A composition and its use in medicaments for the prevention and treatment of hyperuricemia

Abstract

The application belongs to the technical field of biological medicine engineering, and particularly relates to application of hypoxia-inducible factor-1 alpha as a target in development or screening of a drug for preventing and treating hyperuricemia, and proposes that hypoxia-inducible factor-1 alpha as a target is applied in development or screening of a drug for preventing and treating hyperuricemia, and the drug can ubiquitinate and degrade hypoxia-inducible factor-1 alpha. Hypoxia-inducible factor-1 alpha can act on xanthine oxidase for generating uric acid, promote generation of uric acid, or regulate vascular endothelial growth factor through hypoxia-inducible factor-1 alpha, cause damage to intestinal epithelial cells and renal tubular epithelial cells, and then inhibit expression of uric acid excretion transporter protein ATP binding cassette subfamily G member 2, and reduce excretion of uric acid. Therefore, hyperuricemia can be effectively prevented and treated, side effects are reduced, and the life quality of patients is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the application of costus lactone in a drug for preventing and treating hyperuricemia. Background Art

[0002] Hyperuricemia, a pathological condition caused by an imbalance in purine metabolism, is primarily linked to a dual impairment of uric acid production and excretion. Currently, clinical therapeutic agents primarily rely on two mechanisms: one, reducing uric acid production by inhibiting xanthine oxidase (XOD) activity, such as allopurinol and febuxostat; and the other, promoting uric acid excretion by regulating the uric acid transporter ATP Binding Cassette Subfamily G Member 2 (ABCG2), such as benzbromarone and lecithin. However, existing drugs pose challenges such as causing severe hypersensitivity reactions and hepato-renal toxicity.

[0003] Uric acid, as a product of purine metabolism in the human body, is mainly formed by the oxidation of hypoxanthine and xanthine under the catalysis of XOD in tissues such as the liver and intestine. Studies have shown that the intestine is not only a secondary channel for uric acid excretion, but its bacterial metabolites and the local intestinal microenvironment can also indirectly affect uric acid homeostasis by regulating the activity of host metabolic enzymes and the expression of transport proteins, which provides a new direction for the development of multi-target intervention strategies. The ABCG2 protein expressed in the intestine and renal tubular epithelial cells is a key transport protein mediating uric acid excretion. Its functional defects or down-regulation of expression can significantly reduce the efficiency of uric acid excretion. At the same time, patients with hyperuricemia are often accompanied by damage to the intestinal barrier and renal tubular epithelial cells, which affects the expression of ABCG2 and further aggravates the condition. Therefore, coordinated regulation and prevention of the intestine and kidney may be more effective in improving uric acid homeostasis.

[0004] Hypoxia-inducible factor-1α (HIF-1α) is a core transcription factor that regulates cellular responses to hypoxia and participates in the progression of multiple diseases by regulating processes such as angiogenesis, glucose metabolism, and oxidative stress. However, the dynamic regulatory network of HIF-1α in hyperuricemia and its potential as a therapeutic target remain unclear, and existing research has not yet revealed the specific mechanism by which HIF-1α mediates uric acid excretion through intestinal metabolites.

[0005] Based on the above background, the present invention focuses on the regulatory role of HIF-1α, combines the mechanism exploration of the intestinal bacterial metabolite Micheliolide (MCL), and proposes a dual intervention strategy of inhibiting uric acid production and promoting excretion by targeting the ubiquitination degradation of HIF-1α, providing a new idea for the prevention and treatment of hyperuricemia.

[0006] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the inventor studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0007] Based on the above technical problems, one of the objects of the present invention is to provide a composition for preventing and treating hyperuricemia, which comprises costus lactone or a drug for increasing the production of costus lactone, a metabolite of intestinal bacteria.

[0008] According to a preferred embodiment, the drug used to increase the production of costus lactone, a metabolite of intestinal bacteria, is kidney tea.

[0009] According to a preferred embodiment, the composition for preventing and treating hyperuricemia comprises a hypoxia inducible factor-1α inhibitor.

[0010] According to a preferred embodiment, the composition reduces xanthine oxidase levels by regulating uric acid production by xanthine oxidase.

[0011] According to a preferred embodiment, the composition is used to reduce blood uric acid, increase fecal uric acid, or increase urine uric acid. Preferably, the prevention and treatment of hyperuricemia is to reduce blood uric acid. The prevention and treatment of hyperuricemia is to increase fecal uric acid or urine uric acid.

[0012] According to a preferred embodiment, the composition is administered enterally or orally.

