 0.05 mg / mL, and STH-M 200-1000 concentrations were 0.1, 0.2, and 0.4 mg / mL. The cell number was adjusted to 1 × 10⁻⁶ cells / well. 5HK-2 cells were seeded at a density of 1 mL / well in 12-well cell culture plates and incubated at 37°C with 5% CO2. Following the instructions of the ROS detection kit, the original cell culture medium was discarded, the cells were washed twice with PBS, and then incubated with 10 μmol / L DCFH-DA diluent at 37°C for 20 min. The cells were washed three times with PBS, digested and collected, and the ROS fluorescence intensity was assessed by flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0074] like Figure 7 As shown, after stimulation with 4.0 mM uric acid, the fluorescence intensity in the uric acid model group increased, indicating that uric acid induced oxidative stress in HK-2 cells. After treatment with different doses of STH-M and STH-M 200-1000, the low-dose group promoted the production of intracellular ROS, which increased the fluorescence intensity, while the high-dose group showed a decrease in fluorescence intensity.

[0075] The above experimental results indicate that glycosylated skipjack tuna xanthine oxidase inhibitory peptides can alleviate the damage caused by uric acid to HK-2 cells, and the effect is better when the molecular weight of the glycosylated skipjack tuna xanthine oxidase inhibitory peptides is between 200 and 1000.

[0076] The STH-M 200-1000 obtained in Example 2 underwent HK-2 cell transcriptomics analysis based on mRNA sequencing and single-cell sequencing, as follows:

[0077] (1) RNA extraction and detection

[0078] Cells in good growth condition from the uric acid model group and the STH-M 200-1000 repair experimental group were collected, the culture medium was discarded, and the cells were carefully washed twice with 1x PBS (equal volume of enzyme-free water). Trizol lysis buffer was added, and the cells were lysed on ice for 5 min, with repeated pipetting and mixing. The lysis buffer was transferred to cryovials and stored at -80℃ for long-term storage. RNA integrity and concentration were detected using an Agilent 2100 analyzer. PolyA-containing mRNA was specifically captured using oligo(dT) magnetic beads through two rounds of purification. The captured mRNA was fragmented using a magnesium ion fragmentation kit at 94℃ for 5-7 min. The fragmented RNA was synthesized into cDNA using reverse transcriptase, followed by double-strand synthesis. The DNA-RNA complex double strands were converted into DNA double strands, and deoxyribonucleotide triphosphate solution was incorporated into the double strands to complete the ends of the double-stranded DNA. The fragments were blunt-ended; an A base was added to each end to allow them to ligate to adapters with T bases at the ends. Magnetic beads were used to screen and purify the fragments by size. The two strands were digested with uracil-DNA glycosylase, followed by PCR—pre-denaturation at 95°C for 3 min, denaturation at 98°C for a total of 8 cycles of 15 seconds each, annealing at 60°C for 15 s, extension at 72°C for 30 s, and final holding at 72°C for 5 min—to form a library with a fragment size of 300 bp ± 50 bp. Primer sequences used for PCR are shown in Table 2. Illumina Novaseq was used. TM 6000 pairs of end sequencing were performed in PE150 sequencing mode;

[0079] Table 2 PCR primer sequences

[0080]

[0081]

[0082] (2) Screening of differentially expressed genes

[0083] Genes were screened based on both fold change and significance level; the criteria were a fold change FC>=2 or FC<=0.5 (i.e., the absolute value of log2FC>=1) and a q value<0.05 (|log2FC|>=1&q<0.05).

[0084] (3) GO and KEGG analysis of differentially expressed genes

[0085] Functional analysis of differentially expressed genes in each transcriptome was performed using the Gene Ontology (GO) database; enrichment analysis of regulatory pathways involved in differentially expressed genes in each transcriptome was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

[0086] In the comparative analysis between the uric acid model group and the STH-M 200-1000 repair experimental group, a total of 1381 differentially expressed genes were identified, of which 334 were upregulated and 1047 were downregulated. According to the GO annotation classification of differentially expressed genes, the biological processes involved in these genes include signal transduction, transcriptional regulation, multicellular development, RNA polymerase II regulation of transcription, cell adhesion, cell differentiation, G protein-coupled receptor signaling pathways, transmembrane transport, lipid metabolism, ion transport, apoptosis, phosphorylation, protein hydrolysis, cell proliferation, and extracellular matrix organization. The intracellular components involved include the cell membrane, cytoplasm, nucleus, endoplasmic reticulum, mitochondria, cytoskeleton, and cell knots. The molecular functions that differentially expressed genes may perform include protein binding, metal ion binding, nucleotide binding, DNA binding, transferase activity, hydrolase activity, ATP binding, and DNA-binding transcription factor activity.

