Giant salamander bone peptide for relieving renal injury and application thereof

Abstract

The invention discloses andrias davidianus bone peptide for relieving renal injury and application of the andrias davidianus bone peptide, a polypeptide sequence comprises an amino acid sequence shown in any one of SEQ ID NOs: 1-10 or an amino acid sequence shown in any one of SEQ ID NOs: 1-10, and the polypeptide can improve the renal injury. For example, hyperuricemia-induced ferroptosis and fibrosis injury of human kidney proximal tubular HK-2 cells can be relieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptide preparation, and particularly relates to a polypeptide and application thereof. BACKGROUND

[0002] Andrias davidianus, which is the largest amphibian in the world, has extremely high edible and medicinal values and great development and application potentials in the field of life and health. Wild Andrias davidianus is a national second-class protected aquatic animal, but in the past two decades, the artificial breeding technology of Andrias davidianus has gradually matured, and the Andrias davidianus industry has rapidly developed. A large-scale breeding Andrias davidianus industry and market have been formed in many provinces and regions such as Shaanxi, Hunan, Henan and Sichuan, which provides sustainable biological resources for extracting life and health products with medical and health care values from Andrias davidianus meat. SUMMARY

[0003] The present application provides an Andrias davidianus bone peptide for relieving kidney injury and application thereof. The polypeptide can improve kidney injury, and can especially relieve iron death and fibrosis injury of human kidney proximal tubular HK-2 cells induced by high uric acid.

[0004] The specific technical scheme of the present application is as follows:

[0005] 1. Application of a polypeptide in preparation of a medicine for improving kidney injury, wherein the polypeptide sequence comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-10 or an amino acid sequence as shown in any one of SEQ ID NOs: 1-10.

[0006] 2. The application according to item 1, wherein the kidney injury comprises kidney injury caused by high uric acid.

[0007] 3. Application of a polypeptide in improving kidney injury, wherein the polypeptide sequence comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-10 or an amino acid sequence as shown in any one of SEQ ID NOs: 1-10.

[0008] 4. The application according to item 3, wherein the kidney injury comprises kidney injury caused by high uric acid.

[0009] 5. A medicine for improving kidney injury, comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-10 or an amino acid sequence as shown in any one of SEQ ID NOs: 1-10.

[0010] 6. The medicine according to item 5, wherein the kidney injury comprises kidney injury caused by high uric acid.

[0011] INVENTION EFFECT

[0012] The polypeptide described in the present application can improve kidney injury, and comprises an ability to alleviate high uric acid-induced ferroptosis and fibrosis injury of human kidney proximal tubular HK-2 cells. BRIEF DESCRIPTION OF DRAWINGS

[0013] FIGS. 1A-1K is a schematic diagram of the toxicity results of gradient concentration uric acid and giant salamander bone peptides on HK-2 cells, wherein FIG. 1A is a schematic diagram of the toxicity results of different concentrations of uric acid on HK-2 cells; FIG. 1B is a schematic diagram of the toxicity results of different concentrations of RS-7 on HK-2 cells; FIG. 1C is a schematic diagram of the toxicity results of different concentrations of YR-6 on HK-2 cells;

[0014] FIG. 1D is a schematic diagram of the toxicity results of different concentrations of FR-4 on HK-2 cells; FIG. 1E is a schematic diagram of the toxicity results of different concentrations of LE-4 on HK-2 cells; FIG. 1F is a schematic diagram of the toxicity results of different concentrations of HN-12 on HK-2 cells; FIG. 1G is a schematic diagram of the toxicity results of different concentrations of GH-12 on HK-2 cells; FIG. 1H is a schematic diagram of the toxicity results of different concentrations of RK-10 on HK-2 cells; FIG. 1I is a schematic diagram of the toxicity results of different concentrations of GR-9 on HK-2 cells; FIG. 1J is a schematic diagram of the toxicity results of different concentrations of GS-9 on HK-2 cells; FIG. 1K is a schematic diagram of the toxicity results of different concentrations of AR-6 on HK-2 cells.

[0015] FIGS. 2A-2J is a schematic diagram of the inhibition effect of giant salamander bone peptides on uric acid-induced cell injury, wherein FIG. 2A is a schematic diagram of the inhibition effect of RS-7 on uric acid-induced cell injury; wherein FIG. 2B is a schematic diagram of the inhibition effect of YR-6 on uric acid-induced cell injury; wherein FIG. 2C is a schematic diagram of the inhibition effect of FR-4 on uric acid-induced cell injury; wherein FIG. 2D is a schematic diagram of the inhibition effect of LE-4 on uric acid-induced cell injury; wherein FIG. 2E is a schematic diagram of the inhibition effect of HN-12 on uric acid-induced cell injury; wherein FIG. 2F is a schematic diagram of the inhibition effect of GH-12 on uric acid-induced cell injury; wherein FIG. 2G is a schematic diagram of the inhibition effect of RK-10 on uric acid-induced cell injury; wherein FIG. 2H is a schematic diagram of the inhibition effect of GS-9 on uric acid-induced cell injury; wherein FIG. 2Iis a schematic diagram of the inhibitory effect of GR-9 on uric acid-induced cell damage; wherein FIG. 2J is a schematic diagram of the inhibitory effect of AR-6 on uric acid-induced cell damage.

[0016] FIGS. 3A-3B is a schematic diagram of the flow cytometry analysis results of HK-2 cell apoptosis, wherein FIG. 3A is a schematic diagram of AR-6 regulating uric acid-mediated HK-2 cell apoptosis; FIG. 3B is a schematic diagram of the apoptosis rate of different groups of cells.

[0017] FIGS. 4A-4B is a schematic diagram of the GO analysis of the mechanism of AR-6 regulating uric acid-induced kidney damage, wherein FIG. 4A is a schematic diagram of the GO transcriptome analysis chart comparing the model group and the blank control group; FIG. 4B is a schematic diagram of the GO transcriptome analysis chart comparing the AR-6 group and the model group. The model group represents 0.8 mg / mL uric acid treatment, and the AR-6 group represents 400 μg / mL AR-6 and 0.8 mg / mL uric acid treatment of HK-2 cells.

[0018] FIGS. 5A-5B is a schematic diagram of the KEGG analysis of the mechanism of AR-6 regulating uric acid-induced kidney damage, wherein FIG. 5A is a schematic diagram of the KEGG transcriptome analysis results comparing the model group and the blank control group; FIG. 5B is a schematic diagram of the KEGG transcriptome analysis results comparing the AR-6 group and the model group. The model group represents 0.8 mg / mL uric acid treatment, and the AR-6 group represents 400 μg / mL AR-6 and 0.8 mg / mL uric acid co-treatment of HK-2 cells.

[0019] FIG. 6 is a schematic diagram of the metabolite synthesis flux analysis of the gene-scale metabolic model (n=3, represented by median ± 95% CI). The model group represents 0.8 mg / mL uric acid treatment, and the AR-6 group represents 400 μg / mL AR-6 and 0.8 mg / mL uric acid co-treatment of HK-2 cells.

[0020] FIG. 7 is a schematic diagram of the metabolic pathway difference analysis of the gene-scale metabolic model.

[0021] FIGS. 8A-8D is a schematic diagram of the changes in intracellular oxidative stress indicators, wherein FIG. 8A is a schematic diagram of the intracellular SOD enzyme activity indicator; FIG. 8B is a schematic diagram of the intracellular CAT concentration indicator; FIG. 8C is a schematic diagram of the intracellular GSH concentration indicator; FIG. 8Dis a schematic diagram of the intracellular MDA concentration index of the cells. The model group represents 0.8 mg / mL uric acid treatment, and the gradient concentration group represents the corresponding concentration of AR-6 and 0.8 mg / mL uric acid co-treatment of HK-2 cells.

[0022] FIGS. 9A-9B is a schematic diagram of the ROS level visualization analysis of HK-2 cells, wherein FIG. 9A is a schematic diagram of the fluorescence results of AR-6 regulating uric acid-mediated intracellular ROS of HK-2 cells, FIG. 9B is a schematic diagram of the relative levels of ROS in different groups. The model group represents 0.8 mg / mL uric acid treatment, and the gradient concentration group represents the corresponding concentration of AR-6 and 0.8 mg / mL uric acid co-treatment of HK-2 cells.

[0023] FIGS. 10A-10B is a schematic diagram of the fibrosis factor detection results of HK-2 cells, wherein FIG. 10A is a schematic diagram of the α-SMA concentration detection results of HK-2 cells; FIG. 10B is a schematic diagram of the FN-1 concentration detection results of HK-2 cells. The model group represents 0.8 mg / mL uric acid treatment, and the gradient concentration group represents the corresponding concentration of AR-6 and 0.8 mg / mL uric acid co-treatment of HK-2 cells.

[0024] FIGS. 11A-11B is a schematic diagram of the ferroptosis marker detection results of HK-2 cells, wherein FIG. 11A is a schematic diagram of the cell lipid peroxidation results; FIG. 11B is a schematic diagram of the relative levels of Fe + is a schematic diagram of the relative level results. The model group represents 0.8 mg / mL uric acid treatment, the gradient concentration group represents the corresponding concentration of AR-6 and 0.8 mg / mL uric acid co-treatment of HK-2 cells, and Erastin represents the 10 μmol / L ferroptosis inducer Erastin treatment group.

[0025] FIGS. 12A-12D is a schematic diagram of the gene transcription analysis of ferroptosis damage of HK-2 cells, wherein FIG. 12A is a schematic diagram of the Keap-1 gene transcription level results; FIG. 12B is a schematic diagram of the Nrf-2 gene transcription level results; FIG. 12C is a schematic diagram of the HO-1 gene transcription level results; FIG. 12D is a schematic diagram of the ALDH2 gene transcription level results. The model group represents 0.8 mg / mL uric acid treatment, the AR-6 group represents 400 μg / mL AR-6 and 0.8 mg / mL uric acid treatment.

[0026] FIGS. 13A-13F is a schematic diagram of the gene transcription analysis of the ferroptosis action pathway of the cells, wherein FIG. 13Ais a schematic diagram of GPX4 gene transcription level results; FIG. 13B is a schematic diagram of ACSL4 gene transcription level results; FIG. 13C is a schematic diagram of CAT gene transcription level results; FIG. 13D is a schematic diagram of GSS gene transcription level results; FIG. 13E is a schematic diagram of ALOX12 gene transcription level results; FIG. 13F is a schematic diagram of p-53 gene transcription level results. Model group represents 0.8 mg / mL uric acid treatment, AR-6 group represents 400 pg / mL AR-6 and 0.8 mg / mL uric acid treatment group.

[0027] FIGS. 14A-14B is a schematic diagram of cell fibrosis gene transcription analysis, wherein FIG. 14A is a schematic diagram of TGF-β1 gene transcription level results; FIG. 14B is a schematic diagram of SMAD3 gene transcription level results. Model group represents 0.8 mg / mL uric acid treatment, AR-6 group represents 400 pg / mL AR-6 and 0.8 mg / mL uric acid treatment group.

[0028] FIGS. 15A-15D is a schematic diagram of HK-2 cell ferroptosis / fibrosis protein expression results, wherein FIG. 15A is a schematic diagram of Western blot of ferroptosis proteins GPX4 and Nrf2; FIG. 15B is a schematic diagram of quantification table of Western blot of ferroptosis proteins GPX4 and Nrf2; FIG. 15C is a schematic diagram of Western blot of fibrosis proteins TGF-β1 and Smad3; FIG. 15D is a schematic diagram of quantification table of Western blot of fibrosis proteins TGF-β1 and Smad3. Model group represents 0.8 mg / mL uric acid treatment, AR-6 group represents 400 pg / mL AR-6 and 0.8 mg / mL uric acid treatment group.

[0029] FIGS. 16A-16C is a schematic diagram of the effect of Andrias davidianus bone peptide and uric acid on mouse serum kidney injury indicators, wherein FIG. 16A is a schematic diagram of the effect of Andrias davidianus bone peptide and uric acid on mouse serum kidney injury indicator Scr concentration; FIG. 16B is a schematic diagram of the effect of Andrias davidianus bone peptide and uric acid on mouse serum kidney injury indicator BUN concentration; FIG. 16C is a schematic diagram of the effect of Andrias davidianus bone peptide and uric acid on mouse serum kidney injury indicator UA concentration.