[0013] According to a preferred embodiment, the dosage form of the composition is a tablet, capsule, powder, granule, oral solution or pill. Preferably, the composition further comprises an excipient. The excipient can be a carrier or excipient, a disintegrant or solubilizer, a flavoring agent or colorant, or an antioxidant. More preferably, the carrier or excipient is, for example, microcrystalline cellulose, lactose or starch, which is used to stabilize the active ingredient and adjust the formulation form. The disintegrant or solubilizer is, for example, sodium carboxymethyl cellulose or polysorbate, which is used to improve the dissolution rate or bioavailability. The flavoring agent or colorant is, for example, a natural plant extract or steviol glycoside, which is used to improve palatability. The antioxidant is, for example, ascorbic acid, which is used to maintain product stability.

[0014] The beneficial effects of this technical solution are as follows:

[0015] According to Example 1 below, HIF-1α protein is a key target for regulating hyperuricemia. The main pathways of action of this key target are: ① HIF-1α can promote the expression of XOD to increase uric acid production. ② It regulates its downstream vascular endothelial growth factor (VEGF) to increase the permeability of blood vessels and lymphatic vessels, promote inflammatory cell infiltration, increase the level of inflammatory factors, damage intestinal epithelial and renal tubular epithelial cells, and further affect the expression of the uric acid excretion transporter ABCG2 in epithelial cells, reducing uric acid excretion. When the intestinal barrier is damaged, lipopolysaccharides (LPS) enter the blood, further promoting the expression of inflammatory factors. Based on the experimental results in Example 2, it can be seen that the differential metabolite MCL enriched by the present invention can ubiquitinate and degrade HIF-1α, regulating uric acid metabolism.

[0016] Based on this, we can know that KT (Kidney tea) tea bags improve uric acid production and metabolic imbalance by regulating intestinal metabolic pathways. Its core mechanism lies in the regulatory effect of the intestinal metabolite MCL.

[0017] The present invention's research found that KT tea bags can effectively increase the level of the metabolite MCL in the intestine. This substance is directly involved in the key link of uric acid metabolism. It reduces the amount of uric acid produced from the source by inhibiting the activity of xanthine oxidase in the liver and reducing the production of uric acid synthesis precursors. This effect was verified in the gavage animal experiment of the intestinal metabolite MCL, which confirmed that it negatively regulates uric acid production through the intestinal-liver metabolic axis. At the same time, the metabolite MCL can also promote the decomposition and metabolism of uric acid by the intestinal flora, accelerate uric acid excretion, and further alleviate uric acid accumulation in the body. This technology provides a multi-target intervention strategy for uric acid metabolism disorders by dually regulating the dynamic balance of uric acid synthesis and clearance, and avoids the excessive reliance of traditional drugs on the liver and kidney metabolic pathways, reflecting the unique advantages of natural ingredients in coordinating intestinal metabolic regulation.

[0018] One of the objects of the present invention is to provide a composition for use in preparing a health product for reducing uric acid in the body, alleviating kidney tissue inflammation, and alleviating intestinal tissue inflammation, a food for reducing uric acid in the body, alleviating kidney tissue inflammation, and alleviating intestinal tissue inflammation, or a medicine for reducing uric acid in the body, alleviating kidney tissue inflammation, and alleviating intestinal tissue inflammation, wherein the composition includes costus lactone or a substance for increasing the intestinal bacterial metabolite costus lactone.

[0019] The beneficial effects of this technical solution are as follows:

[0020] Based on the experimental data on the colon and kidney tissue structure and morphology of each group of mice after oral administration of MCL, it can be seen that ( Figure 2MCL significantly impacts inflammation in colon and kidney tissues. Table 2 shows that compared to the Mod group, oral administration of MCL significantly decreased serum uric acid levels, while both fecal and urine uric acid levels increased significantly (serum uric acid: 274.07 μmol / L - 168.52 μmol / L; fecal uric acid: 57.03 μmol / L - 71.49 μmol / L; urine uric acid: 35.34 μmol / L - 104.42 μmol / L). These results suggest that increasing the intestinal content of costus lactone can effectively enhance the efficiency of uric acid metabolism in colon and kidney tissues.

[0021] According to a preferred embodiment, the amount of costus lactone supplemented is 2.5-10 mg / kg. Preferably, the amount of costus lactone supplemented is 5 mg / kg.

[0022] One of the objectives of the present invention is to provide a composition for regulating hypoxia inducible factor-1α, which comprises costus lactone.

