Application of forsythin in preparation of medicine for preventing or treating hyperuricemia

Abstract

The invention provides application of forsythin in preparation of a medicine for preventing or treating hyperuricemia, and belongs to the technical field of medical application. According to the present invention, the research results show that the forsythin can reduce the expression levels of uric acid reabsorption proteins GLUT9 and URAT1, and can increase the expression levels of uric acid secretory proteins ABCG2, OAT1 and OAT3; on the basis, the forsythin can be used as a uric acid excretion accelerant, and is further used for preparing the medicine for preventing or treating hyperuricemia caused by uric acid excretion disorder. In addition, the forsythin can also relieve kidney damage caused by hyperuricemia. The application not only provides a new medical application for forsythin, but also provides a new drug choice for hyperuricemia caused by uric acid excretion disorder, and has important guiding significance for prevention and treatment of hyperuricemia and kidney damage caused by hyperuricemia.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical applications, and particularly to the application of phillyrin in the preparation of a medicament for preventing or treating hyperuricemia. Background Art

[0002] Hyperuricemia (HUA) is a metabolic disease caused by purine metabolism disorders or reduced uric acid excretion, and is associated with various diseases such as gout, hypertension, chronic kidney disease, obesity, etc. It has become the fourth "high" after the "three highs" of hypertension, hyperglycemia, and hyperlipidemia. If uric acid production increases or excretion decreases, the serum uric acid level can be elevated, thereby leading to the occurrence of HUA. Given the incidence rate and impact of hyperuricemia, it is necessary to study drugs for preventing or treating HUA.

[0003] Currently, the drugs used clinically to treat HUA can be mainly divided into three categories: drugs for inhibiting uric acid production, drugs for promoting uric acid excretion, and drugs for inhibiting the activity of uricase, corresponding to different types of HUA, but most of the corresponding drugs used clinically have varying degrees of adverse reactions. For example, allopurinol can cause liver function damage and hypersensitivity syndrome, etc., febuxostat can cause abnormal liver function, diarrhea, nausea, headache, vomiting, and rash, etc., benzbromarone can cause fulminant hepatitis, and pegloticase can cause immunogenic drug infusion-related reactions. Therefore, it is still necessary to further explore drugs for the prevention and treatment of HUA to seek new drugs. And among the many factors causing HUA, uric acid excretion disorder is an important factor leading to primary HUA, and the prevention and treatment of HUA caused by uric acid excretion disorder is the current research focus.

[0004] Phillyrin is one of the many components of Forsythia suspensa, and can be used medicinally, with effects such as clearing heat, detoxifying, dissipating nodules and discharging pus. Existing research shows that it has various pharmacological activities such as antioxidant, anti-inflammatory, antiviral, anti-aging, and hypoglycemic effects, but there is currently no relevant application of phillyrin in HUA caused by uric acid excretion disorder. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present application provides the application of phillyrin in the preparation of a medicament for preventing or treating hyperuricemia. The present application discovers through research that phillyrin can down-regulate the expression levels of uric acid reabsorption proteins GLUT9 and URAT1, and up-regulate the expression levels of uric acid secretion proteins ABCG2, OAT1, and OAT3, thereby effectively preventing and treating hyperuricemia caused by uric acid excretion disorder.

[0006] In the first aspect, the embodiments of the present application provide the application of phillyrin in the preparation of a medicament for preventing or treating hyperuricemia.

[0007] In some embodiments, the hyperuricemia includes hyperuricemia caused by impaired uric acid excretion.

[0008] In some embodiments, the uses of forsythoside also include: being used for preparing a medicament for preventing or treating kidney damage caused by hyperuricemia.

[0009] In some embodiments, the medicament for preventing or treating hyperuricemia is a preparation prepared from forsythoside or a pharmaceutically acceptable salt, ester or solvate thereof as an active ingredient, plus a pharmaceutically acceptable carrier or excipient.

[0010] In some embodiments, the dosage form of the preparation is a liquid preparation, a solid preparation or a semi-solid preparation.

[0011] In a second aspect, the embodiments of the present application provide an application of forsythoside in preparing a uric acid excretion promoter.

