Application of piceatannol in preparation of medicine for treating hyperuricemia or kidney injury caused by hyperuricemia

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of piceatannol in preparation of a medicine for treating hyperuricemia or kidney injury caused by hyperuricemia. Research finds that piceatannol can effectively reduce serum uric acid of a hyperuricemia mouse and prevent kidney injury caused by hyperuricemia, so that application of piceatannol in preparation of a medicine for treating hyperuricemia and application of piceatannol in preparation of a medicine for treating kidney injury caused by hyperuricemia are provided. The invention provides a new method for preventing and treating hyperuricemia and kidney injury caused by hyperuricemia.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the use of piceatannol in preparing a medicine for treating hyperuricemia or kidney damage caused by hyperuricemia. Background Art

[0002] Hyperuricemia (HUA) is a metabolic disease characterized by abnormally elevated serum uric acid levels due to uric acid metabolism disorders. In recent years, with the improvement of people's living standards and changes in dietary structure, the overall prevalence of hyperuricemia has shown a significant upward trend. HUA has become the second largest metabolic disease after diabetes. Continuously elevated serum uric acid levels can lead to the deposition of urate crystals in tissues, causing glomerular damage, tubular interstitial fibrosis, and inflammatory cell infiltration, and eventually develop into hyperuricemia nephropathy. In addition, a large number of clinical and epidemiological studies have shown that elevated serum uric acid levels are an independent risk factor for a variety of metabolic diseases such as hypertension, cardiovascular disease, and diabetes.

[0003] At present, the drugs used in clinical treatment of HUA and kidney damage caused by hyperuricemia are mainly divided into three categories: allopurinol and febuxostat that inhibit uric acid production, probenecid and benzbromarone that promote uric acid excretion, and anti-inflammatory drugs such as colchicine that relieve inflammation. However, long-term use of these traditional drugs may cause serious adverse reactions such as nephrotoxicity, hepatotoxicity and hypersensitivity, which limits their clinical application. In recent years, small molecule drugs derived from natural products have attracted much attention due to their high safety and significant efficacy. Studies have shown that a variety of natural compounds have shown good prospects in the treatment of HUA. Piceatannol is a natural stilbene compound widely found in grapes, blueberries and passion fruit, with multiple biological activities such as cardioprotection, anti-diabetes, anti-inflammatory and anti-cancer. However, there are no specific reports on the use of piceatannol in the treatment of hyperuricemia and kidney damage caused by hyperuricemia. Summary of the invention

[0004] One of the purposes of the present invention is to provide a new method for preventing and treating hyperuricemia and kidney damage caused by hyperuricemia.

[0005] In a first aspect, the present invention provides the use of piceatannol in the preparation of a medicament for treating hyperuricemia.

[0006] In a second aspect, the present invention provides the use of piceatannol in the preparation of a medicament for treating kidney damage caused by hyperuricemia.

[0007] In a third aspect, the present invention provides the use of piceatannol in the preparation of a medicament for treating renal insufficiency caused by hyperuricemia.

[0008] In a fourth aspect, the present invention provides the use of piceatannol in the preparation of a medicament for treating renal fibrosis induced by hyperuric acid.

[0009] In a fifth aspect, the present invention provides the use of piceatannol in the preparation of a drug for reducing the uric acid content in the serum of a patient.

[0010] In a sixth aspect, the present invention provides the use of piceatannol in the preparation of a drug for reducing the creatinine content in the serum of a patient.

[0011] In a seventh aspect, the present invention provides the use of piceatannol in the preparation of a medicament for reducing urea nitrogen content in a patient.

[0012] In an eighth aspect, the present invention provides the use of piceatannol in the preparation of a medicament for reducing the activity of xanthine oxidase (XOD) in the liver and / or serum of a patient.

[0013] In a ninth aspect, the present invention provides the use of piceatannol in the preparation of a medicament for reducing a patient's renal index.

[0014] In the application of the present invention, the patient is a hyperuricemia patient.

