Application of urolithin D in preparation of medicine for relieving hyperuricemia

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of urolithin D in preparation of medicine for relieving hyperuricemia. It is found for the first time that the urolithin D can significantly reduce the level of serum uric acid, improve the expression disorder of a kidney urate transporter, efficiently relieve or treat hyperuricemia and related diseases thereof, and relieve kidney injury and fibrosis; and the compound can be used for preparing medicines for treating hyperuricemia and related diseases, is low in cost and good in safety, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of urolithin D in the preparation of drugs to alleviate hyperuricemia. Background Technology

[0002] Urolithin D is a bioactive metabolite derived from ellagitannins, with the molecular formula C2. 13 H8O6, with a molecular weight of 260.2, is a white solid. It is a reversible antagonist of the competitive and selective EphA receptor.

[0003] Current research indicates that urolithin D has the potential to be used in the preparation of drugs for treating autoimmune diseases and immune rejection after organ / tissue transplantation. It also inhibits EphA2 phosphorylation in prostate cancer cells, specifically inhibits the catalytic activity of ppGalNAc-T enzymes, inhibits O-GalNAc glycosylation of tumor-associated proteins, and alters the migration and invasion abilities of colorectal cancer cells. However, there are currently no records of studies on the use of urolithin D in alleviating hyperuricemia. Summary of the Invention

[0004] To address the research and development gaps in existing technologies, this invention provides the application of urolithin D in the preparation of drugs to alleviate hyperuricemia.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides the use of urolithin D in the preparation of medicaments for treating or alleviating hyperuricemia and / or hyperuricemia-related diseases.

[0006] Preferably, the hyperuricemia-related diseases include gout, gouty arthritis, or uric acid nephropathy.

[0007] Preferably, the drug is used to lower serum uric acid levels.

[0008] Preferably, the drug is used to improve disordered expression of renal uric acid transporters.

[0009] Preferably, the drug is used to alleviate kidney damage and fibrosis.

[0010] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0011] Preferably, the drug dosage form provided by the present invention can be prepared into any dosage form acceptable in the clinical field, including but not limited to powder, tablet, pill, granule, capsule, solution, emulsion, suspension or oil.

[0012] The beneficial effects of this invention are as follows: This invention is the first to discover that urolithin D can significantly reduce serum uric acid levels, improve the disordered expression of renal uric acid transporters, and can effectively relieve or treat hyperuricemia and its related diseases, and alleviate kidney damage and fibrosis; it can also be used to prepare drugs for the treatment of hyperuricemia and its related diseases, and is low in cost, safe and has broad application prospects. Attached Figure Description

[0013] Figure 1 Urolithiasis D dose-dependently reduced serum uric acid levels in hyperuricemic mice. (A) Establishment and administration of the hyperuricemic mouse model. (B) Mouse body weight. (C) Kidney organ coefficient. (D) Serum uric acid level. (E) Urinary uric acid (UUA) level. (F) Fractional uric acid excretion (FEUA) level. n ≥ 5. # p <0.05, ## p <0.01, ### p <0.001, compared with the control group; p <0.05, p <0.01, p <0.001, compared to the model group; Figure 2 Urolithiasis-induced renal uric acid transporter expression disorder. Western blot analysis (A) and protein quantification (B) of URAT1, GLUT9, ABCG2, and OAT1 in mouse kidneys. Western blot analysis (C) and protein quantification (D) of URAT1, GLUT9, ABCG2, and OAT1 in HK-2 cells. n = 3. # p <0.05, ## p <0.01, ### p <0.001, compared with the control group; p <0.05, p <0.01, p <0.001, compared to the model group; Figure 3 Urolithin D improves kidney injury in hyperuricemic mice. (A) Kidney appearance. (B) Serum creatinine level. (C) Serum urea nitrogen level. (D) Urinary creatinine level. (E) H&E staining of the kidney. Scale bar: 100 μm. Blue arrow: renal tubule; black arrow: glomerulus. (F) Kidney histopathological score. (G) Masson staining. (H) Quantitative analysis of interstitial collagen deposition. n≥ 5. # p <0.05, ## p<0.01, ### p <0.001, compared with the control group; p <0.05, p <0.01, p <0.001, compared to the model group; Figure 4 Urolithiasis D improves renal fibrosis induced by hyperuricemia. Immunoblotting (A) and quantitative analysis of α-SMA, TGF-β, and E-Cad proteins in the kidneys of hyperuricemic mice (B). Immunoblotting (C) and quantitative analysis of α-SMA, TGF-β, and E-Cad proteins in HK-2 cells (D). n = 3. # p <0.05, ## p <0.01, ### p<0.001, compared with the control group; p <0.05, p <0.01, p <0.001, compared to the model group. Detailed Implementation