[0012] In some embodiments, the uric acid excretion promoter includes a uric acid reabsorption protein inhibitor.

[0013] In some embodiments, the uric acid reabsorption protein includes at least one of GLUT9 and URAT1.

[0014] In some embodiments, the uric acid excretion promoter includes a uric acid secretion protein promoter.

[0015] In some embodiments, the uric acid secretion protein includes at least one of ABCG2, OAT1 and OAT3.

[0016] The beneficial effects of the present application are as follows:

[0017] Through research, it is found in the present application that forsythoside can down-regulate the expression levels of uric acid reabsorption proteins GLUT9 and URAT1, and up-regulate the expression levels of uric acid secretion proteins ABCG2, OAT1 and OAT3. Based on this, forsythoside can be used as a uric acid excretion promoter, and further used for preparing a medicament for preventing or treating hyperuricemia caused by impaired uric acid excretion. Moreover, forsythoside can also relieve kidney damage caused by hyperuricemia. The present application not only provides a new medical use for forsythoside, but also provides a new drug option for hyperuricemia caused by impaired uric acid excretion, which has important guiding significance for the prevention and treatment of hyperuricemia and the kidney damage caused by it. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the present application, the drawings used in the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1 This is a comparison chart of the detection results of the expression levels of uric acid transporters in the kidneys of mice in each group in the embodiments of the present application;

[0020] Figure 2 This is a comparison chart of the appearances of the kidneys of mice in each group in the embodiments of the present application;

[0021] Figure 3 This is a comparison chart of the HE staining results of the kidneys of mice in each group in the embodiments of the present application;

[0022] Figure 4 This is a comparison chart of the serum uric acid levels of mice in each group in the embodiments of the present application;

[0023] Figure 5 This is a comparison chart of the serum creatinine levels of mice in each group in the embodiments of the present application;

[0024] Figure 6 This is a comparison chart of the serum urea nitrogen levels of mice in each group in the embodiments of the present application. Detailed implementation manners

[0025] Hereinafter, embodiments of the technical solutions of the present application will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present application, and thus are only examples and should not be used to limit the protection scope of the present application.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above accompanying drawings are intended to cover non-exclusive inclusion.

[0027] Referring to "embodiments" herein means that specific features, structures or characteristics described in connection with the embodiments may be included in at least one embodiment of this application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein may be combined with other embodiments.

[0028] In the research of this application, it is found that forsythin can down-regulate the expression levels of uric acid reabsorption proteins GLUT9 and URAT1, and up-regulate the expression levels of uric acid secretion proteins ABCG2, OAT1, and OAT3. Based on this, forsythin can be used as a uric acid excretion promoter, and further used to prepare drugs for preventing or treating hyperuricemia caused by uric acid excretion disorders.

[0029] Specifically, the embodiments of the present application provide the use of forsythin in the preparation of a drug for preventing or treating hyperuricemia. Among them, the hyperuricemia includes hyperuricemia caused by impaired uric acid excretion. Forsythin can effectively promote uric acid excretion by downregulating the expression levels of uric acid reabsorption proteins GLUT9 and URAT1 and upregulating the expression levels of uric acid secretion proteins ABCG2, OAT1, and OAT3, and has a good therapeutic effect on hyperuricemia caused by impaired uric acid excretion.

[0030] Furthermore, in some embodiments, the use of forsythin also includes: being used for the preparation of a drug for preventing or treating kidney damage caused by hyperuricemia. Through experiments, it is found that the use of forsythin can not only treat hyperuricemia caused by impaired uric acid excretion, but also effectively reverse kidney damage caused by hyperuricemia, specifically manifested as alleviating kidney swelling, restoring the kidney color, alleviating renal tubule dilation, thinning of the tubule wall, and atrophy of renal tubular epithelium, and reducing the levels of serum urea, creatinine, and blood urea nitrogen.

[0031] In some embodiments of the present application, the drug for preventing or treating hyperuricemia is a preparation prepared from forsythin or a pharmaceutically acceptable salt, ester, or solvate thereof as an active ingredient, plus a pharmaceutically acceptable carrier or excipient. Among them, the dosage form of the preparation is a liquid preparation, a solid preparation, or a semi-solid preparation.