[0087] Based on the GO annotation classification results, GO enrichment analysis was performed to further explore the functional characteristics of differentially expressed genes. It was observed that, compared with the uric acid model group, the differentially expressed genes in the STH-M 200-1000 repair experimental group had the highest enrichment in the categories of regulation of branching structure morphology, negative regulation of chemokine production, sensory neuron axonal fasciculation, and cellular response to sterols. They were mainly enriched in the extracellular matrix and were mainly involved in biological processes such as vitamin A-like metabolism, positive regulation of apoptosis, positive regulation of mitochondrial release of cytochrome c, and positive regulation of mitochondrial release of cytochrome c.

[0088] KEGG signaling pathway enrichment analysis was performed on differentially expressed genes in the STH-M 200-1000 repair experimental group and the uric acid model group. A total of 28 pathways with p < 0.05 were identified, among which Pathways in Cancer, Synaptic vesicle cycle, PPAR signaling pathway, Glycosphingolipid biosynthesis-lacto and neolacto series, and Nicotinate and nicotinamide metabolism were the most statistically significant. This indicates that STH-M 200-1000 exerts its repair effect by regulating key genes in the above pathways.

[0089] Based on KEGG pathway enrichment, 15 key differentially expressed genes were identified by screening pathways in cancer and purine metabolism-related pathways: NQO1 (ENSG00000181019), STAT1 (ENSG00000115415), MMP1 (ENSG00000196611), MAPK1 (ENSG00000100030), CCL2 (ENSG00000108691), NLRP3 (ENSG00000162711), and DUSP1 (ENSG00000120129). DKK1(ENSG00000107984), CCND1(ENSG00000110092), CDKN1B(ENSG00000111276), SERPINE1(ENSG00000106366), NT5E(ENSG00000135318), GUCY1A1(ENSG00000164116), CLDN2(ENSG00000165376), B3GALT5(ENSG00000183778).

[0090] Example 3

[0091] Selection of glycosyl donors for glycosylated skipjack tuna xanthine oxidase inhibitory peptides:

[0092] S1 Take 4 flasks and number them 1-4. Add an aqueous solution containing bonito xanthine oxidase inhibitory peptide with pH adjusted to 7.0 using sodium hydroxide and different glycosyl donors. The specific components in each flask are shown in Table 3.

[0093] S2. Each flask was placed in a water bath and heated as shown in Table 3. After cooling, glycosylated bonito xanthine oxidase inhibitory peptides were obtained.

[0094] Table 3. Reaction conditions for each flask in Example 3.

[0095]

[0096]

[0097] The browning degree and DPPH scavenging rate of glycosylated skipjack tuna xanthine oxidase inhibitory peptides in each numbered flask in Example 3 were detected. The browning degree was determined by spectrophotometry, with absorbance measured at 420 nm. The DPPH scavenging rate was determined using a DPPH free radical scavenging rate detection kit (purchased from Nanjing Jiancheng).

[0098] The four groups of glycosylated skipjack tuna xanthine oxidase inhibitory peptides (Glu3hx30, Fru3hx30, Gal3hx30, and Xyl0.5hx30) had the same mass concentration and degree of browning. The DPPH clearance rates of these four groups were […]. Figure 8 As shown, the DPPH scavenging rates of glycosylated skipjack tuna xanthine oxidase inhibitory peptides were significantly higher than those of the original skipjack tuna xanthine oxidase inhibitory peptides, with Xyl0.5hx30 exhibiting the highest scavenging rate. Considering both process and antioxidant properties, xylose is superior to glucose, fructose, and galactose as a glycosyl donor.