[0014] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0015] Example 1 1. Experimental Methods: 1.1 HK-2 cell culture and modeling HK-2 cells (human renal tubular epithelial cells) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Cells were cultured in MEM medium supplemented with 10% fetal bovine serum and 1% penicillin + streptomycin in a 5% CO2 incubator. A hyperuricemia cell model was established by stimulating HK-2 cells with 100 mg / L uric acid for 48 h.

[0016] 1.2 Establishment and administration of a mouse model of hyperuricemia Male Kunming mice (18-22 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. After 7 days of acclimatization feeding under alternating light and dark conditions, experiments were conducted. The experimental protocol and procedures for the mice were performed in accordance with the approval of the Animal Research Ethics Committee of Shandong Academy of Agricultural Sciences (No. SAAS-2022-SD03).

[0017] Animal experimentation procedures are as follows Figure 1 As shown in Figure A: After 7 days of adaptive feeding, all mice were randomly divided into two groups: a normal control group (n=10) and a model group (n=30). The model group mice were used to establish a hyperuricemia mouse model by gavage administration of 300 mg / kg / d potassium oxonate (PO) and 200 mg / kg / d adenine (AD). To prepare the solution, potassium oxonate and adenine were finely ground in a mortar and then suspended in a 0.5% sodium carboxymethyl cellulose (CMC-Na) solution. The normal control group mice were administered an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage. All mice were administered the solution once daily at 9:00 AM. Two weeks after administration, three mice were randomly selected from each of the normal and model groups to measure their serum uric acid levels. A significantly higher serum uric acid level in the model group compared to the normal group indicated successful establishment of the hyperuricemia mouse model.

[0018] After successfully establishing the hyperuricemia model, the mice were randomly divided into 5 groups (n=10 per group): Model group, benzbromarone (BenZ) group, low-dose urolithin D group (10 mg / kg / d), medium-dose urolithin D group (50 mg / kg / d), and high-dose urolithin D group (100 mg / kg / d). Each group was administered the corresponding drug by gavage once daily for two weeks. Animal grouping and drug administration are shown in Table 1. Table 1 Animal grouping and administration 1.3 Detection of physiological and biochemical indicators in mice Twenty-four hours after the last administration, the mice were fasted for 12 hours (with free access to water). The weight of the mice was weighed and recorded using an electronic balance. After recording the weight, blood samples were collected by enucleation. After standing at room temperature for 30 minutes, the samples were centrifuged at 3000 × rpm for 10 minutes at 4°C. The serum was separated and aliquoted into pre-cooled RNase-free EP tubes and stored at -80°C for later use.

[0019] After blood collection, the experimental mice were euthanized by cervical dislocation, and then abdominal dissection was performed: the left and right kidneys were carefully separated and removed with surgical forceps, weighed and recorded; the capsule on the kidneys was carefully removed and placed in liquid nitrogen for quick freezing; after the tissues were collected, they were transferred to a -80℃ freezer for cryopreservation.

[0020] Kidney organ coefficient: kidney weight (g) / body weight (g). Serum uric acid (SUA), serum creatinine (SCr), blood urea nitrogen (BUN), urinary uric acid (UUA), and urinary creatinine (UCr) levels were measured strictly according to the kit instructions. Uric acid excretion fraction (FEUA): (SCr × UUA) / (SUA × UCR) × 100%.

[0021] 1.4 Western Blot (1) Protein extraction from HK-2 cells and mouse kidney tissue: After cell culture, add an appropriate amount of RIPA lysis buffer (containing 1% protease inhibitor) and lyse on ice for 10 min. Collect the cell lysis buffer, centrifuge at 12000 rpm for 15 min at 4°C, and collect the supernatant for later use.