[0030] FIGS. 17A-17B is a schematic diagram of mouse kidney H&E staining pathology sections, wherein FIG. 17Ais a schematic of H&E staining of kidneys of different groups of mice, FIG. 17B is a schematic of the degree of glomerular abnormalities of kidneys of different groups of mice.

[0031] FIGS. 18A-18B is a schematic of Masson pathological staining sections of kidneys of mice, wherein FIG. 18A is a schematic of Masson staining of kidneys of different groups of mice, FIG. 18B is a schematic of the fibrosis ratio of kidneys of different groups of mice.

[0032] FIG. 19 is a schematic of the appearance of kidneys of experimental mice over time.

[0033] FIG. 20 is a schematic of photographs of kidneys of mice at the end of the experiment.

[0034] FIGS. 21A-21B is a schematic of transmission electron microscopy photographs of kidneys of mice, wherein FIG. 21A is a schematic of kidneys of mice, FIG. 21B is a schematic of the rate of mitochondrial damage of kidneys of different groups of mice.

[0035] FIG. 22 is a schematic of the trend of body weight of mice.

[0036] FIGS. 23A-23B is a schematic of the GO analysis of the mechanisms modulated by AR-6 in vivo to regulate uric acid damage, wherein FIG. 23A is a schematic of the GO transcriptomic analysis comparing the MO group and the CK group; FIG. 23B is a schematic of the GO transcriptomic analysis comparing the HAR-6 group and the MO group.

[0037] FIGS. 24A-24B is a schematic of the KEGG analysis of the mechanisms modulated by AR-6 in vivo to regulate uric acid damage, wherein FIG. 24A is a schematic of the KEGG transcriptomic analysis comparing the MO group and the CK group; FIG. 24B is a schematic of the KEGG transcriptomic analysis comparing the HAR-6 group and the MO group.

[0038] FIGS. 25A-25D is a schematic of the gene transcriptomic analysis of ferroptosis damage in HK-2 cells, wherein FIG. 25A is a schematic of the results of the Keap-1 gene transcript level; FIG. 25B is a schematic of the results of the Nrf-2 gene transcript level; FIG. 25C is a schematic of the results of the GPX4 gene transcript level; FIG. 25D is a schematic of the results of the ACSL4 gene transcript level.

[0039] FIGS. 26A-26B is a schematic of the gene transcriptomic analysis of renal fibrosis, whereinFIG. 26A is a schematic diagram of the results of TGF-β1 gene transcription level; FIG. 26B is a schematic diagram of the results of SMAD3 gene transcription level.

[0040] FIGS. 27A-27C is a schematic diagram of short-chain fatty acid targeted metabolomics analysis, wherein FIG. 27A is a 7 short-chain fatty acid metabolomics clustering heat map; FIG. 27B is a KEGG metabolite molecular network diagram comparing the MO group and the CK group; FIG. 27C is a KEGG metabolite molecular network diagram comparing the HAR-6 group and the MO group.

[0041] FIGS. 28A-28F is a schematic diagram of intestinal microbiota 16s detection analysis, wherein FIG. 28A is a PCoA analysis schematic diagram of 16s detection; FIG. 28B is a schematic diagram of the average relative abundance results of intestinal microbiota at the level of phylum;

[0042] FIG. 28C is a schematic diagram of the average relative abundance results of intestinal microbiota at the level of genus.

[0043] FIG. 29 is a schematic diagram of mouse ileum H&E staining pathology section.

[0044] FIGS. 30A-30B is a schematic diagram of mouse liver H&E staining pathology section, wherein FIG. 30A is a schematic diagram of liver cell staining after different groups; FIG. 30B is a schematic diagram of the degree of inflammatory infiltration of the liver of different groups.

[0045] FIGS. 31A-31B is a schematic diagram of mouse liver Masson pathology staining section, wherein FIG. 31A is a schematic diagram of liver cell staining after different groups; FIG. 30B is a schematic diagram of the fibrosis ratio of the liver of different groups. DETAILED DESCRIPTION

[0046] The embodiments described below are described with reference to the accompanying drawings, in which like numerals indicate like features in all of the several figures. While the application is amenable to various forms of embodiments, it is understood that the application is to cover all such embodiments as fall within the scope of the application. Rather, the embodiments are provided so that this disclosure will be thorough, and will fully convey the scope of the application to those skilled in the art.

[0047] It should be noted that some terms are used throughout the specification and claims which have obtained particular meanings. Those of ordinary skill in the art will appreciate that the same component can be referred to by different names and that such appreciation is within the scope of the present application. The present application is not limited by the names of components.

[0048] As used throughout the specification and claims, the terms "comprise" and "comprising" are to be interpreted as "including but not limited to". The description herein of any aspect or aspect of the application is meant to apply to this aspect of the application per se as well as to such aspects of the application which are different from the aspect described but incorporate the essential features of the aspect described. The description herein of any aspect or aspect of the application is meant to apply to this aspect of the application per se as well as to such aspects of the application which are different from the aspect described but incorporate the essential features of the aspect described. The scope of the application is to be only limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0049] The application provides a polypeptide for use in the preparation of a medicament for improving kidney injury, wherein the polypeptide sequence comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-10 or an amino acid sequence as shown in any one of SEQ ID NOs: 1-10.

[0050] The amino acid sequence of SEQ ID NO: 1 is: AVLVFR (AR-6 for short)

[0051] SEQ ID NO: 2: RGPPGPS (RS-7 for short)

[0052] SEQ ID NO: 3: YLAFLR (YR-6 for short)

[0053] SEQ ID NO: 4: FTLR (FR-4 for short)

[0054] SEQ ID NO: 5: LDFE (LE-4 for short)

[0055] SEQ ID NO: 6: HDEPVTIPNKPN (HN-12 for short)

[0056] SEQ ID NO: 7: GIVLDSGDGVTH (GH-12 for short)

[0057] SEQ ID NO: 8: RGFNGLDGAK (RK-10 for short)

[0058] SEQ ID NO: 9: GPAGPSGPR (GR-9 for short)

[0059] SEQ ID NO: 10: GPRGPOGPS (GS-9 for short)

[0060] In the present application, the cause of the kidney injury is not limited.

[0061] In some embodiments, the cause of the kidney injury comprises high uric acid induced kidney injury.

[0062] In some embodiments, the present application uses high uric acid as a kidney injury modeling condition.

[0063] In the present application, for the above-mentioned polypeptide, based on the screening of the proliferation activity and toxicity of ten Megalobatrachus kneri bone peptides at different concentrations on human kidney proximal tubular HK-2 cells, the safe concentration of Megalobatrachus kneri bone peptides is finally determined, and the peptide segment which has a significant alleviating effect on high uric acid induced HK-2 cell injury is screened out.

[0064] The amino acid sequences of the ten Megalobatrachus kneri bone peptides are shown above.

[0065] In the present application, the amino acid sequence represented by any one of SEQ ID NOs: 1-10 is a sequence obtained according to the method of CN118930613A.

[0066] The present application provides a polypeptide for improving kidney injury, wherein the polypeptide sequence comprises an amino acid sequence represented by any one of SEQ ID NOs: 1-10 or an amino acid sequence represented by any one of SEQ ID NOs: 1-10.

[0067] In some embodiments, the kidney injury comprises high uric acid induced kidney injury.

[0068] The present application provides a drug for improving kidney injury, comprising an amino acid sequence represented by any one of SEQ ID NOs: 1-10 or an amino acid sequence represented by any one of SEQ ID NOs: 1-10.

[0069] In some embodiments, the kidney injury comprises high uric acid induced kidney injury.

[0070] Embodiments

[0071] The materials used in the test and the test method are generally and / or specifically described in the present application. In the following embodiments, % represents wt%, i.e. weight percentage, unless otherwise specified. The reagents or instruments used, if not specified by the manufacturer, are conventional reagent products that can be obtained by purchase.

[0072] Example 1 In vitro screening of Megalobatrachus kneri bone peptide activity against high uric acid induced kidney injury

[0073] 1.1 Cell culture and subculture

[0074] In a biological safety cabinet, DMEM / F12 complete medium containing 10% (by volume) fetal bovine serum, 100 U / mL penicillin and 100 pg / mL streptomycin was configured, and human kidney proximal tubule HK-2 cells (Beina Biological, BNCC339833) were cultured using the complete medium in a constant temperature incubator at 37°C, 95% air and 5% CO2. The medium was changed at a fixed time every day. When changing the medium, the HK-2 cells were washed with 1x phosphate buffered solution (PBS) three times after the waste medium was aspirated. After adding new DMEM / F12 complete medium, the cells were incubated in the constant temperature incubator, and after the HK-2 cells grew on the bottom of the culture dish, the HK-2 cells were passaged. For HK-2 cell passage, the HK-2 cells with removed waste liquid were first washed with PBS, then trypsin (Gibco, 25200072) was added and incubated in a constant temperature incubator at 37°C for 2 min, and DMEM / F12 medium was added to the trypsin at a volume ratio of 1:2 to stop the enzymatic reaction. The culture dish bottom was gently blown with a pipette gun until the adherent cells were completely detached from the dish bottom, and then the cell suspension was transferred to a sterile centrifuge tube and centrifuged at 300g for 1 min. After centrifugation, the supernatant was discarded, and the HK-2 cells were resuspended in fresh DMEM / F12 complete medium, with a cell density of about 5x10 6 Cell / mL, the cell suspension resuspended in DMEM / F12 complete medium was diluted and added to a new sterile culture dish, and the culture dish was placed in a 37°C constant temperature incubator after gentle shaking.

[0075] 1.2CCK8 method for detecting HK-2 cell activity

[0076] To test the effect of uric acid on the proliferation activity and toxicity of HK-2 cells, HK-2 cells in the logarithmic growth phase in a 37°C cell incubator were resuspended and inoculated in a 96-well plate at a density of 10,000 cells per well in 100 pL of suspension, divided into a control group (DMEM / F12 medium treatment) and different concentrations (DMEM / F12 medium containing 50 pg / mL, 100 pg / mL, 200 pg / mL, 400 pg / mL, 800 pg / mL uric acid) of uric acid treatment group, with 5 replicate wells in each group. After drug administration according to the set conditions, it was placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours and 48 hours. Then the medium was removed, CCK-8 detection solution was added, and it was treated in a constant temperature incubator for 2 hours. The absorbance A value of the experimental group, the control group and the related blank control at 450 nm wavelength was detected using a microplate reader, and the HK-2 cell activity X was calculated, with the formula as follows:

[0077]

[0078] To test the effect of polypeptides on the proliferation activity and toxicity of HK-2 cells, HK-2 cells in the logarithmic growth phase in a 37°C cell incubator were resuspended and inoculated in a 96-well plate at a density of 10,000 cells per 100 μL of suspension per well, divided into a control group and different concentrations (DMEM / F12 culture medium containing 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL of Andrias davidianus bone peptide) of Andrias davidianus bone peptide treatment groups, with 5 replicate wells in each group, and the remaining steps were the same as the uric acid cytotoxicity detection method in Example 1.2 above, wherein the Andrias davidianus bone peptide was prepared according to the method described in the CN118930613A example.

[0079] To test the regulatory effect of Andrias davidianus bone peptide on uric acid-induced HK-2 cell toxicity, HK-2 cells in the logarithmic growth phase in a 37°C cell incubator were resuspended and inoculated in a 96-well plate at a density of 10,000 cells per 100 μL of suspension per well, with a control group, a model group (0.8 mg / mL concentration of DMEM / F12 culture medium treatment), and an Andrias davidianus bone peptide treatment group (DMEM / F12 culture medium containing 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL of Andrias davidianus bone peptide and 0.8 mg / mL of uric acid) with 5 replicate wells in each group, and the remaining steps were the same as the uric acid cytotoxicity detection method in Example 1.2.