[0023] One of the objectives of the present invention is to provide a use of a hypoxia-inducible factor-1α inhibitor in regulating intestinal metabolism, wherein intestinal metabolism is regulated by increasing the content of costus lactone, a metabolite of intestinal bacteria.

[0024] One of the purposes of the present invention is to provide an application of kidney tea in increasing the production of costus lactone, a metabolite of intestinal bacteria.

[0025] One of the objectives of the present invention is to provide a use of KT tea bags in the process of lowering uric acid. The present invention further develops the application of KT tea bags in lowering uric acid, thereby regulating the intestinal bacterial metabolite MCL, inhibiting HIF-1α protein expression, downregulating the expression of inflammatory factors in the intestines and kidneys, enhancing the level of uric acid excretion transporters, reducing the content of uricase, regulating uric acid metabolism, and exerting a uric acid-lowering effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of network pharmacology studies on the improvement of hyperuricemia by KT bags prepared by the present invention;

[0027] Figure 2 The experimental results of MCL on the structure and morphology of colon and kidney tissues in each group of mice;

[0028] Figure 3 The results of MCL on the expression of HIF-1α / VEGF signaling pathway proteins in the colon and kidney tissues of each group of mice. DETAILED DESCRIPTION

[0029] In the description of the present invention, terms are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly indicate the number of technical features indicated. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0030] The present invention provides a KT tea bag. The KT tea bag can play a uric acid-lowering role and improve uric acid production and metabolic imbalance from the perspective of intestinal metabolism, thereby achieving the purpose of lowering uric acid.

[0031] Example 1

[0032] This example is a network pharmacology study on the uric acid-lowering effect of KT teabag.

[0033] The KEGG pathway enrichment analysis of 235 intersection targets of KT tea bags in lowering uric acid was performed using the "Cluster Profiler" package of R language software. The results showed that there were 173 signaling pathways for KT tea bags to lower uric acid (q value < 0.05), mainly involving Endocrine resistance, EGFR tyrosine kinase inhibitor resistance, Prostate cancer, AGE-RAGE signaling pathway in diabetic complications, Chemical carcinogenesis - reactive oxygen species, Pancreatic cancer, Bladder cancer, PI3K-Akt signaling pathway, Chemical carcinogenesis - receptor activation, and ErbB signaling pathway. After visualizing the enrichment results, it was found that the HIF-1α / VEGF signaling pathway was the most important in KT tea bags lowering uric acid, such as Figure 1 shown.

[0034] Example 2

[0035] This example is to screen out the intestinal differential metabolites of the KT tea bag prepared by the present invention that exert the effect of lowering uric acid.

[0036] 2.1 Materials and Methods

[0037] Experimental animals and groups: 36 SPF-grade Kunming mice, male, 6 weeks old, weighing 18-20 g, were selected for the experiment.

[0038] After 5 days of adaptive feeding, the rats were randomly divided into blank (Con) group, model (Mod) group, KT low-dose (KTL) group, KT medium-dose (KTM) group, KT high-dose (KTH) group, and positive drug (Allopurinol, ALL) group, with 6 rats in each group.

[0039] Modeling and Administration: A hyperuricemia mouse model was established using a yeast diet plus potassium oxonate (PO) gavage. Except for the Con group, the other five groups were fed a 10% yeast diet combined with oral administration of 300 mg / kg of potassium oxonate suspended in 0.5% sodium carboxymethylcellulose solution. The Con group received the same volume of 0.5% sodium carboxymethylcellulose solution once daily for 14 days. After successful model establishment, one hour after oral gavage, the KTL, KTM, and KTH groups received 750 mg / kg, 1500 mg / kg, and 3000 mg / kg of potassium oxonate solution, respectively. The ALL group received 5 mg / kg of ALL solution by oral gavage. The Con and Mod groups received an equal volume of normal saline (10 mL / kg) once daily for 14 days.

[0040] Colon Sample Collection: Under a sterile environment, mice were dissected on a clean bench and colon tissue was collected. Sterile forceps were used to collect the contents of the colonic segments. Each intestinal sample was individually aliquoted into a 2 mL sterile cryovial, weighed, and numbered. Ensure that the numbers of the experimental mice and samples corresponded to each other to avoid cross-contamination between samples. All samples were quickly frozen in liquid nitrogen and stored at -80°C. Dry ice was used for transport.

[0041] 2.2 Analysis of metabolites

[0042] Table 1 shows the changes in intestinal differential metabolites of the KT tea bag of the present invention in improving hyperuricemia.

[0043] Table 1 shows the changes in intestinal differential metabolites of the KT tea bag involved in the present invention in improving hyperuricemia.