[0099] Example 4

[0100] S1 Take 7 flasks and number them 1-7. Add an aqueous solution containing bonito xanthine oxidase inhibitory peptide with pH adjusted to 7.0 using sodium hydroxide and different glycosyl donors. The specific components in each flask are shown in Table 4.

[0101] S2. Place each flask in a water bath and heat as shown in Table 4. After cooling, dilute to obtain glycosylated bonito xanthine oxidase inhibitory peptide.

[0102] Table 4. Reaction conditions for each flask in Example 4.

[0103] serial number name Glycosyl donor Total mass concentration of glycopeptides Glycopeptide ratio Heating temperature and time Dilution factor 1 Glu30 glucose 100mg / mL 1:2 100℃，1h 30 2 Fru30 fructose 100mg / mL 1:2 100℃，1h 30 3 Gal30 Galactose 100mg / mL 1:2 100℃，1h 30 4 Xyl-0.5h-30 Xylose 100mg / mL 1:2 100℃，0.5h 30 5 Xyl-1h-40 Xylose 100mg / mL 1:2 100℃，1h 40 6 Xyl-1.5h-40 Xylose 100mg / mL 1:2 100℃，1.5h 40 7 Xyl-50℃-5h-40 Xylose 100mg / mL 1:2 50℃，5h 40

[0104] The glycosylated skipjack tuna xanthine oxidase inhibitory peptide from each flask in Example 4 was used for HK-2 cell proliferation assay. The experimental method was the same as in "Detection of the toxicity of glycosylated skipjack tuna xanthine oxidase inhibitory peptide to HK-2 cells". The normal cell group was designated as blank, the model group as UA-672, and the positive drug control group as Feb-8.75, which was treated with 8.75 mg / mL febuxostat. The detection results are as follows: Figure 9As shown, the proliferation rate of HK-2 cells decreased significantly after uric acid treatment, but the proliferation rate rebounded after treatment with the glycosylated skipjack tuna xanthine oxidase inhibitory peptide obtained in Example 4, and the rebound effect was better than that of febuxostat. This indicates that at a total glycopeptide concentration of 3.33 mg / mL, different glycosylated skipjack tuna xanthine oxidase inhibitory peptides all had significant proliferative effects on HK-2 cells.

[0105] Example 5

[0106] Optimization of the glycopeptide ratio of glycosylated skipjack tuna xanthine oxidase inhibitory peptide:

[0107] S1 The pH of the aqueous solution containing bonito xanthine oxidase inhibitory peptide was adjusted to 7.0 with sodium hydroxide and divided into 5 groups. The total concentration of bonito xanthine oxidase inhibitory peptide and xylose in each group was kept at 100 mg / mL, and xylose was added according to the glycopeptide ratio of 1:3, 1:2, 1:1, 2:1, 3:1 (m:m).

[0108] S2 The solution obtained in S1 was heated to 120℃ and maintained for 60 min, and then cooled to obtain the glycosylated bonito xanthine oxidase inhibitory peptide STH-M-Xyl.

[0109] The DPPH scavenging rate, browning degree, and fluorescence value of STH-M-Xyl were obtained from each group in Example 5. The fluorescence value was detected using a fluorescence spectrophotometer at OD420nm.

[0110] The results are as follows Figure 10 As shown, under the conditions of reaction time of 1 h, total mass concentration of 100 mg / mL, temperature of 120 ℃ and pH of 7.0, STH-M-Xyl obtained with glycopeptide ratios of 1:3, 1:2, 1:1, 2:1 and 3:1 all had DPPH scavenging effects. Among them, the STH-M-Xyl prepared with a glycopeptide ratio of 1:2 had a significantly higher DPPH scavenging rate than the other groups, and also inhibited the degree of browning.

[0111] Example 6

[0112] Temperature optimization of glycosylated skipjack tuna xanthine oxidase inhibitory peptide:

[0113] S1 The pH of the aqueous solution containing bonito xanthine oxidase inhibitory peptide was adjusted to 7.0 with sodium hydroxide, and xylose was added to dissolve the peptide according to the total concentration of bonito xanthine oxidase inhibitory peptide and xylose being 100 mg / mL and the glycopeptide ratio being 1:2.