[0080] 1.3 Flow cytometry detection of HK-2 cell apoptosis level

[0081] Annexin V staining with propidium iodide (PI) and fluorescein isothiocyanate (FITC) coupling was used for apoptosis analysis. Briefly, to test the inhibitory effect of Andrias davidianus bone peptide on uric acid-induced HK-2 cell damage, HK-2 cells in the logarithmic growth phase in a 37°C cell incubator were resuspended and inoculated in a 6-well plate at a density of 4×10 5Cells were seeded at a density of 1 x 105cells / well in 6-well plates and incubated in a humidified incubator for 24 h. Control, model (treated with DMEM / F12 medium containing 0.8 mg / mL uric acid), and andouo bone peptide treatment groups (treated with DMEM / F12 medium containing 100, 200, and 400 pg / mL AR-6 and 0.8 mg / mL uric acid) were set up. After treatment, the cells were washed with 1 x PBS and fixed with 70% ethanol. The fixed cells were washed twice with PBS, and then 50 pg / mL RNase A (Sigma-Aldrich) was added and stained with Annexin V-FITC / PI. After incubation at room temperature for 1 h in the dark, flow cytometry analysis was performed using a FACScan flow cytometer (Beckman Coulter, Fullerton, CA, USA) to distinguish between apoptotic cells (Annexin-V positive and PI negative) and necrotic cells (Annexin-V and PI positive). The data were analyzed using FlowJo software.

[0082] 1.4 Experimental results:

[0083] Uric acid and andouo bone peptide cell activity screening results

[0084] From FIGS. 1A-1K As can be seen, the ten andouo bone peptides had no significant inhibitory effect on the survival rate of HK-2 cells, with a decrease of no more than 10% compared with the control group at a concentration of 400 pg / mL for 48 h. However, some andouo bone peptides (RS-7, YR-6, FR-4, LE-4, GH-12, RK-10, GR-9, and AR-6) had a greater cytotoxicity, with a significant decrease in the survival rate of HK-2 cells of more than 15% at a concentration of 800 pg / mL for 48 h. This phenomenon may be related to the high concentration aggregation effect of andouo bone peptides or the destruction of the cell membrane structure by specific amino acid sequences. In summary, high concentrations of uric acid have significant cytotoxicity on HK-2 cells, and the experimental results confirm that 0.8 mg / mL uric acid for 48 h is the modeling condition, and 400 pg / mL is the safe concentration of andouo bone peptides.

[0085] Efficacy screening results of andouo bone peptides in inhibiting uric acid-mediated HK-2 cell damage

[0086] The CCK8 method was used to detect the efficacy of andouo bone peptides in inhibiting high uric acid-induced HK-2 cell damage under the condition of 0.8 mg / mL uric acid treatment for 48 h, and the results are shown in FIGS. 2A-2J

[0087] From FIGS. 2A-2J ​It can be seen that the giant salamander bone peptide HN-12, AR-6 can significantly regulate the activity of HK-2 cells mediated by uric acid at some experimental concentrations FIG. 2A 、 FIG. 2E 、 FIG. 2J ). The giant salamander bone peptide AR-6 has a significant effect on relieving the damage of HK-2 cells induced by high uric acid at 50 μg / mL (p<0.05) and 50 μg / mL or more (p<0.001), and the inhibitory effect increases with the increase of the concentration, and reaches the best damage relief effect at the concentration of 400 μg / mL. The cell activity of AR-6 treated HK-2 cells is close to the level of the control group FIG. 2J ). The giant salamander bone peptide HN-12 has a significant effect on relieving the damage of cells induced by high uric acid at 50 μg / mL (p<0.05) and 50 μg / mL or more (p<0.001), and the relief effect increases with the increase of the concentration of the single peptide, and reaches a significant cell damage relief effect at the concentration of 100 μg / mL FIG. 2E ).

[0088] To further understand the damage of HK-2 cells induced by uric acid and the repair effect of polypeptide, Annexin V-FITC / PI double staining flow cytometry was used to analyze the apoptosis level, and the results are shown in Figures 3A to 3B.

[0089] From FIGS. 3A-3B It can be seen that compared with the control group, after the uric acid modeling, the total cell proportion of HK-2 cells in early and late apoptosis increased by 53.1% (p<0.001), and high uric acid could mediate the apoptosis of HK-2 cells FIG. 3A 、 FIG. 3B ). And under the treatment condition of 100 μg / mL (p<0.01) and 100 μg / mL or more (p<0.001), the proportion of cells in early and late apoptosis decreased significantly with the increase of the treatment concentration, and when the AR-6 treatment concentration was 400 μg / mL, the proportion of cells in early and late apoptosis in the AR-6 treatment group was close to the proportion of normal cells in the control group.

[0090] In summary, high uric acid can induce apoptosis of HK-2 cells, and giant salamander bone peptide (AR-6) can relieve uric acid-mediated apoptosis, and in the safe concentration range of cells, the relief effect of AR-6 on uric acid-induced apoptosis increases with the increase of the concentration.

[0091] Example 2 Mechanism prediction of giant salamander bone peptide regulating high uric acid damage of kidney cells

[0092] 2.1 Cell treatment and RNA extraction

[0093] HK-2 cells were divided into control group (normal culture), model group (0.8 mg / mL uric acid treatment for 48 hours), AR-6 group (0.8 mg / mL uric acid and 0.4 mg / mL AR-6 co-treatment for 48 hours). Each group was set up 3 biological replicates. Total RNA was extracted using TRIzol reagent, and the RNA integrity (RIN≥8.0) was detected by Agilent 2100 Bioanalyzer.

[0094] 2.2 Transcriptomic sequencing and data analysis

[0095] Transcriptomic sequencing was completed by Huada Gene Company. The screening criteria for differentially expressed genes (DEGs) were |log2FC|≥1 and FDR<0.05. GO functional enrichment and KEGG pathway analysis were completed by BGI Cloud Bioinformatics Analysis System of Huada Gene.

[0096] 2.3 Genome-scale metabolic model (GEM) construction

[0097] Based on Human1 metabolic network, transcriptomic data were integrated, and cell phenotype-specific metabolic model was constructed using ftINIT algorithm. The metabolic flux differences of control group, model group and AR-6 group were calculated by flux balance analysis (FBA). The regulation fold of key metabolites (such as arachidonic acid, glutathione, cystine) was screened by flux change rate (FC≥2 or FC≤0.5) and statistical significance (FDR<0.01).

[0098] 2.4 Multi-omics data integration

[0099] Combined with transcriptomic, metabolic flux and western blot (WB) data, weighted gene co-expression network analysis (WGCNA) was used to mine core regulatory modules. The activity of ferroptosis-related pathways (GPX4 / ACSL4 axis, TGF-β / Smad3) was verified by MDA level, mitochondrial membrane potential (JC-1 staining) and quantitative results of fibrosis markers (α-SMA, Collagen I).

[0100] 2.5 Experimental results:

[0101] Transcriptomic mechanism analysis results of AR-6 against uric acid injury

[0102] In order to analyze the downstream injury mechanism of uric acid-mediated oxidative stress, the applicant performed transcriptomic analysis on control group (normal HK-2 cells), model group (0.8 mg / mL uric acid treatment) and AR-6 intervention group (0.8 mg / mL uric acid and 0.4 mg / mL AR-6 co-treatment), and the results are shown in FIGS. 4A-4B , wherein n=3, and the median ±95% CI is represented.

[0103] Depend on FIGS. 4A-4B It can be seen that the AR-6 group had 1009 differentially expressed genes (DEGs, |log2FC|≥1, FDR<0.05) compared with the model group. GO functional enrichment analysis showed that the differentially expressed genes were mainly enriched in energy metabolism (ATP synthesis, p=2.1×10⁻⁶). -4 ) and cell cycle regulation (p=4.3×10-3) related physiological processes.

[0104] To further understand the role of AR-6 in kidney cell pathways, KEGG pathway analysis was performed, and the results are as follows: FIGS. 5A-5B As shown, where n = 3, expressed as median ± 95% CI.

[0105] Depend on FIGS. 5A-5B It can be seen that AR-6 significantly regulates uric acid-mediated ferroptosis (p = 3.2 × 10⁻⁶). -5 ), cell cycle (p=6.5×10), -4 The abnormal expression of the AR-6 gene suggests that AR-6 may alleviate uric acid damage to kidney cells by intervening in the oxidative stress-ferroptosis-fibrosis axis.

[0106] Metabolic model predicts AR-6 anti-damage mechanism results

[0107] Based on transcriptome data, a genome-scale metabolic model (GSM) was integrated for mechanism prediction. Three sets of transcriptome data were imported into the Human1 basal metabolic network, and a hyperuricemia-specific metabolic model of HK-2 phenotype cells was constructed using the ftINIT algorithm. The results are as follows. FIG. 6 , FIG. 7 As shown, where n = 3, expressed as median ± 95% CI.

[0108] Depend on FIG. 6 The metabolic flux comparison showed that AR-6 significantly improved the levels of core metabolites involved in ferroptosis in uric acid-induced renal cells: leucine (Leu) metabolic flux was upregulated by 2.3-fold (FDR = 1.2 × 10⁻⁶). -4 ), glutathione (GSH) metabolic flux increased by 178% (FDR = 1.7 × 10⁻⁶). -4 Cystine transport flux increased by 64% (FDR = 3.8 × 10⁻⁶). -3 ).

[0109] Depend on FIG. 7 It can be seen that the metabolic pathway enrichment analysis shows that AR-6 mainly acts on the following uric acid-induced biological processes in kidney cells: (1) lipid peroxidation pathway: lipid oxidation metabolism was significantly reduced (AR-6 group vs. model group: p = 3.2 × 10⁻⁶). -5); (2) fatty acid beta-oxidation pathway (CPT1A / ACADM): restoring mitochondrial membrane potential to 82% ± 5% of normal levels (AR-6 group vs model group: p = 1.8 x 10 -4 ).

[0110] The above results of multi-omics data integration analysis show that AR-6 interferes with the oxidative stress-iron death cascade of uric acid-induced kidney cells through multiple targets: (1) antioxidant defense system reconstruction: reversing GSH depletion induced by uric acid (GSH / GSSG ratio from 1.2 ± 0.1 to 3.5 ± 0.3, r = -0.89, p < 0.001), restoring cystine / glutamate antiporter (System Xc - ) function (model group activity was 41% ± 5% of the control group, and the AR-6 group recovered to 78% ± 6%); (2) lipid peroxidation inhibition: inhibiting polyunsaturated fatty acid peroxidation mediated by ACSL4 by targeting arachidonic acid metabolism (KEGG:00590) (phosphorylation level decreased by 62% ± 5%).

[0111] From the above, it can be seen that by constructing a high uric acid injury cell-specific metabolic model, the multi-target mechanism of AR-6 is elucidated. AR-6 regulates the key nodes of intracellular glutathione metabolism (KEGG:00480) and arachidonic acid metabolism (KEGG:00590), rebuilds the redox homeostasis, and then blocks the oxidative stress and iron death process.

[0112] Example 3 Mechanism of Megakarya Bone Peptide in Regulating High Uric Acid Injury of Kidney Cells

[0113] 3.1 Biochemical detection

[0114] HK-2 cells in the logarithmic growth phase were resuspended in a 37°C cell incubator, and 2 mL of suspension was inoculated in each well of a 6-well plate at a density of 4 x 10 5 cells, and placed in a constant temperature incubator for 24 hours. The control group, model group (DMEM / F12 medium containing 0.8 mg / mL uric acid), and Megakarya bone peptide treatment group (DMEM / F12 medium containing 400 μg / mL AR-6 and 0.8 mg / mL uric acid) were set up, and treated for 48 hours. According to the kit instructions, the activity levels of GSH (Solabio, CA1410), SOD (Solabio, BC0175), CAT (Nanjing Jiancheng, A007-1-1), MDA (Nanjing Jiancheng, A003-1-2), α-SMA (Huawmei Biological, CSB-E09343h), and FN1 (Shengong, D711079-0048) were tested.