[0015] The present invention establishes a hyperuricemia mouse model by inducing potassium oxonate combined with adenine, and finds that piceatannol can effectively reduce serum uric acid in hyperuricemia mice and prevent kidney damage caused by hyperuricemia.

[0016] Specific findings: 1. Albuminol reduced the serum uric acid (UA) level in hyperuricemia mice.

[0017] 2. Baipisanol achieves the effect of lowering uric acid by inhibiting the activity of xanthine oxidase (XOD) in the serum and liver of the hyperuricemia mouse model.

[0018] 3. Baipisanol reduced the levels of serum urea nitrogen (BUN) and serum creatinine (CRE) in the hyperuricemia mouse model.

[0019] 4. The results of kidney visual images, HE and Masson staining showed that picroside had a protective effect on renal damage in mice with hyperuricemia.

[0020] The beneficial effects of the present invention are at least: The present invention provides a new and effective method for treating hyperuricemia and kidney damage caused by hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the experimental design for mice.

[0022] Figure 2 The trend of weight change.

[0023] Figure 3 The kidney index (A) and liver index (B) are shown.

[0024] Figure 4 Results of serum uric acid (A), serum urea nitrogen (B), and serum creatinine (C).

[0025] Figure 5 The figure shows the effect of piceatannol on the kidney of mice with hyperuricemia. A is a direct view of the kidney, B is a HE-stained section of the kidney, and C is a Masson-stained section of the kidney. The scale bar in the figure represents 20 μm.

[0026] Figure 6 Figure 3 is the effect of piceatannol on XOD in hyperuricemia mice. In the figure, A is the serum XOD activity of hyperuricemia mice, B is the liver XOD activity of hyperuricemia mice, C is the Western blot result of XOD in the liver, and D is the relative expression level of XOD in the liver.

[0027] In each figure (if any), ns means no significant difference. Represents p<0.05 compared with the MC group, Represents p<0.01 compared with the MC group, represents p < 0.001 compared with the MC group, ### represents p < 0.001 compared with the NC group. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0029] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0030] Example 1 (1) Experimental animal grouping and plan Healthy male Kunming mice (22–24 g, 6–8 weeks old) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (License No.: SCXK2020-0001, Shenyang, China).

[0031] Mice were housed in a standard environment (temperature: 23 ± 2 ° C, relative humidity: 50-70%, 12 h light-dark cycle) with free access to food and distilled water. After 1 week of adaptive feeding, 40 experimental mice were randomly divided into 5 groups (n = 8): normal control group (NC), model group (MC), allopurinol group (AP, 10 mg / kg), picolinol low-dose group PIC-30 (30 mg / kg) and picolinol high-dose group PIC-60 (60 mg / kg). Potassium oxamate (PO), adenine, allopurinol, and picolinol were suspended in fresh 0.5% sodium carboxymethylcellulose (CMC-Na) aqueous solution. Mice in the MC, AP, and PIC groups were gavaged with PO (300 mg / kg / ) and adenine (150 mg / kg) at 8:00 am every day to induce a hyperuricemia model. One hour later, the corresponding drugs or solvents were given: the MC group was given 0.5% CMC-Na solution, the AP group was given allopurinol, and the PIC group was given different doses of piceatannol. The NC group was given an equal volume of 0.5% CMC-Na solution at the same time point. The administration lasted for 3 weeks. The schematic diagram of the mouse experimental design is shown in Figure 1 .

[0032] The animals were fasted for 12 hours before the end of the experiment, and blood samples were collected by retro-orbital venous blood sampling. After standing at room temperature for 2 hours, the samples were centrifuged at 3000 r / min for 10 minutes to separate the serum. The liver, kidney and other tissues were quickly separated, frozen in liquid nitrogen and stored at -80°C for testing.

[0033] (2) Evaluation of the effects of piceatannol on body weight, organ indexes, and serum biochemical indicators in hyperuricemia mice The weight of mice was recorded regularly during the experiment. The liver and kidney of mice were obtained on the day of dissection, rinsed with saline, dried with filter paper, photographed and weighed. The organ coefficients of mice were calculated. Kidney coefficient = kidney weight / body weight, liver coefficient = liver weight / body weight.