[0044] Table 1

[0045]

[0046] Note: (1) Compared with the Con group, # P <0.05; (2) Compared with the Mod group, * P <0.05.

[0047] Example 3

[0048] This example is a study on a new approach or new use for improving uric acid metabolism by regulating the intestinal bacterial metabolite MCL.

[0049] 3.1 Materials and Methods

[0050] Experimental Animals and Grouping: Twenty-five male, 6-week-old, SPF Kunming mice weighing 18-20 g were used. After 7 days of adaptive feeding, the mice were randomly divided into three groups: a blank (Con) group, a model (Mod) group, a group expressing the intestinal differential metabolite MCL, a group expressing MCL plus an agonist (dimethyloxallyl glycocine, DMOG), a group expressing MCL plus an inhibitor (lifiguat, YC-1), and a group expressing a positive drug (allopurinol, ALL), with six mice in each group.

[0051] Modeling and Dosing: A hyperuricemia mouse model was established using yeast diet combined with oral gavage. Except for the Con group, the other five groups were fed a 10% yeast diet combined with oral gavage (300 mg / kg) of a 0.5% sodium carboxymethylcellulose solution to establish a hyperuricemia mouse model. The Con group received a standard diet and the same volume of 0.5% sodium carboxymethylcellulose solution once daily for 14 days.

[0052] From days 15 to 28 of the experiment, mice in the MCL+DMOG, MCL, and MCL+YC-1 groups were gavaged with a 5 mg / kg dose, one hour after oral administration. The ALL group was gavaged with 5 mg / kg of ALL. The Con and Mod groups were gavaged with an equal volume of saline (10 mL / kg) once daily for 14 days. Simultaneously, the MCL+DMOG and MCL+YC-1 groups received intraperitoneal injections of 40 mg / kg DMOG and 20 mg / kg YC-1, respectively. Con mice continued to be maintained on a standard diet, while the other five groups continued to be maintained on a 10% yeast diet.

[0053] Blood, colon, kidney, and liver sample collection: Mice were anesthetized using a small animal anesthesia machine, and blood was collected from the abdominal aorta. After standing at room temperature for 30 minutes, the blood was centrifuged at 3000 rpm for 10 minutes. The supernatant serum was extracted and stored at -80°C. After collecting the colonic contents, the colonic tissue was cleaned, and approximately 0.5 cm of the distal colon was cut and fixed in 4% paraformaldehyde solution. Histopathological changes in the mouse colon were examined using HE staining.

[0054] The remaining tissue was briefly stored in liquid nitrogen and then transferred to a -80°C freezer. Liver tissue was obtained from mice under sterile conditions and briefly stored in liquid nitrogen before being transferred to a -80°C freezer. Kidney tissue was obtained from mice under sterile conditions, and the surface connective tissue was removed. The left kidney was fixed in 4% paraformaldehyde solution and then stained with hematoxylin-eosin (H&E). The right kidney was briefly stored in liquid nitrogen and then transferred to a -80°C freezer.

[0055] 3.2 Enzyme colorimetric method for detection of uric acid content in mouse serum, feces, and urine

[0056] Thaw serum, feces, and urine samples slowly on ice, centrifuge, and collect the supernatant. Analyze uric acid levels according to the instructions in the biochemical kit.

[0057] Uric Acid Assay: Incubate the uric acid assay kit at room temperature for 15 minutes to create blank, standard, and sample wells. Add 5 μL of purified water to the blank wells, 5 μL of standard to the standard wells, and 5 μL of serum sample to the sample wells. Add 250 μL of working solution to each well and incubate at 37°C for 10 minutes. Measure the absorbance of each well at 510 nm using an enzyme marker and substitute the value into the formula to calculate the uric acid content.

[0058] The experimental results of MCL on blood uric acid, fecal uric acid, and urine uric acid in each group of mice are shown in Table 2.

[0059] Table 2 shows the effects of MCL on blood uric acid, fecal uric acid, and urine uric acid in each group of mice.

[0060] Table 2

[0061]

[0062] Note: (1) Compared with the Con group, # P<0.05; (2) Compared with the Mod group, * P<0.05.

[0063] As shown in Table 2, compared with the Con group, the blood uric acid content of the mice in the Mod group was significantly increased (P<0.05). Compared with the MOD group, the blood uric acid content of the mice in the MCL+DMOG, MCL, and MCL+YC-1 groups was significantly decreased ( P <0.05, P <0.05, P <0.05). Compared with the Con group, the uric acid content in feces and urine of mice in the Mod group was significantly reduced ( P <0.05, P<0.05). Compared with the Mod group, the uric acid levels in the feces and urine of mice in the MCL and MCL+YC-1 groups were significantly increased ( P <0.05, P <0.05, P <0.05, P <0.05).