[0115] 3.2 Cell iron death metabolism detection

[0116] HK-2 cells in logarithmic growth phase were resuspended in a 37 °C cell incubator and seeded in a 6-well plate at a density of 4 x 10 5 Control, model (treated with DMEM / F12 medium containing 0.8 mg / mL uric acid), and Andrias davidianus bone peptide treatment groups (treated with DMEM / F12 medium containing 400 pg / mL AR-6 and 0.8 mg / mL uric acid) were set up. After 48 hours of treatment, Fe 2+ (Meridian Bioscience, MA0647-1) and lipid peroxidation (Dongjin Chemical, L248-50) levels were tested according to the kit instructions.

[0117] 3.3 ROS detection

[0118] After resuspension of HK-2 cells, control, model (treated with DMEM / F12 medium containing 0.8 mg / mL uric acid), and Andrias davidianus bone peptide treatment groups (treated with DMEM / F12 medium containing 400 pg / mL AR-6 and 0.8 mg / mL uric acid) were set up. After 48 hours of treatment, cells were incubated with 50 mM DCFH-DA for 30 minutes, and DCF fluorescence was detected by confocal microscopy (A1R HD25, Nikon) at excitation and emission wavelengths of 485 and 535 nm, respectively.

[0119] 3.4 Enzyme-linked immunosorbent assay (ELISA)

[0120] Control, model (treated with DMEM / F12 medium containing 0.8 mg / mL uric acid), and Andrias davidianus bone peptide treatment groups (treated with DMEM / F12 medium containing 400 pg / mL AR-6 and 0.8 mg / mL uric acid) were set up. After 48 hours of treatment, ELISA was used to detect the concentrations of alpha-smooth muscle actin (a-SMA) and fibronectin (FN1) in the cell culture supernatant. The kits were purchased from Shenzhen Xingboseng Biological Technology Co., Ltd. and operated according to the instructions. All reactions included more than three biological replicates.

[0121] 3.5 Western blot (WB)

[0122] After HK-2 cells were fully lysed in cell lysis solution, homogenate was centrifuged at 4,000 g for 15 min, and the supernatant was collected after the precipitate was discarded. The total protein concentration was determined by NanoDrop. Then, the lysate was aliquoted into multiple centrifuge tubes, and the SDS-PAGE protein loading buffer (Bi Yun Tian, P0015) was added to dilute the sample, so that the total protein concentration was adjusted to 1-2 mg / mL. Using 4-12% acrylamide gradient Bis-Tris gel, equal amounts of protein samples were added to each lane, and after electrophoretic separation, the proteins were transferred to a methanol pretreated polyvinylidene fluoride (PVDF) membrane. Then, the membrane was blocked with 5% skim milk at room temperature for 30 min, and then the rabbit polyclonal antibodies against TGF-β1, p-SMAD3, GPX4 and Nrf-2 were diluted with blocking buffer and incubated at 4°C overnight. After washing, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody for 2 h (room temperature). Finally, the protein expression was detected by enhanced chemiluminescence (ECL) substrate kit (Bi Yun Tian, P0018S), and the relative protein level was quantitatively analyzed using ImageJ software, and the results were normalized to the GAPDH level.

[0123] 3.6 Real-time fluorescent quantitative PCR (RT-qPCR)

[0124] After the HK-2 cells were resuspended and inoculated in a 6-well plate, they were divided into a control group, a model group and an AR-6 group. The total RNA of the cells was extracted using TRIzol reagent, and its concentration was determined by ScanDrop100 (Analytik Jena, Thuringia, Germany). Then, reverse transcription was performed using Oligo-d(T) and M-mLV reverse transcriptase (Thermo Fisher, USA). Real-time fluorescent quantitative PCR (qRT-PCR) was performed on an Applied Biosystems 7500 real-time PCR system (Foster City, USA) using a PrimeScript RT Master Mix kit (TaKaRa, Dalian). After the PCR products were detected by agarose gel electrophoresis, the bands were analyzed using ImageJ software, and the gray value ratio of the GAPDH band was used as an indicator of the relative expression level of the gene. All primers were synthesized and purified by Guangzhou Shengong Biotechnology Co., Ltd., and the primer sequences are shown in Table 1.

[0125] Table 1 Design of RT-qPCR primers for cell experiments

[0126]

[0127] 3.7 Statistical analysis

[0128] All the above data are expressed as mean ± standard error (SEM) and analyzed using SPSS 19.0 (IBM). Statistical significance was defined as: # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the model group; * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the model group.

[0129] 3.8 Experimental results:

[0130] Results of the regulatory effect of AR-6 on oxidative stress in HK-2 cells

[0131] Based on the transcriptome analysis and metabolic model prediction, AR-6 may alleviate uric acid-induced HK-2 cell ferroptosis and fibrosis damage by regulating energy metabolism and lipid peroxidation pathways. Ferroptosis and fibrosis are often accompanied by oxidative stress damage. Lipid peroxidation is not only a hallmark process of oxidative stress, but also a typical feature of ferroptosis. Oxidative stress becomes a key inducer of ferroptosis and fibrosis by inducing the production of ROS. Therefore, oxidative stress indicators can be used as important markers to judge the occurrence of ferroptosis and fibrosis. The results of oxidative stress indicator detection are shown in Table 5, wherein n = 5, and the median ± 95% CI is represented. FIGS. 8A-8D

[0132] From Table 5, it can be seen that AR-6 significantly reversed the disorder of intracellular oxidative stress-related factors induced by uric acid. In the 0.4 mg / mL AR-6 treatment group, the expression levels of SOD activity (96.7 ± 5.3% vs. 75.2 ± 2.6% in the model group, p < 0.001) and CAT level (175.6 ± 3.8 U / mg prot vs. 82.9 ± 2.5 U / mg prot in the model group, p < 0.01) were restored to near the control group level (p > 0.05). FIGS. 8A-8D FIG. 8A FIG. 8B Notably, 0.1 mg / mL AR-6 could significantly improve the GSH level (3.0 ± 0.3 μmol / mg prot vs. 1.2 ± 0.1 μmol / mg prot in the model group, p < 0.01) and reduce the content of lipid peroxidation product MDA (2.5 ± 0.2 nmol / mg prot vs. 4.8 ± 0.3 nmol / mg prot in the model group, p < 0.001). In summary, AR-6 can significantly alleviate the abnormality of oxidative stress indicators in HK-2 cells mediated by uric acid.

[0133] Results of the regulatory effect of AR-6 on intracellular ROS level

[0134] ​​​Oxidative stress is caused by the accumulation of reactive oxygen species (ROS), leading to an imbalance in the redox state of cells. To further understand the regulatory role of AR-6 in uric acid-mediated oxidative stress, ROS fluorescent probes were used for detection, and the results are as follows: FIGS. 9A-9B As shown, where n = 3, expressed as median ± 95% CI.

[0135] Depend on FIGS. 9A-9B It can be seen that uric acid treatment significantly increased intracellular ROS levels in HK-2 cells (the model group increased by 0.962 times compared with the control group, p<0.001). After AR-6 intervention, ROS levels showed a dose-dependent decrease: the ROS level in the 0.1 mg / mL AR-6 group decreased to 91.5% ± 4% of the model group (p<0.01), and the ROS level in the 0.4 mg / mL AR-6 group decreased to near the control group level (p>0.05), indicating that AR-6 effectively inhibited uric acid-mediated abnormal accumulation of intracellular ROS and improved cellular oxidative stress levels.

[0136] Association analysis results of AR-6 on intracellular oxidative stress and fibrosis

[0137] The results of its correlation analysis are as follows FIGS. 10A-10B As shown, where n = 3, expressed as median ± 95% CI, by FIGS. 10A-10B It can be seen that uric acid induces downstream fibrotic damage by activating the intracellular oxidative stress cascade. AR-6 regulates intracellular GSH metabolism (KEGG:00480) and scavenges lipid peroxidation products (KEGG:00480). FIG. 4B The AR-6 treatment significantly inhibited the abnormal expression of oxidative stress and fibrosis markers (α-SMA, FN1) (r = 0.89, p < 0.001). Furthermore, MDA levels in the AR-6 treatment group were positively correlated with fibrosis factors (r = 0.76, p < 0.01), suggesting a close relationship between oxidative stress damage and the fibrosis process.

[0138] AR-6 inhibits ferroptosis in HK-2 cells.

[0139] Erastin was used as a cellular ferroptosis inducer, via the ferroptosis marker ferrous iron (Fe2+). 2+ The study investigated changes in ferroptosis concentration and the extent of lipid peroxidation, a classic metabolic process, and examined the uric acid-mediated ferroptosis and the regulatory effect of AR-6. The results are as follows: FIGS. 11A-11B As shown.

[0140] Depend on FIGS. 11A-11B It can be seen that free Fe in the intracellular unstable iron pool 2+ AR-6 directly drives the generation of reactive oxygen species (ROS) through the Fenton reaction and exacerbates lipid peroxidation. Notably, AR-6 can significantly alleviate uric acid-induced increases in intracellular lipid peroxidation and Fe...2+ Steady-state imbalance, suggesting that it has a regulatory function on the process of ferroptosis. In addition, compared with the classic ferroptosis inducer Erastin, uric acid exhibits a unique ferroptosis induction effect. Through comparative analysis, the specific mechanism of action of AR-6 in the uric acid-mediated ferroptosis-related pathway is further clarified.

[0141] AR-6 regulates the transcription level of uric acid-mediated intracellular Nrf2 / GPX4 pathway

[0142] In the above "transcriptomic mechanism analysis of AR-6 against uric acid damage" and "metabolic model predicts AR-6 anti-damage mechanism" experimental results, AR-6 may inhibit uric acid-mediated kidney cell damage through ferroptosis and fibrosis pathways, which are predicted by gene transcriptomics and genome-scale metabolic model analysis, and are verified by related phenotype experiments, but the uric acid-induced kidney cell damage and the regulation pathway of AR-6 on uric acid-induced kidney damage are not clear. In order to analyze the regulation of andouhepeptide on ferroptosis, the expression of intracellular ferroptosis-related functional genes was analyzed by RT-qPCR, and the results are shown in FIGS. 12A-12D .

[0143] From FIGS. 12A-12D It can be seen that compared with the control group, the expression of kidney cell ferroptosis activator Keap-1(p<0.001) in the uric acid modeling group was significantly increased( FIG. 12A ), while the expression of ferroptosis regulatory gene Nrf-2(p<0.001), HO-1(p<0.01) was significantly decreased, and the expression of ferroptosis-promoting gene ALDH2(p<0.001) was significantly increased( FIG. 12B , FIG. 12C , FIG. 12D ). From the gene transcription level, it is shown that high uric acid can mediate kidney cell ferroptosis damage. Compared with the model group, the expression of Keap-1 in the AR-6 group was significantly decreased(p<0.001), close to the level of the control group; the expression of ferroptosis regulatory factor Nrf-2(p<0.001), HO-1(p<0.001) was significantly increased, and the transcription level was even higher than that of the control group normal cells; the expression of ferroptosis-promoting gene ALDH2(p<0.001) was significantly decreased. The results show that AR-6 inhibits the abnormal expression of ferroptosis genes mediated by high uric acid in cells, and alleviates the process of intracellular ferroptosis.

[0144] In order to further understand the mechanism of andouhepeptide regulating uric acid-induced ferroptosis damage, RT-qPCR was performed, and the results are shown in 13A-13F.

[0145] From FIGS. 13A-13FAs can be seen, compared with the control group, the expression levels of GPX4 (p<0.01), GSS (p<0.001), and CAT (p<0.001) in the model group were significantly reduced, and the expression levels of GPX4 (p<0.05), GSS (p<0.001), and CAT (p<0.001) in the AR-6 group were significantly increased compared with the model group FIG. 13A , FIG. 13B , FIG. 13C , FIG. 13D ). The expression level of ACSL4 (p<0.001) in the model group was significantly increased compared with the control group, and the expression level of ACSL4 (p<0.01) in the AR-6 group was significantly decreased compared with the model group. Notably, the CAT transcription level in the AR-6 group was close to that in the control group, and the GSS level was even higher than that in the control group. This is likely due to the inhibition of cysteine metabolic pathways by high uric acid directly causing a decrease in glutathione expression, promoting the synthesis of fatty acid enzymes, and thus accelerating the intracellular iron death of cells. AR-6 effectively promotes the synthesis of intracellular glutathione, inhibits the synthesis of fatty acid enzymes, reverses lipid metabolism abnormalities, and thus inhibits the process of uric acid-induced intracellular iron death. Through testing the transcriptional expression levels of ALOX12 and p53 genes, it was found that high uric acid had no significant effect on the expression of ALOX12 (p=ns) and p53 (p=ns) genes, and AR-6 had no significant effect on the expression of ALOX12 (p=ns) and p53 (p=ns) genes FIG. 13E , FIG. 13F ). In summary, high uric acid mediates the occurrence of renal iron death through the GPX4 / ACSL4 pathway rather than the ALOX12 / p53 pathway, and AR-6 regulates the GPX4 / ACSL4 pathway by inhibiting the level of intracellular lipid peroxidation, thereby alleviating uric acid-mediated cellular iron death damage.