[0114] S2 divided the solution obtained in S1 into 7 groups, heated them to 60, 70, 80, 90, 100, 110 and 120℃ respectively and maintained for 60 min, and then cooled to obtain the glycosylated bonito xanthine oxidase inhibitor peptide STH-M-Xyl.

[0115] In Example 6, the DPPH scavenging rate, browning degree, and fluorescence value of STH-M-Xyl obtained in each group were tested. An aqueous solution containing bonito xanthine oxidase inhibitory peptide at pH 7.0 without xylose was heated at 60, 70, 80, 90, 100, 110, and 120°C for 60 min as a control group for browning degree and fluorescence value testing. The results are as follows. Figure 11 As shown, at a glycopeptide ratio of 1:1, a total mass concentration of 100 mg / mL, and a pH of 7.0, the DPPH scavenging rate of the sample under reaction conditions at 120℃ was significantly higher than that of other groups.

[0116] Example 7

[0117] Optimization of heating time for glycosylated skipjack tuna xanthine oxidase inhibitory peptide:

[0118] S1 The pH of the aqueous solution containing bonito xanthine oxidase inhibitory peptide was adjusted to 7.0 with sodium hydroxide, and xylose was added to dissolve the peptides according to the total concentration of bonito xanthine oxidase inhibitory peptide and xylose being 120 mg / mL and the glycopeptide ratio being 1:2.

[0119] S2 divided the solution obtained from S1 into 6 groups, heated it to 120℃ and maintained it for 30, 60, 90, 120, 150 and 180 min respectively, and then cooled to obtain the glycosylated bonito xanthine oxidase inhibitory peptide STH-M-Xyl.

[0120] The DPPH scavenging rate, browning degree, and fluorescence value of STH-M-Xyl obtained in each group of Example 7 were tested, as follows: Figure 12 As shown, under the conditions of total concentration of 100 mg / mL, temperature of 120℃, glycopeptide ratio of 1:2, and pH 7.0, the products obtained at reaction times of 30-180 min all had DPPH scavenging effects. Among them, the DPPH scavenging rate of STH-M-Xyl showed an increasing trend from 30 to 90 min, and its scavenging ability decreased after that.

Claims

1. A method for preparing a marine-derived polypeptide that alleviates gouty kidney injury, characterized in that, Includes the following steps: S1. Adjust the pH of the solution containing skipjack tuna xanthine oxidase inhibitory peptide to 7.0-11.0, and add a glycosyl donor; the skipjack tuna xanthine oxidase inhibitory peptide is obtained by sequentially hydrolyzing skipjack tuna with animal protease and trypsin; the glycosyl donor is one or more of glucose, fructose, xylose and galactose; S2 The solution obtained in S1 is heated at 60~120℃ for Maillard reaction for 30~180 min, and then cooled to obtain marine-derived polypeptide that relieves gouty kidney injury.

2. The preparation method according to claim 1, characterized in that, The glycosyl donor for the marine-derived polypeptide that alleviates gouty kidney injury is xylose.

3. The preparation method according to claim 1, characterized in that, The molecular weight of the marine-derived polypeptide that alleviates gouty kidney injury is no higher than 1000 Da.

4. The preparation method according to claim 3, characterized in that, The molecular weight of the marine-derived polypeptide that alleviates gouty kidney injury is 200-1000 Da.

5. The preparation method according to claim 1, characterized in that, The total mass of the solution in S2 is 90~120 mg / mL.

6. The preparation method according to claim 1, characterized in that, The mass ratio of the glycosyl donor to the bonito xanthine oxidase inhibitory peptide in S1 is (1~3):(1~3).

7. The preparation method according to claim 6, characterized in that, The mass ratio of the glycosyl donor to the skipjack tuna xanthine oxidase inhibitory peptide in S1 is 1:

2.

8. The preparation method according to claim 1, characterized in that, The reaction temperature of the Mérald reaction in S2 is 110~120℃.

9. The preparation method according to claim 5, 6, or 8, characterized in that, The reaction time for the S2 melard reaction is 60-120 min.

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