[0064] 3.3 Colorimetric detection of XOD changes in mouse serum, colon, and liver tissues

[0065] Incubate the XOD kit at room temperature for 15 minutes to create blank and sample wells. Prepare the assay in an EP tube. Add 50 μL of pure water to the blank tube and 50 μL of serum / colon tissue extract to the sample tube. Add 1 mL of Reagents I, II, III, and IV, respectively, 0.05 mL, 0.2 mL, and 0.02 mL, to each tube, mix thoroughly, and incubate each well at 37°C for 20 minutes. Add 1.0 mL of Reagent V to each EP tube. Mix thoroughly and measure the absorbance at 530 nm (A1). Calculate the content of each indicator using the equation.

[0066] Table 3 shows the experimental results of MCL on XOD in the serum, colon tissue, and liver tissue of each group of mice.

[0067] Table 3 shows the effects of MCL on XOD in serum, colon tissue, and liver tissue of mice in each group.

[0068] Table 3

[0069]

[0070] Note: (1) Compared with the Con group, # P <0.05; (2) Compared with the Mod group, * P <0.05

[0071] As shown in Table 3, compared with the Con group, the serum XOD content of mice in the Mod group was significantly increased ( P <0.01), compared with the Mod group, the serum XOD levels in the KTL, KTM, and KTH groups were significantly decreased ( P <0.01, P <0.01, P <0.01). Compared with the Con group, the XOD content in the colon tissue of mice in the Mod group was significantly increased ( P <0.01), and the XOD contents in the colon tissues of mice in the KTL, KTM, and KTH groups also decreased to varying degrees.

[0072] 3.4 H&E staining to observe the morphological and structural changes of mouse colon and kidney tissues

[0073] Tissue samples were removed, dehydrated, and embedded in paraffin. Sections were sequentially placed in environmentally friendly deparaffinization solutions I and II for 20 minutes each, followed by placement in anhydrous ethanol I and II for 5 minutes each, followed by rinsing and dehydration with 75% alcohol and purified water. Frozen sections were rewarmed, fixed, stained with hematoxylin for 5 minutes, dehydrated, and stained with eosin for 5 minutes. The sections were then placed in anhydrous ethanol I, II, and III for 5 minutes each, followed by clearing with xylene I and II for 5 minutes each, and mounted with neutral gum. Images were acquired and analyzed under an upright microscope.

[0074] Figure 2 These are the experimental results of MCL on the structure and morphology of the colon and kidney tissues of each group of mice.

[0075] like Figure 2 As shown in A, H&E staining of colon tissue showed that the colon tissue structure of mice in the blank group was intact and arranged in an orderly manner, without obvious edema and inflammatory cell infiltration. Inflammatory cell infiltration was observed in the model group. After kidney tea intervention, the degree of neutrophil infiltration in the mice in the drug-treated groups decreased to varying degrees.

[0076] like Figure 2 As shown in Figure B, H&E staining of renal tissue revealed that the surface capsule of the kidney tissue in the control group mice was composed of dense connective tissue of uniform thickness. The renal parenchyma consisted of a superficial cortex and a deep medulla, with a distinct cortical-medullary boundary. Glomeruli were evenly distributed in the cortex, with a uniform cell number and matrix within the glomeruli. The renal tubular epithelial cells were round and plump, with regular brush borders. No significant abnormalities were observed in the medulla. The connective tissue between the urinary tubules formed the renal interstitium, which showed no significant proliferation. No obvious inflammatory changes were observed. In the model group mice, a small number of renal tubular epithelial cells showed hydropic degeneration, swollen cells, and loose, lightly stained cytoplasm. Occasional perivascular infiltration of numerous lymphocytes was observed. Treatment with MCL and YC-1 reduced the degree of hydropic degeneration of the renal tubular epithelial cells, the extent of lymphocyte infiltration, and the degree of congestion.