[0146] Results of AR-6 regulating the transcriptional level of the TGF-β1 / SMAD3 pathway

[0147] As shown in FIGS. 14A-14B , FIGS. 14A-14B As can be seen, at the transcriptional level, the expression levels of the fibrosis-activating genes TGF-β1 (p<0.001) and SMAD3 (p<0.001) in the model group were significantly increased compared with the control group, and the expression levels of the fibrosis-activating genes TGF-β1 (p<0.001) and SMAD3 (p<0.001) in the AR-6 group were significantly decreased compared with the model group, and the level of SMAD3 was even lower than that in the control group. The results show that high uric acid can mediate the expression of fibrosis-activating genes in HK-2 cells in the kidney, and AR-6 can effectively inhibit the disorder of fibrosis factors in HK-2 cells induced by high uric acid.

[0148] AR-6 modulates uric acid-mediated ferroptosis-fibrosis pathway protein levels results

[0149] To further verify the mechanism of AR-6 regulating uric acid-induced ferroptosis and fibrosis injury. The expression levels of key proteins of ferroptosis and fibrosis were determined, and the results are shown in FIGS. 15A-15D .

[0150] From FIGS. 15A-15D It can be seen that the expression levels of ferroptosis key proteins Nrf2 (p<0.001), GPX4 (p<0.001) in the model group were significantly lower than those in the control group FIG. 15B ), and the expression levels of Nrf2 (p<0.001), GPX4 (p<0.001) in the AR-6 group were significantly higher than those in the model group FIG. 15C ). The expression levels of kidney fibrosis key proteins TGF-β1 (p<0.001), p-SMAD3 (p<0.001) in the model group were significantly higher than those in the control group FIG. 15B ), and the expression levels of TGF-β1 (p<0.001), p-SMAD3 (p<0.001) in the AR-6 group were significantly lower than those in the model group FIG. 15D ). The results show that high uric acid can inhibit the expression of ferroptosis regulatory proteins in HK-2 cells, increase the level of kidney fibrosis proteins in HK-2 cells, and AR-6 can alleviate this process, protect HK-2 cells from ferroptosis injury, and improve the abnormal level of fibrosis proteins.

[0151] Example 4 Mechanism verification of giant salamander bone peptide regulating uric acid-induced kidney injury in mice

[0152] 4.1 Animal experiment

[0153] 82 specific pathogen-free (SPF) male C57BL / 6 mice (6-8 weeks, 18-21 g) were fed in SPF facilities for one week before the experiment started, with a light / dark light cycle of 12 / 12 (25±1) ℃; (55±5) % humidity.

[0154] All mice were randomly divided into 7 groups: normal control group (CK), model group (MO), positive drug group (VC), low AR-6 group and high AR-6 group (LAR-6 group and HAR-6 group), low concentration giant salamander mixed peptide group and high concentration giant salamander mixed peptide group (LMIX group and HMIX group) (n=10 in each group), a total of 70.

[0155] The test period was 28 days. During the modeling process, the mice in the control group were orally administered with 200 μL of 0.25% CMC-Na. The other groups were all mixed and gavaged with 200 μL of oxypurinol (1 g / (kg·d)) and adenine (80 mg / (kg·day)) once every two days, starting at 9:00 am, for 4 weeks, to induce a mouse kidney high uric acid injury model. The mice in the model group, the positive group, and the AR-6 group were orally administered with treatment 1 hour after gavage with oxypurinol and adenine, and the mice in the control group were orally administered with 200 μL of 0.25% carboxymethylcellulose sodium (CMC-Na), once a day, starting at 11:00 am, for 4 weeks. The body weight was recorded every week, and the gavage amount was calculated and adjusted according to the body weight measured immediately before administration.

[0156] Since one mouse needed to be sacrificed every 7 days during the experiment to observe the kidney structure to detect the modeling and the effect of the positive drug, the experiment lasted for a total of 28 days, and 4 additional mice were needed for the control group, the model group, and the positive control group, for a total of 14 mice per group, and a total of 82 mice. The grouping information (a total of 82) and the treatment method are as follows:

[0157] Control group: 14 mice per group, gavaged with 0.25% CMC-Na twice a day, 100 μL each, with a 2-hour interval;

[0158] Model group: 14 mice per group, gavaged with 100 μL of modeling agent every day, and gavaged with 100 μL of CMC-Na solution every day 2 hours later;

[0159] Positive control group: 14 mice per group, gavaged with 100 μL of modeling agent every day, and gavaged with 100 μL of 100 mg / kg ascorbic acid solution every day 2 hours later;

[0160] Low AR-6 group (50 mg / kg): 10 mice per group, gavaged with 100 μL of modeling agent every day, and gavaged with 100 μL of low-concentration AR-6 solution every day 2 hours later;

[0161] High AR-6 group (200 mg / kg): 10 mice per group, gavaged with 100 μL of modeling agent every day, and gavaged with 100 μL of high-concentration AR-6 solution every day 2 hours later;

[0162] Low mixed peptide group (200 mg / kg): 10 mice per group, gavaged with 100 μL of modeling agent every day, and gavaged with 100 μL of low-concentration gastric mixed peptide solution every day 2 hours later;

[0163] High mixed peptide group (800 mg / kg): 10 mice per group, gavaged with 100 μL of modeling agent every day, and gavaged with 100 μL of high-concentration gastric mixed peptide solution every day 2 hours later;

[0164] The animal experiment grouping information is shown in Table 2.

[0165] Table 2 Animal experiment grouping information

[0166]

[0167] All animal experiment protocols of the present application have been approved by the Ethics Review Committee on Animal Experiments of Peking University Shenzhen Graduate School, reported and approved, and the ethics approval number is: 11512. All animal experiment operations were performed at the Peking University Shenzhen Experimental Animal Center.

[0168] 4.2 Animal sacrifice

[0169] After 28 days of experiment, the experiment was terminated, and the experimental animals were sacrificed by CO2 asphyxiation. The specific operation was as follows: at the end of the experiment, after 12 hours of fasting, each mouse was placed in an euthanasia box, carbon dioxide gas was introduced, and the experimental animals were observed. The life signs of the animals were confirmed to be completely disappeared by methods such as pinching the toe reflex of the animals, and it was determined that there was no breathing and no heartbeat. The carcass was removed from the euthanasia box. Then whole blood was taken by eye blood collection technique, and immediately after the whole blood was taken, the animals were dissected (at the dissection room of the Peking University Experimental Animal Center), and the kidneys, liver, colon, pancreas and adipose tissue of the experimental animals were removed and cut open. Part of them were placed in a -80°C refrigerator for biochemical analysis, part were fixed in a paraformaldehyde solution for pathological analysis, and another part were used for electron microscope detection. The blood samples of the experimental animals were placed in a -80°C refrigerator after centrifugation for biochemical and omics analysis. After dissection, the carcasses of the experimental animals were placed in the experimental animal carcass placement place of the experimental animal center. All mice involved in the animal experiment met the ARRIVE guidelines and were approved by the Tsinghua University Experimental Animal Use and Management Committee and the Ethics Review Committee of Peking University Shenzhen Graduate School.

[0170] During the experiment, the body weight of the mice was recorded every week, and at 0, 7, 14, 21 d, one mouse of the control group, the model group and the positive control group was sacrificed according to the animal ethics standard. After eye blood collection, the levels of kidney injury indicators SCr, BUN and UA were detected, and the kidney structure was observed to determine the effect of the modeling agent and the positive drug.

[0171] 4.3 Serum biochemical detection

[0172] After the eye blood of the mice was collected, the fresh blood was placed in a test tube without anticoagulant. After standing at room temperature for 30-60 minutes, the blood was coagulated. Centrifugation was performed at a speed of 2000-3000 rpm for 5-10 minutes, and the supernatant was the serum. The levels of serum urea nitrogen (BUN), serum creatinine (SCr) and uric acid (UA) were detected using commercial detection kits purchased from Jiancheng Bioengineering Institute (Nanjing, China) and Biyun Tian Biological Co., Ltd. (Nanjing, China), and all detection steps were performed according to the kit instructions.

[0173] 4.4 Histopathology section

[0174] The kidney, liver, ileum tissues of mice were fixed with 4% paraformaldehyde (PFA), 10% paraffin-embedded, and cut into 4 pm thick for hematoxylin-eosin (H&E) staining. The H&E staining of kidney was detected by microscope, and the images were analyzed by computer digital image analysis (K-Viewer).

[0175] The kidney, liver tissues of mice were fixed with 4% paraformaldehyde (PFA), 10% paraffin-embedded, and cut into 4 pm thick sections which were stained with Masson after deparaffinization and rehydration. The stained sections were observed under an optical microscope at 20 or 80 times magnification. Quantitative analysis, 10 fields were randomly selected from each sample section, at least 3 samples per group, the blue (collagen fibers), red (cytoplasmic muscle fibers) fibrosis area was detected by ImageJ software (NIH), respectively.

[0176] 4.5 Transmission electron microscopy

[0177] Fresh kidney tissue samples were rinsed with pre-cooled PBS, and 1 mm 3 Cortex tissue blocks were immediately immersed in pre-cooled (4°C) 2.5% glutaraldehyde (0.1 M phosphate buffer, pH 7.4) for primary fixation for 24 hours; then transferred to 1% osmium tetroxide (same buffer system) for post-fixation for 2 hours. The gradient dehydration process is as follows: 50%, 70%, 90% and absolute ethanol for 15 minutes each, propylene oxide replacement for 2 times (10 minutes / time). After the sample was immersed in a mixture of Epon 812 and propylene oxide (1:1 volume ratio) for 4 hours, it was embedded in fresh resin and polymerized at 60°C for 48 hours. The embedded block was cut into 70 nm thick sections using an ultramicrotome (Leica EMUC7), and double-stained with 3% uranyl acetate ethanol solution (room temperature, avoid light, stain for 30 minutes) and Reynolds' lead citrate (stain for 15 minutes). After staining, the sections were rinsed with double distilled water for 3 times, naturally dried, and placed on a copper grid (200 mesh) for observation and image collection using a Hitachi H-7650 transmission electron microscope (accelerating voltage 80 kV). Image analysis was independently completed by two blind pathologists, and the percentage of damaged mitochondria in each mitochondrial image was quantified based on ImageJ software (NIH).

[0178] 4.6 Kidney transcriptome analysis

[0179] The kidney tissue samples (n = 3 for each group) were randomly selected from the CK group (control group), MO group (high uric acid model group), and HAR-6 group (high concentration AR-6 peptide gavage group) mice, and were entrusted to Pishon Technology Co., Ltd. for cDNA library construction, Pishon Gene Cloud Analysis Platform high-throughput sequencing, and bioinformatics analysis. Based on the transcriptome sequencing data, strict statistical standards (P < 0.05 and |log2FC| > 1) were used as significant thresholds for systematic screening of differentially expressed genes (DEGs) in the comparison between the MO group and the CK group, and the comparison between the HAR-6 group and the MO group. To further elucidate the biological significance of DEGs, Gene Ontology (GO) analysis was used to annotate the differential genes, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis method was used to systematically explore the potential biological processes and key metabolic pathways involved in the differential expression genes.

