Marine fish uric acid-reducing peptide with kidney protection activity as well as preparation method and application of marine fish uric acid-reducing peptide

Abstract

The invention discloses a marine fish uric acid lowering peptide with kidney protection activity as well as a preparation method and application of the marine fish uric acid lowering peptide. The marine fish uric acid reducing peptide Trp-Gly-Val-Pro-Glu, WGVPE for short, with kidney protection activity is prepared, and the marine fish uric acid reducing peptide Trp-Gly-Val-Pro-Glu, WGVPE for short, can remarkably reduce the blood uric acid level in a body, promote uric acid excretion and improve pathological damage of the kidney. In addition, the pentapeptide provided by the invention can be used in common food, food additives and functional food for preventing and treating hyperuricemia and kidney injury or health care products or medicines for relieving hyperuricemia and kidney injury, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of protein engineering, and in particular to a marine fish uric acid-lowering peptide with kidney-protecting activity, and a preparation method and application thereof. Background Art

[0002] The prevalence of a high-sugar, high-fat, and high-purine diet in modern society has led to the development of a variety of metabolic disorders, among which purine metabolism disorders can significantly elevate serum uric acid levels. Sustained high uric acid levels can trigger the deposition of urate crystals in the renal parenchyma, ultimately leading to kidney damage, characterized by abnormal renal structure and function and impaired homeostatic regulation. Studies have shown that insufficient uric acid excretion is the primary factor causing elevated uric acid levels, and the kidney, as the core site of uric acid metabolism, plays a crucial role in maintaining uric acid homeostasis. However, renal injury is prevalent worldwide, particularly in patients with diabetes, chronic kidney disease, cardiovascular disease, and hyperuricemia. Data from a prospective epidemiological survey of renal injury show that the cumulative incidence of renal injury within 24 hours of admission to the intensive care unit (ICU) is as high as 57.3%. However, effective treatments for renal injury have not yet been applied in clinical studies. Therefore, in-depth research on prevention and treatment strategies for renal injury is urgently needed.

[0003] Currently, clinical intervention for renal injury primarily involves medications (such as nonsteroidal anti-inflammatory drugs and xanthine oxidase inhibitors). However, long-term medication use can easily lead to side effects such as liver and kidney damage, gastrointestinal discomfort, and allergies. Therefore, the development of natural and safe food-derived bioactive ingredients as intervention strategies for renal injury has become a research hotspot. Existing studies have shown that a variety of food-derived protein bioactive peptides can ameliorate renal injury and thereby exert uric acid-lowering activity. For example, intervention with marine fish protein peptides can alleviate potassium oxonate-induced renal pathological damage in rats, improve renal function, and promote uric acid excretion. Studies have found that intervention with giant salamander bone peptides can alleviate renal injury in mice with chronic kidney disease (CKD) and restore renal metabolic function, as demonstrated by lowering serum uric acid, creatinine, and urea nitrogen levels. Studies have also found that intervention with yellowfin tuna peptides can alleviate renal injury induced by potassium oxonate combined with yeast extract in mice, downregulate the expression of the renal fibrosis factor TGF-β1, and promote uric acid metabolism. Although research on renal protective peptides has made some progress, reports of peptides with clear renal protective activity remain limited. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a marine fish uric acid-lowering peptide with kidney protective activity.

[0005] Another object of the present invention is to provide the use of the above-mentioned marine fish uricosuric peptide with kidney protective activity.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A marine fish uric acid-lowering peptide with kidney protective activity, whose amino acid sequence is Trp-Gly-Val-Pro-Glu, abbreviated as WGVPE.

[0008] The marine fish uricosuric acid-lowering peptide with kidney protection activity is obtained through a chemical synthesis method.

[0009] The use of the marine fish uric acid-lowering peptide with kidney protective activity in the preparation of drugs for treating / preventing kidney diseases.

[0010] The use of the above-mentioned marine fish uric acid-lowering peptide with kidney protective activity in the preparation of uric acid-lowering drugs.

[0011] Use of the marine fish uric acid-lowering peptide with kidney protective activity in the preparation of drugs for the treatment / prevention of hyperuricemia.

[0012] The use of the marine fish uric acid-lowering peptide with kidney protective activity in the preparation of food or health products with uric acid-lowering or kidney protective effects.