[0077] 3.5 ELISA to detect the effects of IL-6, IL-18, IL-1β, TNF-α, and LPS in mouse serum, colon tissue, and kidney tissue

[0078] Remove approximately 20 mg of tissue and rinse with PBS to remove any blood and impurities. Then, add 9 times the tissue mass in PBS and homogenize the tissue using a tissue grinder. Centrifuge at 4°C for 15 minutes, aspirate the supernatant, and keep it on ice. ELISA kit instructions were used to assay for IL-6, IL-18, IL-1β, TNF-α, and LPS levels. The specific steps are as follows:

[0079] (1) Set up the plate layout: Pre-set the positions of the standard wells, sample wells, and blank wells. (2) Remove the required strips: After the kit has been placed at room temperature for 20 minutes, remove the strips. (3) Add samples: Add samples in sequence according to the pre-set positions of each well according to the instructions; add 10 μL of sample and 40 μL of sample diluent to the sample wells; add 50 μL of different concentrations of standard wells; and do not add reagents to the blank wells. (4) Add antibodies: Add 100 μL of horseradish peroxidase (HRP)-labeled antibody to the standard wells and sample wells respectively; no antibody is added to the blank wells. (5) Incubate: Seal the reaction wells with the sealing film provided in the kit; and incubate in a water bath for 60 minutes. (6) Wash the plate thoroughly: Remove the strips from the water bath, pour out the liquid, pat dry on absorbent paper, then fill each well with the pre-prepared washing solution, let it stand for a few minutes, pour out the washing solution, pat dry on absorbent paper, and repeat this process 5 times to wash the plate thoroughly; the first 3 washes should be left for 30 seconds each, and the last 2 washes should be 2 minutes. (7) Color development: Add 50 μL of substrate A and substrate B to each well; incubate in a water bath for 15 minutes. (8) Stop the reaction: Add 50 μL of stop solution to each well to stop the reaction. After incubation, add stop solution directly to each well. (9) Detect OD value on the machine: Use an enzyme-linked microplate reader to detect within 15 minutes; select a wavelength of 450 nm and measure the OD value of each well. (10) Draw a standard curve: Use the concentration of the standard as the horizontal axis and the corresponding OD value as the vertical axis to draw a standard curve; calculate the concentration value of each sample according to the curve equation.

[0080] The experimental results of MCL on interleukin-6 (IL-6), interleukin-18 (IL-18), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and LPS in the serum, colon tissue, and kidney tissue of each group of mice are shown in Table 4. Table 4 shows the effects of MCL on IL-6, IL-18, IL-1β, TNF-α, and LPS in mouse serum by enzyme-linked immunosorbent assay.

[0081] Table 4

[0082] Note: (1) Compared with the Con group, # P <0.05; (2) Compared with the Mod group, * P <0.05.

[0083] As shown in Table 4, compared with the Con group, the levels of serum inflammatory factors IL-6, IL-18, IL-1β, and TNF-α in the Mod group were significantly increased ( P <0.05), LPS content increased significantly ( P <0.05). Compared with the Mod group, the serum IL-18, IL-1β, and TNF-α levels of mice in the MCL and MCL+YC-1 groups were significantly decreased ( P <0.05), serum IL-6 levels in the MCL group were significantly decreased ( P <0.05), LPS levels in the MCL+DMOG, MCL, and MCL+YC-1 groups decreased significantly ( P <0.05).

[0084] Table 5 shows the effects of IL-6, IL-18, IL-1β, TNF-α, and LPS on mouse colon tissue by enzyme-linked immunosorbent assay.

[0085] Table 5

[0086]

[0087] Note: (1) Compared with the Con group, # P <0.05; (2) Compared with the Mod group, * P <0.05.

[0088] As shown in Table 5, compared with the Con group, the levels of serum inflammatory factors IL-6, IL-18, IL-1β, and TNF-α in mice in the Mod group were significantly increased ( P <0.05), LPS content increased significantly ( P <0.05). Compared with the Mod group, the levels of serum inflammatory factors IL-6, IL-18, IL-1β, and TNF-α in the MCL+DMOG, MCL, and MCL+YC-1 groups decreased to varying degrees ( P <0.05), and LPS content also showed a downward trend ( P <0.05).

[0089] Table 6 shows the effects of IL-6, IL-18, IL-1β, TNF-α, and LPS on mouse kidney tissue by enzyme-linked immunosorbent assay.

[0090] Table 6

[0091]

[0092] Note: (1) Compared with the Con group, # P <0.05; (2) Compared with the Mod group,* P <0.05.

[0093] As shown in Table 6, compared with the Con group, the levels of IL-6, IL-18, IL-1β, and TNF-α in the kidney tissue of mice in the Mod group were significantly increased ( P <0.05), LPS content increased significantly ( P <0.05). Compared with the Mod group, the levels of IL-6, IL-18, IL-1β, and TNF-α in the kidney tissues of mice in the MCL+DMOG, MCL, and MCL+YC-1 groups decreased to varying degrees ( P <0.05), and LPS content also showed a downward trend ( P <0.05).