[0180] 4.7 Real-time fluorescent quantitative PCR (RT-qPCR)

[0181] After thawing the mouse kidney cells, tissue disruption was performed. Total RNA of tissue homogenate was extracted using TRIzol reagent, and its concentration was determined by ScanDrop100 (Analytik Jena, Thuringia, Germany). Subsequently, reverse transcription was performed using Oligo-d(T) and M-mLV reverse transcriptase (Thermo Fisher, USA). Real-time fluorescent quantitative PCR (qRT-PCR) was performed on an Applied Biosystems 7500 real-time PCR system (Foster City, USA) using a PrimeScript RT Master Mix kit (TaKaRa, Dalian). After agarose gel electrophoresis of the PCR products, the bands were analyzed using ImageJ software, and the gray value ratio of the GAPDH band was used as an indicator of the relative expression level of the gene. All primers were synthesized and purified by Guangzhou Shengong Biotechnology Co., Ltd. The design of RT-qPCR primers for animal experiments is shown in Table 3.

[0182] Table 3 Design of RT-qPCR primers for animal experiments

[0183]

[0184] 4.8 Intestinal flora detection

[0185] The intestinal flora of the fecal samples of the mice in the CK group (control group), the MO group (model group), and the HAR-6 group (intervention group) was detected by 16S rDNA high-throughput sequencing technology (completed by PatSnap Technology Co., Ltd.). First, the structural characteristics of the intestinal flora of each group were evaluated by a diversity analysis. Then, a difference analysis between groups was performed, and further analysis was performed by LEfSe (Linear discriminant analysis Effect Size) multi-level species difference discriminant analysis (LDA score threshold > 2.0, p < 0.05) to identify the characteristic microbial groups with significant differences between groups.

[0186] 4.9 Mouse fecal short-chain fatty acid test

[0187] In this study, the colorectal feces of the mice in the CK group, the MO group, and the HAR-6 group (n = 3 in each group) were randomly selected and entrusted to PatSnap Technology Co., Ltd. for a mouse fecal short-chain fatty acid test, a short-chain fatty acid metabonomics clustering analysis was performed, and a KEGG molecular metabolic network diagram of the two groups of MO vs. CK and HAR-6 vs. MO was constructed.

[0188] 4.10 Statistical analysis

[0189] All the above data are expressed as mean ± standard error (SEM), and analyzed using SPSS 19.0 (IBM). Statistical significance is defined as P* < 0.05, P** < 0.01, or P*** < 0.001.

[0190] 4.11 Experimental results:

[0191] AR-6 inhibits kidney injury in hyperuricemic mice

[0192] The key indicators of Scr, BUN, and UA, which are important indicators of kidney injury in mice serum after 28 days, were detected, and the results are shown in FIGS. 16A-16C .

[0193] From FIGS. 16A-16C It can be seen that the expression levels of the kidney injury factors Scr and BUN in the model group (MO) were significantly increased by 2.13 times (p < 0.001) and 3.27 times (p < 0.001) compared with the control group (CK), indicating that high uric acid induced kidney injury in the model animals FIG. 16A , FIG. 16B ) and the level of UA was significantly increased by 0.73 times (p < 0.01) FIG. 16CThe results indicate that the hyperuricemia model was successfully established. Compared with the MO group, the positive drug group (VC) showed significant decreases in Scr, BUN, and UA levels of 0.53-fold (p<0.01), 0.64-fold (p<0.001), and 0.39-fold (p<0.01), respectively. These results demonstrate that VC can alleviate hyperuricemia-mediated kidney damage and reduce uric acid levels.

[0194] The results of gavage analysis of renal parameters in mice treated with AR-6 showed that, compared with the MO group, the low-dose AR-6 monopeptide group (LAR-6) exhibited a 0.23-fold (p<0.01), 0.31-fold (p<0.01), and 0.22-fold (p<0.01) decrease in Scr, BUN, and UA levels, respectively. Conversely, the high-dose AR-6 monopeptide group (HAR-6) showed a significant 0.49-fold (p<0.001), 0.73-fold (p<0.001), and 0.33-fold (p<0.01) decrease in Scr, BUN, and UA levels, respectively, compared with the MO group. Furthermore, the renal injury-related physiological and biochemical parameters decreased accordingly with increasing AR-6 gavage concentration. These results indicate that hyperuricemia induces severe structural damage to the renal tubules and glomeruli, leading to abnormal expression of renal injury factors. AR-6 monopeptide can effectively alleviate this process, and the inhibitory effect on renal injury increases with increasing AR-6 concentration. The animal experimental results are consistent with the cell experimental results.

[0195] The H&E pathological section images reflect the effects of long-term hyperuricemia damage and AR-6 and positive drug gavage on the kidneys of mice with long-term hyperuricemia damage. The results are as follows: FIGS. 17A-17B As shown.

[0196] Depend on FIGS. 17A-17B As can be seen from the H&E staining results, in the control group, the glomerular endothelial cells were regularly arranged and structurally intact, without interstitial inflammatory cell infiltration or basement membrane thickening. The glomeruli were pink in color, and the cells showed no obvious shrinkage. The tubular and collecting duct cells were neatly and tightly arranged, with no abnormal material deposition in the lumen. The cytoplasm of the tubular and glomerular cells was uniformly colored, and the nuclei showed no significant abnormalities. In contrast, in the H&E-stained pathological sections of the MO group, compared with the CK group, the glomerular structure of the MO group was abnormal, showing obvious shrinkage, and the glomerular cells were enlarged. There was obvious interstitial inflammatory cell infiltration and basement membrane thickening around the glomeruli (marked by arrows). The tubular and collecting duct cells were loosely arranged, with obvious red foreign bodies in the lumen and large areas of vacuoles. The renal cell nuclei were significantly enlarged. FIG. 17B ).

[0197] Compared with the MO group, the VC group had reduced glomerular structure atrophy, interstitial inflammatory cell infiltration, and basement membrane thickening. The cell swelling condition was alleviated, and the renal tubular and collecting duct cells were more closely arranged. Compared with the MO group, the LAR-6 group and the HAR-6 group had more closely arranged renal tubular and collecting duct cells, reduced glomerular atrophy, interstitial inflammatory cell infiltration, and basement membrane thickening, and reduced large area vacuoles in the kidney. The kidney cells were uniformly distributed. In summary, high uric acid mediated glomerular sclerosis and inflammatory cell infiltration, causing damage to the structure of the glomerulus and renal tubule. AR-6 can effectively protect the structure of the glomerulus and renal tubule in the kidney and inhibit uric acid-mediated damage.

[0198] In addition, Masson staining was performed on the kidneys of mice with long-term high uric acid damage and AR-6 and positive drug gavage, and the results are shown in FIGS. 18A-18B

[0199] As can be seen from FIGS. 18A-18B , the CK group had no collagen fiber aggregation phenomenon, and the collagen fiber level was low. The cytoplasmic muscle fibers were uniformly distributed, and the cell nucleus structure was normal. In the MO group Masson staining pathological section, compared with the CK group, collagen fibers appeared to be aggregated (arrow mark), and the collagen fiber level increased by 1.89 times (p<0.001). The cytoplasmic muscle fibers were arranged abnormally, and the structure was not uniform. In the positive drug VC group mouse kidney Masson staining pathological section, compared with the MO group, the VC group had no collagen fiber aggregation phenomenon, and the total collagen fiber level decreased by 0.71 times (p<0.001). The cytoplasmic muscle fibers were arranged neatly and regularly. In the AR-6 gavage group mouse kidney Masson staining pathological section, compared with the MO group, the LAR-6 group had significantly reduced collagen fiber aggregation (arrow mark), and the total collagen fiber level decreased by 0.21 times (p<0.05). The cytoplasmic muscle fibers were arranged more neatly. The HAR-6 group had no collagen fiber aggregation phenomenon, and the total collagen fiber level decreased by 0.68 times (p<0.001). The cytoplasmic muscle fibers were arranged more evenly and regularly, and the cell nucleus structure was normal. The staining results were close to those of the positive drug VC group.

[0200] In summary, long-term high uric acid levels can induce abnormal increase in collagen fiber levels in mouse kidneys, abnormal cytoplasmic muscle fiber structure, uneven distribution, and further fibrosis damage. AR-6 can regulate the collagen fiber level in the proximal tubule of mouse kidneys, maintain the structure of cytoplasmic muscle fibers, and alleviate the fibrosis damage of the kidney.

[0201] The morphological characteristics of kidney damage mainly include tissue congestion, edema, and color change. By regularly observing the kidney structure of experimental mice, the modeling effect and the effect of positive drugs were analyzed, and the results are shown in​FIG. 19 As shown in FIG. 6, wherein the black scale is 1 cm.

[0202] From FIG. 19 It can be seen that at the beginning of the experiment, the kidneys of the CK group, the MO group and the VC group were dark brown, regular in shape, and had no congestion and edema. With the increase of the days of gavage, the kidneys of the MO group gradually appeared edema, and the congestion in the kidneys was obvious. After 14 days of the beginning of the experiment, the color of the kidneys of the MO group changed significantly compared with the CK group. The color of the kidneys of the MO group changed from red to bright red, and with the extension of the gavage time, the degree of edema and the degree of congestion deepened, and the bright red color increased. At the beginning of the experiment for 21 days, the surface of the kidneys of the MO group was rougher compared with the CK group. Compared with the MO group, the VC group did not appear congestion and edema with the increase of the days of gavage. Compared with the CK group, the state of the kidneys of the VC group was close, and there was no significant difference. Moreover, after 14 days of the beginning of the experiment, with the extension of the gavage time, the kidneys did not appear the phenomenon of changing from dark red to bright red, and the surface of the kidneys was smooth and the structure was complete. In summary, the high uric acid model of the MO group was successful, high uric acid could cause the destruction of the glomerular filtration membrane structure and interstitial congestion and edema, and then induce serious kidney damage. The positive drug VC could effectively reduce the uric acid level, inhibit the destruction of the glomerular structure of the mouse kidney by high uric acid and edema, protect the kidney from the damage induced by uric acid, and the results were consistent with the detection results of the serum kidney injury indicators.

[0203] To further verify the effect of high uric acid damage in mice and the role of AR-6 in inhibiting kidney damage, the kidney structure of the mice after 28 days of the beginning of the experiment was observed, and the results are shown in FIG. 8. FIG. 20 As shown in FIG. 8, wherein n = 10, and the black scale is 1 cm.

[0204] From FIG. 20It can be seen that in the kidney photos of the 28th day of the experiment, the kidney color of the MO group is significantly different from that of the CK group, the kidney color of the MO group mouse changes from red to bright red, the degree of edema is serious, the congestion is obvious, and the kidney surface is rough, and the kidney appears obvious light transmission phenomenon. While the kidney color of the VC group is dark red compared with the MO group, the degree of kidney edema and congestion is significantly reduced, and the kidney structure is more regular and the surface is smoother. By observing the photos of AR-6 gavage mice, it was found that the kidney edema of LAR-6 and HAR-6 groups was lower than that of the MO group, the congestion was relieved, the kidney structure was more regular and the surface was smoother, and with the increase of the concentration of polypeptide gavage, the degree of kidney congestion was lower and the kidney color was more inclined to the dark red healthy state. Therefore, AR-6 can inhibit the hyperuricemia-induced kidney congestion and edema, protect the kidney from the damage of hyperuricemia, and the protection effect is proportional to the concentration of AR-6. The results are consistent with the detection results of serum kidney damage indicators, which verify the protection effect of AR-6 on the kidney of model mice.

[0205] The dynamic changes of kidney cell mitochondrial structure are key morphological and functional indicators for evaluating oxidative stress and iron death related damage. The TEM photos of the kidney of the mice at the end of the experiment were analyzed to detect the changes of mitochondrial morphology and structure, and the results are shown in FIGS. 21A-21B

[0206] As can be seen from FIGS. 21A-21B It can be seen that the mitochondria of the MO group (high uric acid modeling group) mice are more damaged than the mitochondria of the CK group, and the proportion of damaged mitochondria in the MO group is significantly increased by 5.14 times (p<0.001) compared with the CK group, and the MO group has significant mitochondrial cristae rupture (yellow arrow mark) and membrane structure swelling (black arrow mark). The results show that high uric acid can induce mitochondrial cristae rupture, membrane structure swelling or shrinkage of kidney cells, induce oxidative stress and mitochondrial dysfunction, and further cause mitochondrial electron transport chain abnormalities, abnormal increase of ROS level, and further induce mitochondrial DNA damage and membrane potential collapse. The kidney of the HAR-6 group mice gavaged with high-dose AR-6 has complete mitochondrial structure, and the membrane structure swelling phenomenon is significantly reduced, and the mitochondrial cristae rupture disappears. The proportion of damaged mitochondria in the HAR-6 group is significantly reduced by 42.3% (p<0.001) compared with the MO group. The results show that AR-6 can inhibit the abnormality of kidney mitochondrial structure induced by high uric acid, inhibit oxidative stress, relieve mitochondrial electron transport chain abnormalities and dysfunction, mitochondrial DNA damage and membrane potential collapse, and further inhibit uric acid-mediated kidney damage.