[0032] In some embodiments of the present application, the effects of forsythin are all dose-dependent. The dosage of forsythin or a pharmaceutically acceptable salt, ester, or solvate thereof is preferably 40 - 120 mg / kg in terms of forsythin, and more preferably 120 mg / kg.

[0033] Since forsythin can downregulate the expression levels of uric acid reabsorption proteins GLUT9 and URAT1 and upregulate the expression levels of uric acid secretion proteins ABCG2, OAT1, and OAT3, the embodiments of the present application also provide the use of forsythin in the preparation of a uric acid excretion promoter. Among them, the uric acid excretion promoter includes a uric acid reabsorption protein inhibitor and / or a uric acid secretion protein promoter. The uric acid reabsorption protein includes at least one of GLUT9 and URAT1, and the uric acid secretion protein includes at least one of ABCG2, OAT1, and OAT3. Based on this, forsythin can be used as a GLUT9 inhibitor, a URAT1 inhibitor, an ABCG2 promoter, an OAT1 promoter, and an OAT3 promoter, effectively expanding the medical uses of forsythin.

[0034] In the above manner, the present application not only provides a new medical use for forsythoside, but also provides a new drug option for hyperuricemia caused by uric acid excretion disorders, which has important guiding significance for the prevention and treatment of hyperuricemia and the kidney damage caused by it.

[0035] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present application, and should not be construed as a limitation of the present application. For those technologies or conditions not specified in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0036] Embodiment

[0037] In order to verify the actual effect of forsythoside, the following experiments were carried out in the present application:

[0038] Experiment 1: Method for establishing, administering drugs and obtaining samples of hyperuricemia mouse models

[0039] Forty-two SPF-grade male Kunming mice (weighing 22 - 24 g, purchased from the Experimental Animal Center of Hubei University of Medicine, certificate number: No. 42000900001362) were randomly divided into a blank control group (Ctrl), a blank drug administration group (the administration dose of forsythoside was 120 mg / kg, denoted as Ctrl + PHI 120), a model group (HUA), a febuxostat group (establishing a HUA model and administering febuxostat at a dose of 10 mg / kg, denoted as HUA + Feb 10), a low-dose forsythoside group (establishing a HUA model and administering forsythoside at a dose of 40 mg / kg, denoted as HUA + PHI 40), and a high-dose forsythoside group (establishing a HUA model and administering forsythoside at a dose of 120 mg / kg, denoted as HUA + PHI 120), with 7 mice in each group. Each mouse was housed separately in a cage, allowed to drink and eat freely, and the room temperature was maintained at 25 ± 2 °C, and the relative humidity was 50% - 70%.

[0040] A HUA mouse model was established by intraperitoneal injection of hypoxanthine (300 mg / kg) combined with potassium oxonate (300 mg / kg) for 7 consecutive days. At 3 pm every day, the blank control group and the model group were given normal saline by gavage, and the other groups were given the corresponding drugs by gavage. Starting from the 8th day, at 9 am every day, the blank control group and the blank drug administration group were given normal saline by intraperitoneal injection, and the other groups were given a mixed solution of potassium oxonate and hypoxanthine by intraperitoneal injection. At the same time, at 3 pm every day, the blank control group and the model group were given normal saline by gavage, and the other groups were given the corresponding drugs by gavage for 7 consecutive days. On the 14th day, food was withheld but water was provided. 2 hours after the last gavage administration, blood was collected by eye socket puncture, centrifuged at 4000 rpm at 4 °C for 10 min, the supernatant was taken and stored at -80 °C for later detection. Two kidneys were taken and weighed. The outer membrane of one side of the kidney was peeled off, and the middle part was cut and fixed in 4% paraformaldehyde for observing the pathological changes of the kidney. All operations were carried out on ice. The gavage and intraperitoneal injection of the above groups were carried out at 0.4 mL / 20 g.

