Application of astragalus smicus glycoside in treatment of hyperuricemia

Abstract

The invention discloses a new application of astragalus smicus glycoside, namely an application of astragalus smicus glycoside in preparation of a medicine for treating hyperuricemia. Reabsorption inhibition experiments of in-vitro transporter show that the molecule has pharmacological activity for treating the hyperuricemia; an in-vitro cell experiment result shows that the astragalus smicus glycoside has an inhibiting effect on GLUT9, and the mechanism of the astragalus smicus glycoside for resisting the hyperuricemia provides a candidate mechanism for research and development of drugs for treating the hyperuricemia.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to the application of astragalus glycoside in the preparation of a drug for treating hyperuricemia. Background Art

[0002] Clinically, hyperuricemia is divided into two types: primary hyperuricemia and secondary hyperuricemia. Primary hyperuricemia refers to abnormal expression of related enzymes in the process of uric acid anabolism (purine metabolism), which leads to excessive synthesis of uric acid; or abnormal expression of uric acid transporters in the intestines and kidneys leads to the disruption of the secretion-reabsorption balance of uric acid, and ultimately leads to severe insufficient uric acid excretion. Secondary hyperuricemia refers to the phenomenon of a transient significant increase in serum uric acid levels after the intake of a large amount of uric acid or its synthetic precursors (such as fructose, purine, alcohol, etc.), which are absorbed into the circulatory system with the small intestine; in addition, under pathological conditions, renal insufficiency can lead to abnormal filtration and reabsorption of uric acid in the kidneys, resulting in abnormal uric acid excretion function. But overall, the pathological characteristics of this patient group are still mainly insufficient renal excretion of uric acid, and only a small number of patients show excessive uric acid synthesis.

[0003] GLUT9 (Glucose Transporter Member 9) is located on chromosome 4. The encoding gene is called SLC2A9 (Solute Carrier Family 2 Member 9). It is mainly distributed in tissues such as the kidney and liver. Due to the structural differences in its amino terminus, the difference is only in the first 29 residues of the N-terminal domain, so it is divided into two subtypes (long and short subtypes). In addition to the function of glucose transport, GLUT9 can also perform the physiological function of transporting uric acid, and promote the reabsorption of uric acid in the renal tubules by exchanging glucose and fructose. The up-regulation of GLUT9 expression in the kidney will enhance the reabsorption of uric acid in the renal tubules, thereby increasing the serum uric acid concentration. Therefore, the overexpression index of GLUT9 protein is often used as one of the bases for judging hyperuricemia in clinical practice. GLUT9, as a key transporter that affects the excretion of uric acid in the kidney, is also a potential hot target for a variety of uric acid-lowering therapies. In addition, there are data showing that the uric acid transport capacity of GLUT9 is strongly correlated with gender, especially in the female hyperuricemia patient group, where mutations in the GLUT9 gene are common. Summary of the invention

[0004] The present invention provides a new use of astragaloside, that is, its use in the preparation of a drug for treating hyperuricemia. Astragaloside can act on transcriptional regulation and reduce the expression of transporter protein GLUT9 by regulation to treat hyperuricemia. The chemical structure of astragaloside is as follows: .

[0005] The ingredient (or active ingredient) of the drug of the present invention is astragaloside, and one or more pharmaceutically acceptable excipients may be added to improve the absorption effect of the drug or facilitate use, and to prepare a suitable dosage form, such as capsules or pills, powders, tablets, granules, oral liquids and injections, etc.

[0006] The present invention verifies that astragaloside has anti-hyperuricemia activity through an in vitro reabsorption inhibition experiment; in a cell model under the pathological state of hyperuricemia induced by uric acid, GLUT9 protein expression is significantly increased, while after astragaloside treatment, GLUT9 is significantly reduced; the present invention provides a reference for the research and development of anti-hyperuricemia therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 This is the cell pharmacokinetic curve of the inhibition of uric acid reabsorption by astragalus glycosides; Figure 2 It is a bar graph quantifying the inhibition of uric acid reabsorption by astragalus glycosides; Figure 3 The Western Blot detection results of GLUT9 protein expression level under pathological conditions of cell models (upper figure) and its quantitative bar graph (lower figure). DETAILED DESCRIPTION

[0008] The present invention is further described below by means of the accompanying drawings and examples, but the scope of protection of the present invention is not limited to the described contents. The methods in the present embodiments are all operated according to conventional methods unless otherwise specified, and the reagents used are all conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified, and the culture media are all commercially available products and are used according to the instructions.