[0013] The present invention has the following advantages and effects compared to the prior art:

[0014] The present invention has produced a marine fish uric acid-lowering peptide WGVPE with kidney-protective activity, which can significantly reduce blood uric acid levels, promote uric acid excretion, and improve pathological kidney damage. In addition, the pentapeptide of the present invention can be used in common foods, food additives, functional foods, or health products or medicines to prevent and treat hyperuricemia and kidney damage, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the mass spectrum of pentapeptide WGVPE;

[0016] Figure 2 Figure 2 is a graph showing the cell survival rate and antioxidant enzyme activity after uric acid-damaged HK-2 cells were treated with pentapeptide WGVPE. A is the cytotoxicity test of pentapeptide WGVPE, B is the change in cell survival rate after pentapeptide WGVPE intervention, and C and D are the changes in SOD and GSH-Px activity after pentapeptide WGVPE intervention, respectively.

[0017] Figure 3 The following are graphs showing the uric acid levels and renal function of rats in each group: A is a graph showing changes in blood uric acid levels in rats; B is a graph showing changes in blood creatinine levels in rats; C is a graph showing changes in urine creatinine levels in rats; and D is a graph showing changes in urine uric acid levels in rats.

[0018] Figure 4Figure 3 is a graph showing the renal injury of rats in each group, where A is the renal collagen volume fraction, and BE are graphs showing changes in the levels of renal injury markers KIM-1, IL-18, NAG, and Cys-C, respectively.

[0019] Figure 5 This is a microscopic picture of kidney tissue sections in Example 3. DETAILED DESCRIPTION

[0020] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0021] If specific experimental conditions are not specified in the following embodiments, conventional experimental conditions or those recommended by the reagent company will generally be used. Materials and reagents used were commercially available unless otherwise specified.

[0022] Example 1 Screening and synthesis of active peptides

[0023] Our research group screened and identified several potentially active peptides from marine fish hydrolysates. To further characterize their properties, peptides were synthesized using conventional solid-phase synthesis methods and used in subsequent experiments. The specific steps were as follows: Using a Wang resin-based solid-phase synthesis resin incorporating Fmoc protecting groups and amino acid residues, the column was swollen with DCM for 30 minutes and then evacuated to dryness. The resin was washed three times with DMF, a 20% piperidine / DMF solution was added, and nitrogen was bubbled through the column for 30 minutes. The solvent was removed and the column was washed six times with DMF. Subsequent amino acids containing Fmoc protecting groups were added to the resin, dissolved in DMF, and DIEA was added. A cleavage buffer was added to remove the resin and amino acid side chain protecting groups. The resin and cleavage buffer were then evacuated, and ether was added to fully precipitate the peptides. This process was repeated six times by centrifugation, and the column was evacuated to dryness. Mass spectrometry analysis was performed. Purification method: Use HPLC to analyze the main peak time of 5% to 95% concentration of crude peptide, dissolve it in acetonitrile and water, clarify it, filter it, and separate it by HPLC-MS to obtain the pure product and determine the molecular weight of the peptide.

[0024] After characterization and verification, the peptide WGVPE was successfully synthesized with a purity of >95%. The mass spectrum is shown in Figure 1 shown.

[0025] Example 2: Renal protective effect of pentapeptide WGVPE on HK-2 cells damaged by uric acid

[0026] 2.1 Cell culture

[0027] HK-2 cells were cultured in DMEM / F12 medium supplemented with 10% FBS (v / v) and 1.1% 100 IU / mL penicillin-streptomycin (v / v). Cells were incubated at 37°C in a 5% CO2 incubator. When cells reached 80% to 90% of the flask volume, they were digested with 0.25% trypsin (containing EDTA) and centrifuged (1100 rpm for 4 minutes). The cells were typically passaged at a 1:3 ratio.