[0041] Experiment 2: Detection of the expression levels of renal uric acid reabsorption proteins GLUT9, URAT1 and uric acid secretion proteins ABCG2, OAT1, OAT3 in the kidneys obtained in Experiment 1

[0042] Add 500 μL of lysis buffer (5 μL of cocktail and 495 μL of RIPA) to every 50 mg of tissue. The tissue was quickly minced with ophthalmic scissors on ice and homogenized 3 times, stopping for 10 s each time, then sonicated 10 times and placed in an ice bath for 30 min. Centrifuge at 12000 g at 4 °C for 15 min, and take the supernatant into a new EP tube to obtain the total tissue protein product. Dilute the protein sample 10-fold and measure the protein concentration by the BCA method. Adjust the protein concentration of each group to the same level with distilled water, add 2×loading buffer (containing 2% β-mercaptoethanol), mix well, seal with a sealing film, boil in boiling water for 10 min, and then place in an ice bath for 5 min. Aliquot according to the protein concentration and the amount of protein required for each electrophoresis, and store at -80 °C. Take 20 - 30 μg of tissue protein sample, perform gel electrophoresis, transfer membrane, blocking, primary antibody incubation, membrane washing, secondary antibody incubation, membrane washing, ECL color development, and use an ImageQuant LAS 4000 gel imager for densitometry analysis. The results are as Figure 1 shown.

[0043] Figure 1Among them, from left to right are the blank control group, blank drug administration group, model group, febuxostat group, low-dose phillyrin group, and high-dose phillyrin group. Compared with the blank control group, the expressions of uric acid reabsorption proteins GLUT9 and URAT1 in the model group were significantly increased, while the expressions of uric acid secretion proteins ABCG2, OAT1, and OAT3 were significantly decreased, indicating that the mice in the model group may have hyperuricemia due to uric acid excretion disorders. Compared with the model group, both the low-dose phillyrin group and the high-dose phillyrin group could significantly down-regulate the expression levels of URAT1 and GLUT9 proteins in the renal tissues of HUA mice, while significantly up-regulating the expression levels of ABCG2, OAT1, and OAT3 proteins in the renal tissues of HUA mice, showing a dose-dependent relationship. It indicates that phillyrin can inhibit uric acid reabsorption and promote uric acid secretion at the same time, thus playing a role in promoting uric acid excretion. It can be used as a uric acid excretion promoter and can also be applied to hyperuricemia caused by uric acid excretion disorders.

[0044] Experiment 3: Examine the kidneys obtained in Experiment 1

[0045] Take pictures of the mice's kidneys for comparison, and the results are as Figure 2 shown. It can be seen from Figure 2 that the kidneys of the mice in the model group were significantly swollen and the color became lighter. As the dose of phillyrin increased, the degree of kidney swelling in the mice decreased, and the kidney color gradually recovered, indicating that phillyrin can effectively relieve the kidney damage caused by hyperuricemia.

[0046] After fixing the kidney tissues of the mice in 4% paraformaldehyde fixative for 24 hours, dehydrate, clear, infiltrate with wax and embed in paraffin in a tissue embedding machine. Use a rotary microtome to section continuously (4 μm thickness), stain with HE, and after dewaxing, washing with water, differentiating, bluing, eosin staining, dehydrating, clearing and mounting, observe the pathological changes of the kidney tissues under a microscope. After HE staining, the cell nuclei are stained blue-purple by hematoxylin, and the cytoplasm, muscle fibers, collagen fibers, etc. are stained red by eosin. The results are as Figure 3 shown. It can be seen from Figure 3 that compared with the blank control group, the renal tissues of the model group and the febuxostat group both showed renal tubular dilation (as shown by the black arrow in Figure 3 ), thinning of the tube wall and atrophy of renal tubular epithelium (as shown by the red arrow in Figure 3 ), while there were no obvious abnormalities in the high-dose phillyrin group, indicating that high-dose phillyrin can effectively reverse the abnormal pathological morphology of the renal tissues of mice caused by hyperuricemia.