[0094] 3.6 RT-qPCR detection of relative mRNA expression of Claudin-1, Zonulaoccludens-1 (ZO-1), Occludin, HIF-1α, VEGF, and ABCG2 in mouse colon and kidney tissues

[0095] (1) Weigh 30-40 mg of tissue sample into a 1.5 mL sterile EP tube, add steel beads and 500 μL RNAisoPlus;

[0096] (2) Prepare tissue homogenate using a bead mill (speed: 60 rpm / min; time: 5 min);

[0097] (3) Add 0.2 mL of chloroform and let stand on ice for 2 min;

[0098] (4) Centrifuge the sample (4°C, 12,000 rpm, 15 min) and aspirate the supernatant with a pipette.

[0099] (5) Add an equal amount of isopropanol to the supernatant, let it stand at -20°C for 10 min, and then centrifuge for 15 min;

[0100] (6) Add 75% ethanol and centrifuge for 5 minutes;

[0101] (7) Discard the supernatant and repeat step (6);

[0102] (8) Air-dry in a fume hood, dissolve in DEPC water, and store at -80°C;

[0103] (9) Thaw total RNA on ice for cDNA synthesis;

[0104] (10) Removal of genome;

[0105] (11) Reverse transcription (12) Amplification on a microarray.

[0106] Experimental results of MCL on the transcription levels of Claudin-1, ZO-1, and Occludin mRNA in the colon tissues of each group of mice.

[0107] Table 7 shows the effects of MCL on the transcription levels of Claudin-1, ZO-1, and Occludin mRNA in the colon tissues of mice in each group.

[0108] As shown in Table 7, to confirm the effect of MCL on the HIF-1α / VEGF signaling pathway, RT-qPCR was used to detect the effect of MCL on the mRNA expression of intestinal barrier tight junction proteins in mice with hyperuricemia induced by PO combined with yeast diet. The mRNA expression of intestinal barrier tight junction proteins Claudin-1, ZO-1, and Occludin in the Mod group was decreased compared with that in the Con group ( P >0.05), and its expression increased after intervention with MCL, pathway-related inhibitors and agonists.

[0109] Table 7

[0110]

[0111] Note: (1) Compared with the Con group, # P <0.05; (2) Compared with the Mod group, * P <0.05.

[0112] 3.7 MCL experimental results on the transcriptional levels of uric acid transporter ABCG2 mRNA in the colon and kidney tissues of mice in each group

[0113] Table 8 shows the effects of MCL on the mRNA transcription level of UA transporter ABCG2 in mouse colon and kidney tissues.

[0114] Table 8

[0115]

[0116] Note: (1) Compared with the Con group, # P <0.05; (2) Compared with the Mod group, * P <0.05.

[0117] As shown in Table 8 , compared with the Con group, the relative transcription levels of ABCG2 mRNA in the colon and kidney tissues of the Mod group were decreased ( P <0.05), compared with the Mod group, the relative transcription level of ABCG2 mRNA in the kidney tissue MCL group and MCL+YC-1 group was significantly increased (P <0.05), the relative transcription level of ABCG2 mRNA in colon tissue increased to varying degrees after intervention with MCL and inhibitors ( P <0.05). This result suggests that MCL can enhance the expression of UA transporter ABCG2 to enhance UA excretion.

[0118] Table 9 shows the effects of MCL on the mRNA transcription levels of the HIF-1α / VEGF signaling pathway in the colon and kidney tissues of each group of mice.

[0119] Table 9

[0120]

[0121] Note: (1) Compared with the Con group, # P <0.05; (2) Compared with the Mod group, * P <0.05.

[0122] As shown in Table 9, compared with the Con group, the relative transcription levels of HIF-1α and VEGF mRNA in the colon tissue Mod group were significantly increased ( P <0.05); compared with the Mod group, the relative transcription levels of HIF-1α and VEGF mRNA in the colon tissue KTM and KTH groups were significantly decreased ( P <0.05), the relative transcription level of VEGF mRNA in the KTL group was significantly decreased ( P <0.05). Compared with the Con group, the relative transcription levels of HIF-1α and VEGF mRNA in the kidney tissue of the Mod group were significantly increased ( P <0.05); compared with the Mod group, the relative transcription levels of HIF-1α and VEGF mRNA in the renal tissue KTL, KTM, and KTH groups were significantly decreased ( P <0.05).

[0123] 3.8 Immunohistochemical detection of HIF-1α / VEGF pathway protein expression in mouse kidney and colon tissues

[0124] Mouse tissue samples were taken and fixed with 4% paraformaldehyde overnight.