[0028] 2.2 CCK-8 assay for cell viability

[0029] After digestion, the cells were counted as 1×10 5 Cells were seeded at a density of 100 μg / mL in a 96-well plate. After culturing for 24 h, the supernatant was discarded and the cells were divided into the following groups for drug administration: (1) Control group (Con): cultured in serum-free medium; (2) Blank group: no cells, the same as the control group; (3) Model group (UA): cultured in serum-free medium containing 650 μg / mL uric acid solution; (4) WGVPE control group: cultured in serum-free medium containing 50 μg / mL WGVPE, and only the effect of WGVPE on cells was measured; (5) WGVPE intervention group: cultured in serum-free medium containing 650 μg / mL uric acid solution and 50 μg / mL WGVPE. After culturing for 24 h, 10 μL CCK-8 reagent was added to each 100 μL culture medium in a 96-well plate according to the instructions. The plate was placed in a 37°C constant temperature incubator for 2 h, and then the cell viability was measured at 450 nm. The cell viability was calculated as follows: Cell viability (%) = 100 × (OD 样品组 -OD 空白组 ) / (OD 对照组 -OD 空白组 ).

[0030] The results are as follows Figure 2 As shown, Figure 2 The CCK-8 assay in Figure A showed that the cell viability of the WGVPE control group was between 90% and 110%, indicating that the WGVPE control group did not exhibit cytotoxicity at a concentration of 50 μg / mL, with no significant difference from the control group. Therefore, this concentration can be used in subsequent experiments. Figure 2 As shown in Figure B, after intervention with 650 μg / mL uric acid, the cell survival rate of the model group was significantly reduced to about 50% (p<0.05). After intervention with 50 μg / mL WGVPE, the cell survival rate of the WGVPE intervention group was significantly increased compared with the model group (p<0.05), indicating that WGVPE intervention can significantly improve the cell survival rate after uric acid damage.

[0031] 2.3 Antioxidant enzyme (SOD and GSH-Px) activity assay

[0032] Refer to the experimental settings in 2.2 and count the cells after digestion, with a value of 1×10 5 Cells were seeded at a density of 100 μg / mL in 6-well plates, with no blank control. 24 hours after administration, cells were washed twice with pre-chilled sterile PBS, and an appropriate amount of lysis buffer (RIPA:PMSF = 99:1) was added. Lysis was continued on ice for 30 minutes. After lysis, cells were scraped off in one direction using a cell scraper, and the cell lysate was collected. The cells were centrifuged at 12,000 rpm and 4°C for 15 minutes. The supernatant was aspirated and the protein concentration was determined using a BCA kit. Enzyme activity was determined according to the instructions of the antioxidant enzyme (SOD, GSH-Px) kit.

[0033] The results are as follows Figure 2 As shown in Figures C and D, the activities of intracellular antioxidant enzymes (SOD and GSH-Px) in the model group were significantly reduced. After intervention with 50 μg / mL WGVPE, the activities of antioxidant enzymes (SOD and GSH-Px) in the WGVPE intervention group were significantly increased (p<0.05), indicating that WGVPE intervention can play a protective role in the kidneys by alleviating uric acid-induced oxidative stress.

[0034] Example 3 Evaluation of the in vivo renal protection and uric acid-lowering activity of the pentapeptide WGVPE

[0035] 3.1 Animal husbandry

[0036] 6-8 week old SD rats were adaptively fed for one week and randomly divided into 4 groups (n=8), namely control group (NC), model group (MC), positive control group (BenZ) and WGVPE group.

[0037] Potassium oxonate and adenine were dispersed in a 0.5% (w / w) sodium carboxymethylcellulose solution for tube feeding. The control group was gavaged with 0.5% sodium carboxymethylcellulose solution every morning, while the other groups were gavaged with potassium oxonate (1.5g / kg) and adenine (50mg / kg) for 7 consecutive days to induce a hyperuricemia renal injury model.

[0038] After the hyperuric acid renal injury model was successfully established, the experiment was officially started. For the next 28 days, rats in the model group, BenZ group, and WGVPE group were gavaged with potassium oxonate (1.5 g / kg) and adenine (50 mg / kg) in the morning. In the afternoon, the model group rats received only normal saline, the BenZ group was given benzbromarone (10 mg / kg), the WGVPE group was given 40 mg / kg of the solid-phase synthesized pentapeptide in Example 1, and the NC group was gavaged with normal saline.

[0039] 3.2 Sample collection and processing

[0040] During the experiment, orbital blood was collected from rats every week and the supernatant was obtained by centrifugation.