[0034] Blood samples were collected by retro-orbital venous blood sampling. After standing at room temperature for 2 hours, the samples were centrifuged at 3000 r / min for 10 minutes to separate serum. Serum uric acid (UA) levels were determined by enzyme colorimetry, and serum urea nitrogen (BUN) and serum creatinine (CRE) levels were determined by urease method and creatinine oxidase method, respectively. All tests were performed strictly according to the instructions of the kit.

[0035] Experimental results: In order to evaluate the effect of piceatannol on hyperuricemia in vivo, the present invention used PO combined with adenine to induce a hyperuricemia mouse model. After 3 weeks of hyperuricemia modeling treatment, the weight of MC mice decreased significantly, and after treatment with 10 mg / kg allopurinol and 30mg / kg and 60mg / kg piceatannol, the weight of mice recovered to varying degrees compared with the MC group ( Figure 2). Compared with the NC group, the renal index of mice in the MC group was significantly increased, and piceatannol treatment significantly reversed this increase ( Figure 3 There was no significant difference in liver index among the groups ( Figure 3 B in the figure).

[0036] In addition, compared with the NC group (91.28 ± 6.04 μM), the serum uric acid level in the MC group was significantly increased to 214.89 ± 4.40 μM, indicating the successful establishment of a hyperuricemia mouse model. At the doses of 30 mg / kg and 60 mg / kg piceatannol, piceatannol significantly and dose-dependently reduced the serum uric acid level to 116.49 ± 5.14 μM and 103.36 ± 5.22 μM ( Figure 4 A in the figure). It is worth noting that the serum uric acid level in the AP-treated group was lower than that in the normal control group, a result consistent with previous studies. Serum creatinine (CRE) and urea nitrogen (BUN) are commonly used clinical indicators of renal function assessment, which can indirectly reflect the filtration capacity of the glomerulus. Figure 4 As shown in Figures B and C, allopurinol and piceatannol treatment significantly reduced serum CRE and BUN levels and improved renal insufficiency compared with the MC group. In summary, piceatannol treatment has a good protective effect on the kidneys of hyperuricemia mice.

[0037] (3) Effects of piceatannol on the kidneys of mice with hyperuricemia Fresh kidney tissues collected after the experiment were fixed in 4% paraformaldehyde solution for 24 h, dehydrated with gradient ethanol, and embedded in paraffin to prepare 3 μm thick serial sections. After dewaxing and hydration, the sections were stained with hematoxylin-eosin (H&E) and Masson's trichrome (Masson) staining, respectively. The pathological changes of kidney tissues were observed and photographed at a magnification of 400× using a DP26 optical microscope (Olympus Corporation, Tokyo, Japan).

[0038] Experimental results: Figure 5 As shown in A, after hyperuricemia induction, the kidneys of mice in the MC group were yellow-brown and accompanied by edema, indicating significant renal damage. In contrast, after treatment with allopurinol (AP group) and piceatannol (PIC-30, PIC-60 groups), the appearance of the mouse kidneys was significantly improved, especially in the AP group and PIC-60 group, which almost returned to normal. HE staining results ( Figure 5 The improvement was further verified in Figure B. The MC group showed obvious tubular dilatation, glomerular atrophy, and inflammatory cell infiltration. The renal tissue structure of the AP and PIC-60 groups was close to normal. Although the PIC-30 group had mild lesions, the injury score was significantly better than that of the MC group.

[0039] To further evaluate the effects of each treatment on renal fibrosis, Masson staining analysis was performed ( Figure 5 C in the figure). The renal interstitial fibrosis of mice in the MC group was severe, and the fibrosis area increased significantly. After treatment with the AP group and high-dose piceatannol (PIC-60), the fibrous deposition was significantly reduced, especially in the PIC-60 group, where almost no fibrosis remained, showing an excellent anti-fibrosis effect. The PIC-30 group also showed a certain improvement effect, but the effect was not as good as that of the AP group and the PIC-60 group.