[0207] ​Body weight loss is an important clinical indicator of organ damage in model animals, and its mechanism involves metabolic, inflammatory, and fluid regulation imbalance. The body weight of the mice was recorded every week until the end of the experiment, and the results are shown in FIG. 22 , where n = 10, expressed as median ± 95% CI.

[0208] From FIG. 22 It can be seen that the average body weight of the MO group mice decreased by 8.3% (p < 0.05) on day 21 compared to the CK group. The average body weight of the VC group was significantly increased by 7.1% on day 21 and by 8.3% on day 28 compared to the MO group (p < 0.05). The average body weight of the LAR-6 and HAR-6 groups was also significantly increased by 8.8% (p < 0.05) and 13.3% (p < 0.01) on day 21, and by 8.6% (p < 0.05) and 16.7% (p < 0.01) on day 28 compared to the MO group, respectively, and the body weight increase level was proportional to the AR-6 gavage concentration. Notably, the weight detection by the LMIX group found that the body weight of the LMIX group was significantly increased by 17.5% (p < 0.05) on day 28 compared to the MO group.

[0209] Analysis of the mechanism of AR-6 in vivo regulation of uric acid injury

[0210] To further analyze the mechanism of AR-6 in vivo inhibition of uric acid injury, the mouse kidney was subjected to transcriptome testing and omics analysis. The GO enrichment analysis results systematically revealed significant differences in molecular function between the experimental groups, and the results are shown in FIGS. 23A-23B , where n = 3.

[0211] From FIGS. 23A-23B It can be seen that the GO enrichment analysis results show that the model group (MO) presents significant differences in multiple functional categories compared to the control group (CK) FIG. 23A ). In the biological process (BP) aspect, the "extracellular matrix organization" related genes are significantly enriched; in the cell component (CC) level, the "extracellular matrix" related genes are up-regulated; the molecular function (MF) analysis shows that the "type 2 fibroblast growth factor receptor binding" related genes are highly active. These results indicate that uric acid may promote collagen deposition acceleration by abnormally activating the fibroblast proliferation signaling pathway, especially the MAPK / ERK pathway, thereby driving the progression of kidney fibrosis. This finding is highly consistent with the known pathological features of kidney fibrosis, providing molecular-level evidence for the mechanism of uric acid-induced kidney injury.

[0212] The results of the drug intervention group (HAR-6) further elucidate the kidney protection mechanism of AR-6 FIG. 23B ) At the molecular level, AR-6 exhibits multi-target regulation characteristics: the significant enrichment of "negative regulation of hh target transcription factor activity" in BP analysis (-log10(p) = 7) reveals its inhibitory effect on the Hedgehog signaling pathway, which may effectively inhibit the abnormal activation of myofibroblasts; at the same time, the expression profile changes of "response to alcohol" related genes confirm the potential of the drug to alleviate oxidative stress damage. It is particularly noteworthy that although the enrichment signal of the "extracellular matrix" pathway can still be detected in the CC analysis, the regulation intensity of "type 2 fibroblast growth factor receptor binding" at the MF level is significantly weakened, and this dose-dependent effect fully proves that AR-6 can dose-dependently block the excessive generation of ECM induced by uric acid. Based on these systematic experimental evidences, we can conclude that AR-6 achieves precise intervention on the pathological process of the kidney through a carefully designed dual-acting mechanism, both antagonizing the ferroptosis process by regulating the iron metabolism related gene network and delaying the fibrosis progression through a multi-pathway synergistic inhibition strategy.

[0213] To further understand the action pathway of AR-6 in uric acid-mediated kidney injury, KEGG pathway analysis was performed, and the results are shown in FIGS. 24A-24B , where n = 3.

[0214] As can be seen from FIGS. 24A-24B , compared with the control group (CK), the PPAR signaling pathway (Rich factor = 0.12, p = 0.004) and the MAPK signaling pathway (p = 0.008) are significantly enriched in the model group (MO) FIG. 24A . The abnormality of the PPAR pathway suggests that uric acid exacerbates oxidative stress and lipid peroxidation by inhibiting antioxidant defense (such as down-regulation of SOD, GPX4 expression), thereby activating ferroptosis; the enrichment of the MAPK pathway is related to the overexpression of pro-fibrotic factors such as TGF-β, driving ECM deposition (such as collagen I / III). After drug group (HAR-6) intervention FIG. 24BThe PPAR pathway remained enriched (Rich factor = 0.10, p = 0.012), but this was accompanied by a significant upregulation of the oxidative phosphorylation pathway (p = 0.021), suggesting that AR-6 may enhance antioxidant capacity (e.g., promoting the Nrf2 / HO-1 pathway) by activating PPARγ, while simultaneously repairing mitochondrial function (restoring ATP synthesis and reducing ROS accumulation), thereby inhibiting key ferroptosis events (e.g., ACSL4 downregulation and GPX4 upregulation). Furthermore, the enrichment of the MAPK pathway in the MO group was significantly reduced in the HAR-6 group (p = 0.008 to 0.15), indicating that AR-6 inhibits TGF-β / Smad3 phosphorylation by blocking the MAPK / ERK signaling axis, reducing fibroblast activation and collagen synthesis (downregulation of COL1A1 and FN1 expression). In summary, AR-6 exerts its nephroprotective effect through a dual mechanism: 1) it regulates the PPAR-oxidative phosphorylation axis to improve iron metabolism homeostasis and antagonize ferroptosis; 2) it inhibits the MAPK-TGF-β signaling network to block the fibrosis process.

[0215] AR-6 alleviates dysregulation of the Nrf2 / GPX4 pathway transcriptional level in the kidneys of hyperuricic mice.

[0216] To further validate the in vivo mechanism of action of AR-6 predicted by transcriptome analysis, RT-qPCR tests were performed on typical ferroptosis and fibrosis genes in mouse kidneys. The results are as follows: FIGS. 25A-25D As shown.

[0217] Depend on FIGS. 25A-25D As can be seen from the gene transcription results, compared with the CK group, the expression levels of renal cell ferroptosis activator Keap-1 (p<0.001) and ACSL4 (p<0.001) were significantly increased in the MO group. FIG. 25A , FIG. 25D The expression levels of ferroptosis regulatory genes Nrf-2 (p<0.001) and GPX4 (p<0.001) were significantly decreased, indicating at the gene transcription level that high uric acid mediates ferroptosis damage in kidney cells. FIG. 25B , FIG. 25C The results were consistent with transcriptome analysis. Compared to the MO group, the HAR-6 group showed a significant decrease in Keap-1 expression (p<0.001), approaching the level of the control group; the expression levels of ferroptosis regulators Nrf-2 (p<0.001) and HO-1 (p<0.001) were significantly increased, with transcriptional levels significantly higher than those of the mixed giant salamander bone peptide, even exceeding those of normal cells in the control group; the expression level of the ferroptosis-promoting gene ACSL4 (p<0.001) was also significantly decreased in the HAR-6 group. These results indicate that AR-6 inhibited the abnormal expression of ferroptosis genes mediated by intracellular hyperuricemia, alleviating the intracellular ferroptosis process.

[0218] AR-6 modulates the transcription level of uric acid-mediated TGF-β1 / SMAD3 pathway

[0219] Transcriptome analysis predicted that AR-6 could alleviate the occurrence of fibrosis by regulating kidney TGF-β fibrosis initiation genes, so RT-qPCR tests were performed on typical fibrosis genes, and the results are shown in FIGS. 26A-26B .

[0220] From FIGS. 26A-26B It can be seen that the expression levels of fibrosis activation genes TGF-β1 (p<0.001) and SMAD3 (p<0.001) in the MO group were significantly increased compared with the control group; the expression levels of fibrosis activation genes TGF-β1 (p<0.001) and SMAD3 (p<0.001) in the HAR-6 group were significantly decreased compared with the MO group, which was significantly better than the mixed Andrias davidianus bone peptide, and the level of fibrosis gene was close to the level of the CK group. The results show that high uric acid can mediate the expression of mouse kidney fibrosis activation genes, and AR-6 can effectively alleviate the disorder of fibrosis factors mediated by high uric acid.

[0221] Metabolome predicts AR-6 mechanism for relieving uric acid-induced kidney damage in vivo results

[0222] In order to further analyze the protective effect of AR-6 on the kidney of high uric acid mice, the feces of mice were subjected to metabolomic analysis, and the results are shown in FIGS. 27A-27C .

[0223] From FIGS. 27A-27C It can be seen that according to the results of short-chain fatty acid targeted metabolomic analysis, it was found that compared with the CK group, 7 kinds of short-chain fatty acids (SCFAs) in the MO group showed significant metabolic disorder (Caproic acid, Valeric acid, etc. The signal intensity difference is 2.3-4.1 times), revealing that hyperuricemia leads to intestinal flora metabolic dysfunction, and exacerbates the imbalance of kidney microenvironment through the intestinal-kidney axis Figure 27A ). It is worth noting that after AR-6 intervention, the SCFAs spectrum of the HAR-6 group was significantly adjusted to the level of the CK group (such as Butyric acid recovered 85.6%, p<0.01) Figure 27A ). According to the KEGG metabolite molecular network analysis of the HAR-6 group and the MO group Figure 27C), AR-6 intervention significantly reversed the metabolic disorder induced by high uric acid. The down-regulated short-chain fatty acids (SCFAs) and related metabolites (such as acetic acid, butyric acid, propionic acid, with log2FC negative value) in MO group were dose-dependently up-regulated in HAR-6 group, especially the "propionic acid metabolism" pathway, suggesting that AR-6 might restore the intestinal-renal metabolic axis homeostasis by promoting SCFAs production or enhancing the activity of its receptors (such as FFAR2 / GPR43). At the same time, the abnormal activation of pathological pathways (such as "taurine and hypotaurine metabolism" "alcoholic liver disease") in MO group was attenuated in HAR-6 group, indicating that drug intervention effectively alleviated oxidative stress and lipid metabolism imbalance. Combined with the metabolic network characteristics of MO group and CK group, hyperuricemia might destroy the intestinal barrier integrity and weaken the antioxidant capacity (such as limited glutathione synthesis), thereby activating ferroptosis (such as enhanced lipid peroxidation reaction, GPX4 inhibition) and TGF-β / Smad-driven fibrosis process. While AR-6 might achieve kidney protection through the following dual mechanisms by restoring SCFAs levels: 1) activating the SCFAs-FFAR2 pathway to up-regulate antioxidant genes (such as GPX4, Nrf2) and inhibit lipid ROS accumulation and ferroptosis; 2) indirectly blocking the TGF-β signal activation induced by endotoxin by improving intestinal barrier function, thereby reducing the abnormal response of fibrosis-related pathways (such as EMT process).

[0224] Results of intestinal flora predicting the mechanism of AR-6 in vivo to alleviate uric acid-induced kidney injury

[0225] Intestinal microflora is one of the important factors for regulating fecal metabolites. In order to further analyze the protective effect of AR-6 on hyperuricemic mice, intestinal flora detection was performed. Based on PCoA analysis of intestinal microflora 16s detection, the results are shown in Figures 28A-28F

[0226] As can be seen from Figures 28A-28F , the flora distribution of the MO group deviates from that of the CK group, indicating that high uric acid stress leads to intestinal microecological imbalance; while the flora distribution of the HAR-6 group partially recovers to the vicinity of the CK group, indicating that AR-6 gavage can partially reverse the flora disorder induced by high uric acid and restore homeostasis.