[0041] Two days before the rats were sacrificed, the rats were placed in metabolic cages to collect 24-hour urine, and urine and serum samples were obtained for comprehensive evaluation; after the rats were sacrificed, kidney tissues were obtained for detailed histopathological examination and biochemical analysis.

[0042] 3.3 Detection of blood uric acid, blood creatinine, urine creatinine, and urine uric acid content

[0043] Commercial kits were used to detect the levels of serum uric acid, serum creatinine, urine creatinine, and urine uric acid.

[0044] The experimental results are as follows Figure 3 As shown, Figure 3 Figure A shows the changes in blood uric acid levels in rats of each group. Compared with the control group (51.87±9.61μmol / L), the blood uric acid level in the model group (204.40±6.90μmol / L) increased significantly. After pentapeptide intervention, the blood uric acid level (144.60±24.28μmol / L) decreased significantly (p<0.05).

[0045] Figure 3 Figures B and C show the changes in blood creatinine and urine creatinine levels in rats. Compared with the control group, the blood creatinine level in the model group increased significantly, while the urine creatinine level decreased significantly (p<0.05), reflecting the impaired renal function of the rats. However, pentapeptide intervention significantly decreased the blood creatinine level and increased the urine creatinine level (p<0.05).

[0046] Figure 3 Figure D shows the changes in uric acid in urine. Compared with the model group, the uric acid level in the WGVPE group was significantly increased by 50.84% ​​(p<0.05), indicating that pentapeptide intervention promoted uric acid excretion.

[0047] Based on the above results, it can be seen that pentapeptide WGVPE treatment can maintain normal kidney function and promote uric acid metabolism.

[0048] 3.4 Detection of renal collagen volume fraction and damage markers KIM-1, IL-18, NAG, and Cys-C

[0049] Renal tissue was fixed in 4% paraformaldehyde, then routinely dehydrated with ethanol, embedded in paraffin, and sectioned at 4 μm thickness. After dewaxing and rehydration, the sections were stained with hematoxylin-eosin (H&E) or Masson's trichrome. Pathological changes in renal tissue were observed under a light microscope at 200x magnification. Semi-quantitative analysis was performed using Image J to quantitatively measure the renal interstitial collagen volume fraction. Collagen volume fraction (%) = collagen area / total tissue area * 100%. The levels of kidney injury markers KIM-1, IL-18, NAG, and Cys-C were also measured using ELISA kits.

[0050] The experimental results are as follows Figure 4 As shown, Figure 4 A is the collagen volume fraction, which can reflect the presence of collagen deposition and renal interstitial fibrosis in rat kidneys. The collagen volume fraction in the kidneys of rats in the model group was significantly increased (p<0.05); however, after WGVPE intervention, the collagen volume fraction decreased by 15.78%.

[0051] Figure 4 Figures B to E show the changes in the levels of kidney injury markers. Compared with the control group, the levels of kidney injury markers KIM-1, IL-18, NAG, and Cys-C in the model group were significantly increased (p<0.05), while after WGVPE intervention, the levels of the above kidney injury markers decreased by 22.76%, 25.54%, 26.07%, and 27.68%, respectively.

[0052] Based on the above results, it can be seen that pentapeptide WGVPE treatment can effectively improve kidney damage in rats.

[0053] In summary, the pentapeptide WGVPE screened from marine fish in the present invention has kidney protection effects, and can reduce blood uric acid levels and promote uric acid excretion by maintaining normal kidney function. It does not have potential liver toxicity and can be used to prepare foods and health products for preventing and treating hyperuricemia and kidney damage.

[0054] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A marine fish uricosuric peptide with kidney protective activity, characterized by: Its amino acid sequence is Trp-Gly-Val-Pro-Glu, abbreviated as WGVPE.

2. Use of the marine fish uricosuric acid-lowering peptide with kidney protective activity according to claim 1 in the preparation of a drug for treating / preventing kidney disease.

3. Use of the marine fish uric acid-lowering peptide with kidney protective activity according to claim 1 in the preparation of uric acid-lowering drugs.

4. Use of the marine fish uricosuric peptide with kidney protective activity according to claim 1 in the preparation of a drug for treating / preventing hyperuricemia.

5. Use of the marine fish uric acid-lowering peptide with kidney protective activity according to claim 1 in the preparation of food or health products with uric acid-lowering or kidney-protecting effects.

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