[0047] Experiment 4: Detect the levels of uric acid, creatinine, and blood urea nitrogen in the serum obtained in Experiment 1

[0048] Uric acid, creatinine, and urea nitrogen detection kits (all purchased from Nanjing Jiancheng Bioengineering Institute, with product numbers C012-2, C011-1, and C013-2 respectively) were used to detect the levels of serum uric acid, serum creatinine, and serum urea nitrogen in mice according to the methods and requirements in the kit instructions. The results are respectively as Figures 4 - 6 shown.

[0049] It can be seen from Figures 4 - 6 that compared with the blank control group, ### P < 0.001, indicating that the levels of serum uric acid, creatinine, and urea nitrogen in the model group were extremely significantly statistically significant, suggesting that the HUA model was successfully established. Compared with the model group, *** P < 0.001. Both the low-dose and high-dose phyllanthoside groups could significantly reduce the levels of serum uric acid, creatinine, and urea nitrogen, and showed a dose-dependence, indicating that phyllanthoside could effectively relieve the symptoms of hyperuricemia and improve renal function.

[0050] In summary, the present application provides the use of phyllanthoside in the preparation of drugs for preventing or treating hyperuricemia, belonging to the technical field of pharmaceutical applications. Through research, it was found that phyllanthoside could down-regulate the expression levels of uric acid reabsorption proteins GLUT9 and URAT1, and up-regulate the expression levels of uric acid secretion proteins ABCG2, OAT1, and OAT3. Based on this, phyllanthoside could be used as a uric acid excretion promoter, and thus be used to prepare drugs for preventing or treating hyperuricemia caused by uric acid excretion disorders. Moreover, phyllanthoside could also relieve the kidney damage caused by hyperuricemia. The present application not only provides a new pharmaceutical use for phyllanthoside, but also provides a new drug option for hyperuricemia caused by uric acid excretion disorders, and has important guiding significance for the prevention and treatment of hyperuricemia and the kidney damage caused by it.

[0051] It should be noted that the present application is not limited to the above embodiments. The above embodiments are only examples, and embodiments with the same composition and the same function and effect as the technical idea within the technical solution scope of the present application are all included in the technical scope of the present application. In addition, within the scope not departing from the gist of the present application, various modifications that those skilled in the art can think of and other ways constructed by combining some constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. Use of forsythiaside in the preparation of drugs for preventing or treating hyperuricemia.

2. The use of forsythiaside according to claim 1 in the preparation of a medicament for preventing or treating hyperuricemia, characterized in that: The hyperuricemia includes hyperuricemia caused by uric acid excretion disorder.

3. The use of forsythiaside according to claim 1 in the preparation of a medicament for preventing or treating hyperuricemia, characterized in that: The use of the forsythiaside also includes: being used for preparing a medicine for preventing or treating kidney damage caused by hyperuricemia.

4. The use of forsythiaside according to claim 1 in the preparation of a medicament for preventing or treating hyperuricemia, characterized in that: The drug for preventing or treating hyperuricemia is a preparation prepared by using forsythiaside or its pharmaceutically acceptable salt, ester or solvate as an active ingredient and adding a pharmaceutically acceptable carrier or excipient.

5. The use of forsythiaside according to claim 1 in the preparation of a medicament for preventing or treating hyperuricemia, characterized in that: The dosage form of the preparation is a liquid preparation, a solid preparation or a semisolid preparation.

6. Application of Forsythiaside in the preparation of uric acid excretion promoter.

7. The use of forsythiaside in the preparation of a uric acid excretion promoter according to claim 6, characterized in that: The uricosuric acid excretion promoter includes a uric acid reabsorption protein inhibitor.

8. The use of forsythiaside in the preparation of a uric acid excretion promoter according to claim 7, characterized in that: The uric acid reabsorption protein includes at least one of GLUT9 and URAT1.

9. The use of forsythiaside in the preparation of a uric acid excretion promoter according to claim 6, characterized in that: The uric acid excretion promoter includes a uric acid secretion protein promoter.

10. The use of forsythiaside in the preparation of a uric acid excretion promoter according to claim 9, characterized in that: The uric acid secretion protein includes at least one of ABCG2, OAT1, and OAT3.

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