[0040] In summary, allopurinol and piceatannol have significant protective effects on renal damage and fibrosis in hyperuricemia mice. In particular, piceatannol showed the best therapeutic effect at high doses, providing strong support for its use as a potential therapeutic agent.

[0041] (4) Effects of piceatannol on the expression and activity of xanthine oxidase in hyperuricemia mice The liver and kidney tissues collected after the experiment were added with pre-cooled physiological saline at a mass-volume ratio of 1:9, and the tissues were homogenized under ice bath conditions. Centrifuged at 3000 r / min for 10 min at 4°C, and the supernatant was collected. The tissue protein concentration was determined using the BCA protein quantification kit. The XOD activity of liver tissue was detected using a commercial kit.

[0042] Liver tissue was lysed in RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors for 30 min. The supernatant was collected by centrifugation at 4°C and 12,000 rpm for 10 min, and the protein concentration was determined using a BCA kit. 80 μg of denatured protein samples were separated by 10% SDS-PAGE electrophoresis and transferred to a 0.2 μm PVDF membrane. The membrane was blocked with 5% skim milk powder / TBST for 2 h and then incubated with primary antibodies at 4°C overnight. After washing with TBST, the membrane was incubated with the corresponding secondary antibody labeled with horseradish peroxidase (1:4000 dilution, S0001, Affinity) at room temperature for 1 h. The ECL luminescence kit was used for color development, and images were collected on a Tanon 5200 chemiluminescence imaging system (Tianneng Technology, Shanghai, China). The grayscale value of the bands was semi-quantitatively analyzed using Gelpro32 software (MediaCybernetics, Silver Spring, MD, USA) with β-actin as the internal reference.

[0043] Experimental results: The liver is the main organ for purine metabolism, and the activity and expression of XOD directly determine the rate and total amount of uric acid production. Therefore, inhibiting liver XOD is an important method for treating hyperuricemia. Figure 6As shown in A and B in Figure , the liver and serum XOD activities of mice in the MC group (19.14 ± 0.53 U / g protein and 14.65 ± 0.21 U / L, respectively) were significantly higher than those in the control group (13.13 ± 0.57 U / g protein and 8.54 ± 0.16 U / L, respectively) (### p<0.001), indicating that hyperuricemia significantly activated the XOD enzyme activity. The piceatannol-treated groups (PIC-30 and PIC-60) showed a dose-dependent XOD inhibition effect, in which the PIC-30 group showed a decrease in both serum and liver XOD activities, but the inhibitory effect was slightly weaker than that of allopurinol. The PIC-60 group significantly reduced XOD activity, with serum and liver XOD activities falling to 12.38 ± 0.30 U / L and 11.87 ± 0.56 U / g protein, respectively ( p<0.001), close to the normal level, comparable to the AP group, showing a strong inhibitory effect. Western blot results ( Figure 6 C and D) showed that after allopurinol treatment, the expression of XOD protein in the liver was significantly decreased, and the relative expression level decreased to 0.40 ± 0.01 ( p<0.001), which is consistent with its inhibitory effect in reducing XOD enzyme activity, further confirming the mechanism by which allopurinol reduces uric acid production by downregulating XOD expression. The piceatannol-treated groups also showed a dose-dependent inhibitory effect, among which the XOD protein expression in the PIC-30 group was relatively high, at 0.58 ± 0.04, but significantly lower than that in the MC group ( p<0.05). The PIC-60 group further significantly inhibited the expression of XOD, and the relative expression level dropped to 0.47 ± 0.02 ( p<0.05), close to the level of the AP group, which was consistent with the result of decreased XOD activity.

[0044] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Application of piceatannol in the preparation of drugs for treating hyperuricemia.

2. The use of piceatannol in the preparation of drugs for treating kidney damage caused by hyperuric acid.

3. The use of piceatannol in the preparation of drugs for treating renal insufficiency caused by hyperuricemia.

4. The use of piceatannol in the preparation of drugs for treating renal fibrosis caused by hyperuric acid.

5. Use of piceatannol in the preparation of a drug for reducing xanthine oxidase activity in the liver and / or serum of a patient.

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