[0227] In the MO group, the relative abundance of Firmicutes (related to SCFAs production) decreased, while the abundance of Proteobacteria (potential pathogenic bacteria) increased, suggesting that hyperuricemia might destroy the intestinal barrier integrity and induce inflammatory response; in the HAR-6 group, the abundance of Firmicutes recovered, and the abundance of Proteobacteria decreased, indicating that the drug improved intestinal homeostasis by regulating flora composition Figure 28B ​). The abundance of Bacteroidetes was significantly increased (p<0.05) and the F / B ratio was decreased in the MO group, which was consistent with the characteristics of metabolic disorders; the F / B ratio was partially restored in the HAR-6 group (p<0.05), suggesting that the drug might alleviate uric acid toxicity by regulating energy metabolism-related flora Figure 28D ). The abundance of Lactobacillus (probiotics) and Lachnospiraceae (butyric acid-producing bacteria) was significantly reduced, while the abundance of Escherichia (opportunistic pathogens) was enriched in the MO group; the above trends were partially reversed in the HAR-6 group, especially the abundance of Lactobacillus, suggesting that AR-6 might promote SCFAs production by restoring the abundance of beneficial bacteria, thereby inhibiting uric acid-induced systemic inflammation and kidney damage Figure 28C ). The abundance of Akkermansia (mucin-degrading bacteria) was increased in the MO group, which might exacerbate intestinal barrier damage; while the abundance of Bifidobacterium (anti-inflammatory probiotics) was significantly increased (p<0.01) in the HAR-6 group, suggesting that AR-6 might reduce endotoxin translocation and kidney inflammatory response by enhancing the protective function of intestinal mucosa Figure 28E ). Linear discriminant effect size analysis showed that Gammaproteobacteria and Enterobacterales were significantly enriched (LDA score>4.0) in the MO group, suggesting that hyperuricemia might promote the proliferation of opportunistic pathogens; while Clostridiales and Lachnospiraceae became the marker taxon (LDA score>3.5) in the HAR-6 group, which could activate the Nrf2 / HO-1 pathway by secreting SCFAs such as butyric acid, thereby inhibiting lipid peroxidation and ferroptosis Figure 28F ). In summary, AR-6 provides a microbiome basis for inhibiting uric acid-mediated kidney damage by targeting intestinal flora homeostasis and rebuilding metabolic-immune regulatory networks.

[0228] Results of AR-6 inhibiting intestinal damage in hyperuricemic mice

[0229] Intestinal flora plays a key role in maintaining intestinal mucosal homeostasis, and its imbalance often indicates damage to the structural integrity of the intestinal tract. Since hyperuricemia also mediates metabolic organ damage related to the kidney, such as the liver and ileum, the regulatory effect of andrographolide on body weight may be achieved by inhibiting hyperuricemia-induced damage to other organs related to the kidney. Therefore, it is necessary to analyze the inhibitory effect of andrographolide on damage to organs related to the kidney, such as the liver and ileum, and the results are shown in Table 2. Figure 29 ​

[0230] By Figure 29 As can be seen in the CK group of small intestine H&E staining pathology sections, ileum cell villus structure is complete, regular, good uniformity, crypt structure is complete, no large area of epithelial cell shedding and lamina propria inflammatory cell infiltration. Compared with the ileum of CK group mice, the ileum of MO group mice villi appear significant atrophy or fracture, most of the villus structure is abnormal (arrow mark), MO group ileum villus crypt structure is disorder, MO group ileum epithelial cell shedding and lamina propria inflammatory cell infiltration. The results show that high uric acid can destroy the ileum epithelial villus structure, cause villus fracture, crypt structure disorder, and a large number of inflammatory infiltration, and cause serious ileum tissue damage. In the VC group of ileum H&E staining pathology sections, compared with the MO group, the ileum epithelial villus structure abnormality of the VC group is relieved, the phenomenon of villus fracture or loss is significantly reduced, the crypt structure is more complete, and the inflammatory cell infiltration is significantly reduced. The results show that the positive drug VC can inhibit the destruction of high uric acid to the ileum appearance structure, reduce the crypt structure disorder, the occurrence of tissue inflammation infiltration, and inhibit the damage of high uric acid to the ileum tissue. In the H&E staining sections of the ileum of mice gavaged with mixed andaman barb bone peptides, the ileum epithelial cell structure of mice gavaged with mixed andaman barb bone peptides is more complete, and the ileum villus of LAR-6 group, HAR-6 group, LMIX group and HMIX group is significantly lower than that of MO group. After gavage with mixed andaman barb bone peptides and AR-6 single peptide, the ileum villus is arranged in order, the uniformity is good, the crypt structure is more complete, the epithelial cell shedding and lamina propria inflammatory cell infiltration are significantly reduced, and the inhibition effect on ileum villus structure damage is proportional to the increase of andaman barb bone peptide concentration. The results show that andaman barb bone mixed peptide and AR-6 single peptide can inhibit the ileum epithelial villus structure damage caused by high uric acid, inhibit inflammatory cell infiltration and epithelial cell shedding, and protect the ileum from damage. In summary, high uric acid can mediate ileum epithelial villus cell structure damage, induce severe intestinal villus loss and inflammatory cell infiltration, and further induce ileum cell damage. Andaman barb bone mixed peptide and AR-6 single peptide can gradiently inhibit high uric acid mediated ileum epithelial villus structure damage, reduce the number of neutrophils and lymphocytes in lamina propria, protect crypt structure, and inhibit the damage of high uric acid to ileum.

[0231] Andaman barb bone peptide inhibits liver damage in high uric acid mice

[0232] By analyzing the H&E staining pathology sections of experimental mice, the efficacy of andaman barb bone peptide in inhibiting uric acid induced liver damage was detected, and the results are shown in Figures 30A-30B

[0233] By Figures 30A-30B ​As can be seen, in the CK group, the kidney H&E staining section showed less neutrophil infiltration, uniform cell nucleus size, uniform cytoplasm staining, less liver cavity, and intact liver cell structure. Compared with the CK group, the MO group showed a large number of neutrophil infiltration (arrow mark), abnormal cell nucleus enlargement, uneven cytoplasm staining, more intercellular cavity, and obvious liver structure damage. The results showed that the liver cells were induced by high uric acid to cause inflammatory infiltration, which triggered liver damage. Compared with the MO group, the VC group showed significantly reduced neutrophil infiltration (arrow mark), normal central vein structure, more uniform cell nucleus size, uniform cytoplasm staining, reduced intercellular cavity, and intact liver cell structure. The results showed that the positive drug VC could reduce high uric acid-mediated inflammatory infiltration, thereby inhibiting liver damage. In the liver H&E staining section of the mice gavaged with Megalobatrachus knochi peptides, the LAR-6 group, the HAR-6 group, the low-dose mixed peptide group (LMIX), and the high-dose mixed peptide group (HMIX) showed significantly less neutrophil infiltration in the liver than the MO group, uniform cytoplasm staining, intact central vein structure, less liver cavity, and more complete liver cell structure. Moreover, as the gavage concentration increased, the liver neutrophil infiltration decreased, the cytoplasm staining increased, and the mixed Megalobatrachus knochi peptide and AR-6 monopeptide gavage concentration was inversely proportional to the degree of liver inflammatory infiltration and damage. Notably, the HMIX group and the HAR-6 group had similar effects on relieving inflammatory infiltration (p<0.05). The results showed that the mixed Megalobatrachus knochi peptide and AR-6 monopeptide could gradiently inhibit the occurrence of high uric acid-mediated liver inflammatory infiltration and structural damage. In summary, high uric acid can mediate severe inflammatory infiltration of liver cells, thereby inducing liver damage. Megalobatrachus knochi peptides can inhibit high uric acid-mediated inflammatory response, protect the central vein structure, and inhibit liver damage. High-concentration AR-6 monopeptide has the best regulatory effect on liver inflammatory infiltration.

[0234] The efficacy of mixed Megalobatrachus knochi peptides in inhibiting uric acid-induced liver damage was detected by analyzing the Masson staining pathological sections of the experimental mice, and the results are shown in Figures 31A-31B .

[0235] Figures 31A-31B ​As can be seen in the CK group, the liver Masson staining pathology section shows that the liver cell collagen fiber level is low, the cytoplasm muscle fiber and collagen fiber are evenly distributed, and the fibrosis level of the cells around the central vein is low. Compared with the CK group, the liver of the MO group has a significant increase of 4.23 times (p<0.001) in the liver cell collagen fiber level, the cytoplasm muscle fiber and collagen fiber are unevenly distributed, and there is significant fibrosis around the central vein. The MO group has more cavities in the liver cells. The MO group has poor homogeneity of the liver cell nucleus and cytoplasm, and has a significant decrease in the matrix density. The results show that high uric acid induces the liver cells to produce a large amount of collagen fiber, causing abnormal structure of the liver cells, decrease in the mechanism density, and causing serious liver damage. In the VC group, the liver Masson staining pathology section shows that the collagen fiber in the liver cells of the VC group is significantly reduced by 73.1% (p<0.001) compared with the MO group, and the muscle fiber and collagen fiber in the liver cells are evenly distributed, and the fibrosis level of the cells around the central vein is low. The results show that the positive drug VC can inhibit the abnormal collagen fiber level and cytoplasm muscle fiber and collagen fiber distribution caused by high uric acid, and inhibit the occurrence of fibrosis. In the liver H&E staining section of the mice administered with the mixed Andrias davidianus bone peptides, the liver collagen fiber level of the mice administered with the Andrias davidianus bone is low, and the collagen fiber level of the LAR-6 group, HAR-6 group, LMIX group and HMIX group is significantly reduced by 24.5% (p<0.01), 66.1% (p<0.001), 18.7% (p<0.05) and 39.8% (p<0.001) respectively compared with the MO group. The muscle fiber and collagen fiber in the liver of the LMIX group and HMIX group are evenly distributed, the fibrosis level of the cells around the central vein is low, and the inhibition effect of kidney fibrosis is proportional to the increase of the concentration of the mixed Andrias davidianus bone peptides. The results show that the mixed Andrias davidianus bone peptides can inhibit the abnormal collagen fiber level and cytoplasm muscle fiber and collagen fiber distribution caused by high uric acid, and inhibit the occurrence of fibrosis, and the inhibition effect of the HMIX group is close to that of the HAR-6 group. In summary, high uric acid can mediate the liver cells to have serious collagen fiber expression, and then cause irregular distribution of the cytoplasm muscle fiber and collagen fiber, and then induce liver fibrosis damage. The mixed Andrias davidianus bone peptides and AR-6 single peptide can both gradiently inhibit the fibrosis damage mediated by high uric acid, reduce the fibrosis level around the central vein, inhibit the liver damage, and the AR-6 has the best regulation effect at a high concentration.

[0236] The description of the present disclosure is given for the purpose of illustration and description, but not exhaustive or limiting the present disclosure to the disclosed form. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are chosen and described in order to better illustrate the principles and practical application of the present disclosure, and to enable those of ordinary skill in the art to understand the present disclosure in order to design various embodiments with various modifications for specific use.

Claims

1. The application of a polypeptide in the preparation of drugs to improve kidney injury, wherein, The polypeptide sequence comprises an amino acid sequence as described in any one of SEQ ID NOs:1-10 or an amino acid sequence as shown in any one of SEQ ID NOs:1-10.

2. The application according to claim 1, wherein the kidney injury includes kidney injury caused by hyperuricemia.

3. The application of a polypeptide in improving kidney injury, wherein, The polypeptide sequence comprises an amino acid sequence as shown in any one of SEQ ID NOs:1-10 or an amino acid sequence as shown in any one of SEQ ID NOs:1-10.

4. The application according to claim 3, wherein the kidney injury includes kidney injury caused by hyperuricemia.

5. A medicament for improving kidney injury, comprising an amino acid sequence as shown in any one of SEQ ID NOs:1-10 or an amino acid sequence as shown in any one of SEQ ID NOs:1-10.

6. The drug according to claim 5, wherein the kidney injury comprises kidney injury caused by hyperuricemia.

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