[0022] Accurately weigh 50 mg of kidney tissue using a balance, and add RIPA lysis buffer (containing 1% protease inhibitor) at a weight (g):volume (mL) ratio of 1:9. Homogenize the tissue thoroughly in a glass homogenizer until fully homogenized. The entire process should be performed on ice. Centrifuge the prepared tissue homogenate at 12000 rpm for 15 min at 4°C and collect the supernatant for later use.

[0023] (2) Detection and quantification of protein content: The protein content in cells and tissues was determined strictly according to the instructions of the protein quantification (BCA) kit. The protein concentration of each sample was diluted to a uniform concentration by adding 5× Loading buffer and PBS, and heated in a metal bath at 100℃ for 10 min, and stored at -80℃ for later use.

[0024] (3) Gel preparation: Before the experiment, clean the glass plate with tap water and rinse it several times with deionized water, then dry it in an oven for later use. Clamp the 1.5 mm glass plate tightly onto the gel preparation mold (note that the shorter plate is on the outside and the longer plate is on the inside), and prepare the PAGE gel according to the instructions of the One-Step PAGE Gel Fast Preparation Kit (10%) (Nanjing Novizan). After the gel solidifies, proceed with the subsequent experiments.

[0025] (4) Electrophoresis: Assemble the prepared PAGE gel (short plate inward, long plate outward) and place it in the electrophoresis tank (note the difference between positive and negative electrodes). Pour in 1× electrophoresis buffer diluted with deionized water, slowly pull out the plastic comb, and slowly add 30 µg of protein sample to each well. Connect the electrophoresis apparatus, adjust the voltage to 120 V, and perform electrophoresis at a constant voltage.

[0026] (5) Transfer: Prepare 1× transfer buffer in advance at a volume ratio of 7 (deionized water): 2 (anhydrous methanol): 1 (10× transfer buffer), and pre-cool at 4 ℃ for later use. Activate the PVDF membrane by soaking it in anhydrous methanol for 2 min, and then equilibrate it in 1× transfer buffer for 5 min. Arrange the membrane on the transfer clamp in the following order: sponge-two layers of filter paper-PAGE gel-membrane-two layers of filter paper-sponge (gel on the black clamp side, membrane on the white clamp side). After clamping the clamp tightly, place it in the electrophoresis tank and slowly pour in the transfer buffer. Adjust the current to 250 mA and perform the transfer at a constant current in an ice-water bath. Set the transfer time according to the molecular weight of the target band.

[0027] (6) Milk sealing: Add 5% milk sealing solution (5 g skim milk powder dissolved in 100 mL of 1 × TBST) to the incubation box, place the PVDF membrane in the incubation box, and seal it at room temperature on a shaker for 1 h.

[0028] (7) Binding primary antibody: After blocking, discard the blocking solution, wash the PVDF membrane 3 times with 1 × TBST for 10 min each time. After the last wash, discard the washing solution, add the primary antibody corresponding to the target protein (according to the antibody instructions, dilute the antibody corresponding to the target protein to the appropriate ratio with 1 × TBST in advance), and incubate overnight at 4°C.

[0029] (8) Binding to secondary antibody: After the primary antibody incubation is completed, wash the PVDF membrane three times with 1 × TBST for 10 min each time. Add the corresponding secondary antibody (select the appropriate secondary antibody according to the primary antibody instructions, and dilute the secondary antibody to the appropriate ratio with 1 × TBST in advance according to the secondary antibody instructions), and incubate on a shaker at room temperature for 2 h.

[0030] (9) Development: After the secondary antibody incubation, the PVDF membrane was washed 3 times with 1 × TBST for 10 min each time. ECL luminescent solution was prepared at a ratio of 1:1, mixed and slowly added to the PVDF membrane, placed in a chemiluminescence imaging system for development, and photographed for record.

[0031] (10) Image analysis: Image-Pro Plus software was used to perform grayscale analysis on the target protein bands, and GraphPad Prism 9.5.1 software was used to draw the graphs.

[0032] 1.5 Pathological examination of kidney and intestinal tissues (1) Sectioning: Kidney and intestinal tissues from each treatment group were fixed in 4% paraformaldehyde solution for 48 h. The kidneys and intestinal tissues were removed from the fixative and cut according to the largest cross-section. The cut mouse kidney and intestinal tissue blocks were dehydrated using 80%, 90%, 95%, 100% ethanol I, 100% ethanol II, and 100% ethanol III, respectively. Subsequently, they were soaked in xylene I for 45 min, xylene II for 45 min, paraffin I for 1 h, and paraffin II for 16 h. Following the principle of specimen facing downwards, the mouse kidney and intestinal tissues were embedded in paraffin and placed at room temperature to allow the paraffin blocks to cool and solidify before being refrigerated at -20℃. Paraffin sections were prepared using a rotary microtome, with a section thickness of 4 μm, and placed in a 65℃ incubator for 6–12 h. After packaging, they were stored at room temperature.