[0125] (1) Dewaxing of paraffin sections: Place the sections in environmentally friendly dewaxing solution I for 10 min, then in environmentally friendly dewaxing solution II for 10 min, then in environmentally friendly dewaxing solution III for 10 min, then in anhydrous ethanol I for 5 min, then in anhydrous ethanol II for 5 min, then in anhydrous ethanol III for 5 min, then in distilled water.

[0126] (2) Antigen retrieval: Place the tissue sections in EDTA antigen retrieval buffer (pH = 9.0) and microwave for antigen retrieval. After cooling naturally, place the slides in PBS buffer (pH = 7.4) and decolorize on a decolorizing shaker.

[0127] (3) Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS according to the proportion on the slice, and place the slice flat in a humidified box and incubate at 4°C overnight.

[0128] (4) Adding secondary antibody: Place the slide in PBS (pH = 7.4) and wash on a decolorizing shaker three times, 5 minutes each time. After the sections are slightly dried, add a secondary antibody (HRP-labeled) of the same species as the primary antibody to cover the tissue and incubate at room temperature for 50 minutes.

[0129] (5) DAB staining: Place the slide in PBS (pH = 7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the sections are slightly dried, add freshly prepared DAB staining solution to the circle. The staining time is controlled under a microscope. The positive color is brown-yellow. Rinse the sections with tap water to stop the staining.

[0130] (6) Re-staining of cell nuclei: Re-stain with hematoxylin for 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, blue with hematoxylin bluing solution, and rinse with running water.

[0131] (7) Dehydration and sealing: Place the slices in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and make them transparent. Take the slices out of the xylene and let them dry slightly, then seal them with sealing glue.

[0132] (8) Microscopic examination: Place the sample under a microscope for analysis.

[0133] Table 10 shows the effect of MCL on the protein expression levels of the HIF-1α / VEGF signaling pathway in the colon and kidney tissues of each group of mice.

[0134] Table 10

[0135]

[0136] Note: (1) Compared with the Con group, # P <0.05; (2) Compared with the Mod group, * P <0.05.

[0137] As shown in Table 10, Figure 3As shown in A and B, compared with the Con group, the relative expression levels of HIF-1α and VEGF proteins in the colon tissue of mice in the Mod group were significantly increased ( P <0.05). These results indicate that the intestinal HIF-1α / VEGF signaling pathway is enhanced in hyperuricemia mice induced by 10% yeast diet combined with PO gavage.

[0138] Compared with the Mod group, the relative expression levels of HIF-1α and VEGF proteins in the colon tissue of the MCL+YC-1 group were significantly decreased ( P <0.05), and the expression level of VEGF protein in the colon tissue of mice in the MCL group was significantly decreased ( P <0.05). This result indicates that MCL can reduce the expression of HIF-1α / VEGF signaling pathway in colon tissue.

[0139] As shown in Table 10, Figure 3 As shown in C and D, compared with the Con group, the relative expression levels of HIF-1α and VEGF proteins in the kidney tissue of the Mod group mice were significantly increased ( P <0.05). These results indicate that the HIF-1α / VEGF signaling pathway in the kidneys of hyperuricemia mice induced by 10% yeast diet combined with PO gavage is enhanced.

[0140] Compared with the Mod group, the relative expression levels of HIF-1α and VEGF proteins in the kidney tissue of the MCL+YC-1 group were significantly decreased ( P <0.05) indicating that MCL can reduce the expression of HIF-1α / VEGF signaling pathway in colon tissue.

[0141] It should be noted that the above-described specific embodiments are illustrative only. Those skilled in the art may devise various solutions based on the disclosure of the present invention, and such solutions fall within the scope of the present invention and are intended to be protected by the present invention. Those skilled in the art should understand that the present description and its accompanying drawings are intended to be illustrative only and are not intended to limit the scope of the claims. The scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing and treating hyperuricemia, characterized in that: The composition comprises costus lactone and rificiguat.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition is for enteral administration or oral administration.

3. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition is used for reducing blood uric acid, increasing fecal uric acid or increasing urine uric acid.

4. Use of the pharmaceutical composition in the preparation of a medicament for preventing and treating hyperuricemia, characterized in that: The pharmaceutical composition comprises costus lactone and rificiguat.

5. The use according to claim 4, characterized in that The amount of costus lactone supplied is 2.5-10 mg / kg.

6. The use according to claim 4, characterized in that The medicine is used to reduce uric acid in the body, relieve kidney tissue inflammation, and relieve intestinal tissue inflammation.

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