[0009] Example 1: Experiment on the inhibition of uric acid reabsorption by astragalus glycosides at the cellular level HK-2 cells were cultured in MEM (containing NEAA) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO. 2Culture in an incubator. When the cell confluence reaches 70-80%, perform a subculture or plate experiment on the cells. When plating, remove the culture medium from the HK-2 cells used in the experiment, rinse with a PBS buffer solution with a pH of 7.2-7.4, remove the PBS, and digest with 0.25% trypsin (containing 0.1% EDTA) for about 30 seconds. When the cells are in a quicksand state, add three times the volume of trypsin to terminate the digestion. Place in a 5mL centrifuge tube and centrifuge at 800rpm for 3 minutes. After centrifugation, discard the supernatant and add 1mL of complete culture medium to mix to obtain a cell suspension. Take out the blood cell counting plate and cover glass from the alcohol tank, dry them, use a pipette to suck about 10μL of cell suspension, slowly drip it from one side of the blood cell counting plate, and then use a pipette to suck about 10μL of cell suspension, slowly drip it from the other side of the blood cell counting plate until the gap between the cover glass and the counting plate is filled with liquid; put it under a microscope and count the total number of cells in the four corners in the direction of the arrow (each corner has 16 small squares). According to the cell density and the required total number of cells M, the required volume of cell suspension V=M / C is obtained. The required culture medium V'=10mL-V is calculated. Take V' volume of culture medium and V somatic cell suspension and mix them evenly; take a 96-well plate, plate 5000 cells in each well, and the total volume of complete culture medium in each well is 100 μL; after 24 hours of culture, set up the model group (80 μg / mL uric acid treatment), astragalus glycoside group (100 μg / mL astragalus glycoside), and positive drug group (10 μg / mL probenecid), and pretreat with each group of drugs for 24 hours. After that, replace them with MEM complete culture medium containing 80 μg / mL uric acid. Take the supernatant after culture at 0, 3, 6, 10, 15, 21, and 30 min, centrifuge at 800 rpm for 3 min, and use the uric acid detection kit to detect the residual uric acid concentration.

[0010] Results Figure 1 , 2 From the figure, it can be seen that compared with the model group, the positive drug group has a tendency to inhibit uric acid reabsorption; compared with the model group and the probenecid group, the effect of the astragalus glycoside group in inhibiting uric acid reabsorption is more significant, indicating that astragalus glycoside does have anti-hyperuricemia activity.

[0011] Example 2: Using Western Blot technology to detect the effect of astragalus glycosides on GLUT9 protein expression under uric acid stimulation The cultured HK-2 cells were aspirated to remove the culture medium, rinsed with PBS buffer solution with a pH of 7.2-7.4, and digested with 0.25% trypsin (containing 0.1% EDTA) for about 30 seconds. When the cells were in a quicksand state, three times the volume of trypsin was added to terminate the digestion. The cells were placed in a 5 mL centrifuge tube and centrifuged at 800 rpm for 3 minutes. After centrifugation, the supernatant was discarded, and 1 mL of complete culture medium was added to mix to obtain a cell suspension. After counting the cell suspension, a 6-well plate was taken and HK-2 cells were plated in the 6-well plate. 2.5×10 5 cells, and the total culture medium volume in each well was 1.7 mL; when the cell confluence reached 70-80%, the cells were set as blank group (no treatment), solvent blank group (200 μg / mL uric acid + 0.1% DMSO), model group (200 μg / mL uric acid), astragalus glycoside gradient group (200 μg / mL uric acid and 0.1, 1, 10, 50, 100 μg / mL astragalus glycoside co-treated), and positive drug group (10 μg / mL probenecid + 200 μg / mL uric acid). The above experimental groups were collected after 24 hours of induction; Results Figure 3 As can be seen from the figure, under the stimulation of uric acid, the expression level of GLUT9 protein in HK-2 cells in the model group was significantly increased, and after the administration of astragalus glycoside, the expression level of GLUT9 protein showed a dose-dependent change with the concentration of astragalus glycoside. At a concentration of 100 μg / mL, the astragalus glycoside group was significantly different from the model group, and the expression level of GLUT9 protein was significantly reduced. This shows that astragalus glycoside can indeed achieve the purpose of treating hyperuricemia by inhibiting the expression of GLUT9 protein.

Claims

1. Application of astragalus glycosides in the preparation of drugs for the treatment of hyperuricemia.

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