[0033] (2) H&E staining: The prepared sections were soaked in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 80% ethanol for 5 min, and then rinsed and soaked in tap water for 1 min for dewaxing and rehydration. Immerse the slides in hematoxylin staining solution at room temperature for 5 minutes, then rinse with tap water for 1 minute. Next, immerse the slides in 1% hydrochloric acid alcohol solution for a few seconds, then rinse with tap water until the tissue turns blue. Finally, immerse the slides in eosin staining solution for 3-5 minutes, then rinse with tap water to remove any excess stain. After staining, treat the slides with 80% ethanol for 0.5 minutes, 95% ethanol I for 0.5 minutes, 95% ethanol II for 0.5 minutes, anhydrous ethanol I for 0.5 minutes, anhydrous ethanol II for 0.5 minutes, xylene I for 3 minutes, and xylene II for 3 minutes. Then, remove the slides, mount them with neutral resin, and observe the tissue sections under an inverted microscope (Nikon TS2, Japan) at 200x magnification.

[0034] 2 Results and Analysis 2.1 Urolithin D dose-dependently reduced serum uric acid levels in hyperuricemic mice Figure 1 A illustrates the construction and drug administration process of a mouse model of hyperuricemia. The trend of mouse body weight change is as follows: Figure 1 As shown in BC, the body weight of mice in the model group decreased significantly. Benzbromarone and urolithiasis D could restore body weight and kidney organ coefficient to varying degrees. Compared with the control group (84.25 ± 38.56 μmol / L), the serum uric acid level in the model group was significantly increased to 194.44 ± 44.79 μmol / L, while benzbromarone could reduce it to 119.44 ± 30.98 μmol / L. Figure 1The presence of urolithin D indicates that the hyperuricemia model was successfully established. Urolithin D dose-dependently reduced serum uric acid levels in hyperuricemic mice. The serum uric acid levels in the low, medium, and high dose urolithin D groups decreased to 75.56 ± 16.00 µmol / L, 56.48 ± 14.66 µmol / L, and 50.93 ± 11.87 µmol / L, respectively. Figure 1 D). Notably, serum uric acid levels in mice treated with urolithin D were significantly lower than those in the benzbromarone group. Compared to the control group, urinary uric acid levels in hyperuricemic mice were significantly decreased, and urolithin D had no significant regulatory effect on these levels. Figure 1 E). Furthermore, benzbromarone and urolithiasis D significantly restored the abnormal decrease in uric acid excretion fraction caused by hyperuricemia (E). Figure 1 F). These data indicate that urolithin D can restore serum uric acid levels in hyperuricemic mice by promoting renal uric acid excretion.

[0035] 2.2 Urolithiasis-induced renal uric acid transporter expression disorder by urolithiasis D Renal uric acid excretion depends on specific uric acid transporters. Therefore, this invention uses a hyperuricemic mouse model and a uric acid-induced HK-2 cell model to investigate the effects of urolithin D on the expression of these transporters. Figure 2 AB analysis showed that in in vivo experiments, the expression of uric acid reabsorption proteins URAT1 and GLUT9 in the kidneys of model mice was significantly upregulated, while the expression of uric acid secretion proteins ABCG2 and OAT1 was significantly decreased. This result indicates that uric acid reabsorption is enhanced in the kidneys of hyperuricemic mice, while uric acid excretion is reduced, leading to elevated serum uric acid levels. Compared with the model group, after intervention with urolithin D, the expression of URAT1 and GLUT9 in the kidneys of mice was significantly downregulated in a dose-dependent manner, while the expression of ABCG2 and OAT1 was significantly upregulated in a dose-dependent manner. These results suggest that urolithin D can promote renal uric acid excretion in hyperuricemic mice and restore their blood uric acid levels by regulating the expression of uric acid reabsorption and uric acid secretion proteins in the kidneys.

[0036] Renal uric acid transporters are mainly expressed in the renal tubules; therefore, human renal tubular epithelial cells HK-2 were used for in vitro experiments to further verify the effect of urolithin D on uric acid transporter expression. The results are as follows: Figure 2 As shown in CD, treatment with 100 mg / L uric acid for 48 h significantly increased the protein expression of URAT1 and GLUT9 in HK-2 cells, while significantly decreasing the expression of ABCG2 and OAT1, indicating successful model construction. Urolithin D significantly improved this change in a clear concentration-dependent manner, further demonstrating that urolithin D can improve hyperuricemia by regulating the expression of renal uric acid transporters.

[0037] In conclusion, urolithiasis-derived uric acid can effectively promote renal uric acid excretion and exert a uric acid-lowering effect by improving the disordered expression of renal uric acid transporters induced by hyperuricemia.

[0038] 2.3 Urolithiasis D alleviates kidney damage and fibrosis caused by hyperuricemia. Kidney damage and fibrosis are clinical complications of hyperuricemia, which can accelerate its progression. Therefore, this invention investigates the effects of urolithiasis D on kidney damage and fibrosis induced by hyperuricemia.

[0039] Figure 3 A shows the changes in the appearance of the kidneys. The kidneys of hyperuricemic mice are pale white with obvious white granular texture; while in the urolithin D and benzbromarone treatment groups, the kidneys regained their red and smooth appearance. Figure 3 BC analysis showed that in the hyperuricemic mouse model, serum creatinine and serum urea nitrogen levels were significantly elevated, while urinary creatinine (UCr) levels were significantly decreased, indicating severe renal function impairment in hyperuricemic mice. Urolithin D and benzbromarone reversed these abnormal changes to varying degrees. Histopathological results showed severe glomerular atrophy (indicated by black arrows) and renal tubular dilation (indicated by blue arrows) in the hyperuricemic model group mice, and both of these pathological changes were significantly improved by urolithin D and benzbromarone. Figure 3 E). Histopathological scores further support these views ( Figure 3 F). The above results indicate that urolithiasis D can effectively alleviate kidney damage and dysfunction induced by hyperuricemia.

[0040] Kidney damage is often accompanied by severe renal fibrosis. Masson staining results showed that there were large amounts of collagen fiber deposits in the renal tubular interstitium of hyperuricemic mice (shown in blue areas), a pathological feature that was significantly improved by urolithin D and benzbromarone. Figure 3 GH). Western blotting was used to further detect the expression of key markers of fibrosis in the kidneys of hyperuricemic mice: α-smooth muscle actin (α-SMA), transforming growth factor β (TGF-β), and E-cadherin (E-Cad). Figure 4 The results show (). Figure 4 (AB) The expression of α-SMA and TGF-β was significantly increased and the expression of E-Cad was significantly decreased in the kidneys of hyperuricemic mice, indicating severe renal fibrosis in these mice. Urolithin D could dose-dependently restore the expression levels of α-SMA, TGF-β, and E-Cad, indicating that urolithin D can improve hyperuricemia-induced renal fibrosis. Subsequently, the effect of urolithin D on renal fibrosis caused by hyperuricemia was further verified in vitro using a uric acid-induced HK-2 cell model. The results are as follows: Figure 4As shown in CD, in the uric acid-induced HK-2 cell model, the expression of α-SMA and TGF-β was significantly increased, while the expression of E-Cad was significantly decreased. Urolithin D dose-dependently reversed this abnormal change, a result consistent with in vivo findings. This further indicates that urolithin D has the effect of improving renal fibrosis caused by hyperuricemia.

[0041] In conclusion, urolithiasis D can effectively alleviate renal dysfunction and renal fibrosis caused by hyperuricemia, thereby restoring renal uric acid excretion.

Claims

1. The use of urolithin D in the preparation of a medicament for treating or alleviating hyperuricemia and / or hyperuricemia-related diseases.

2. The application according to claim 1, characterized in that, The diseases associated with hyperuricemia include gout, gouty arthritis, or uric acid nephropathy.

3. The application according to claim 1, characterized in that, The drug is used to lower serum uric acid levels.

4. The application according to claim 1, characterized in that, The drug is used to improve disordered expression of uric acid transporters in the kidneys.

5. The application according to claim 1, characterized in that, The drug is used to alleviate kidney damage and fibrosis.

6. The application according to any one of claims 1-5, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

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