Use of crocin I in preparation of drugs for preventing and treating hyperuricemia

Abstract

The application discloses application of Crocin I in preparation of a medicine for preventing and / or treating hyperuricemia, and the molecular formula of the Crocin I is C44H64O24, and the molecular weight is 976.97. The application regulates the expression of the renal uric acid transporters URAT1, GLUT9, OAT1 and ABCG2 by targeting the expression of the hormone receptors ER and AR of the Crocin I, is beneficial to promoting the excretion of uric acid, and the Crocin I can down-regulate the expression of the uric acid reabsorption related transporters URAT1 and GLUT9, and simultaneously up-regulate the expression of the uric acid excretion related transporters OAT1 and ABCG2, so as to regulate the renal uric acid metabolism from two directions of reducing the uric acid reabsorption and promoting the uric acid excretion, and is beneficial to reducing the blood uric acid level. Therefore, the Crocin I can be used for preparing the medicine for preventing or treating the hyperuricemia and the renal injury induced by the hyperuricemia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a novel pharmaceutical use of crocin I in the preparation of drugs for the prevention and treatment of hyperuricemia. Background Technology

[0002] Hyperuricemia is a common metabolic disease caused by purine metabolism disorder, leading to elevated serum uric acid levels. Long-term hyperuricemia is closely related to various complications such as gouty arthritis, kidney damage, and cardiovascular disease, severely impacting patients' quality of life. Currently, commonly used uric acid-lowering drugs mainly include xanthine oxidase inhibitors (such as allopurinol and febuxostat) and uric acid excretion promoters (such as benzbromarone). However, these drugs have adverse reactions in clinical application, including liver and kidney damage, severe allergic reactions, and gastrointestinal discomfort. Some patients discontinue treatment due to poor tolerance, and clinical needs have not been fully met. Therefore, developing uric acid-lowering drugs with novel mechanisms of action, natural sources, and good safety profiles is of significant clinical importance.

[0003] Saffron (Crocus sativus L.) is a perennial herb belonging to the genus Crocus in the family Iridaceae. Its stigmas are a traditional and valuable Chinese medicinal material, with the main active components being carotenoid compounds such as crocin. Crocin has been proven to possess various biological activities, including anti-inflammatory and antioxidant effects. Existing literature reports that azafrine saponins derived from saffron exhibit in vitro anti-inflammatory activity, and related research has elucidated the dual mechanism by which sex hormones regulate uric acid metabolism: on the one hand, it relies on ERα and AR nuclear translocation to regulate the expression of uric acid transport proteins; on the other hand, it promotes uric acid production by directly binding to and activating XO. Simultaneously, it reveals the functional differentiation of ERα and ERβ in the regulatory mechanism. For the first time, specific DNA sequences of the promoters of SLC22A12, SLC2A9, and ABCG2 were clearly identified, with ERα binding to SLC22A12 and SLC2A9 with a high affinity at the pM level, and AR binding to SLC2A9 and SLC22A12 with a high affinity at the nM level. This provides molecular evidence for the regulation of uric acid transport genes by sex hormone receptors. EST downregulates the expression of uric acid reuptake proteins URAT1 and GLUT9 in a concentration-dependent manner through ERα nuclear translocation, while upregulating the expression of uric acid secretion protein ABCG2. DHT upregulates the expression of URAT1 and GLUT9 and downregulates the expression of ABCG2 in a concentration-dependent manner through AR nuclear translocation. A novel regulatory mode of ERβ mediating posttranscriptional degradation of GLUT9 through the mTOR pathway was elucidated, providing key molecular evidence for the development of novel uric acid-lowering drugs targeting sex hormone receptors; however, there are no reports on the use of crocin I for uric acid-lowering therapy targeting sex hormone receptor expression. Summary of the Invention

[0004] This invention provides a new use for crocin I, namely, in the preparation of drugs for the prevention and / or treatment of hyperuricemia, to overcome the shortcomings of existing uric acid-lowering drugs, such as numerous adverse reactions, poor tolerance in some patients, and insufficient fulfillment of clinical needs.

[0005] Crocin I is a carotenoid glycoside (a gentiobiose ester of crocin acid), a commercially available product, with the following chemical structure: ; The active ingredient (or active ingredient) of the drug described in this invention is crocin I. One or more pharmaceutically acceptable excipients may also be added to improve the absorption effect of the drug or facilitate its use, and to prepare a suitable dosage form, such as capsules or pills, powders, tablets, granules, oral liquids and injections, etc., that is, to prepare a pharmaceutically suitable dosage form.

[0006] The present invention has the following beneficial effects: 1. This invention targets and regulates the expression of sex hormone receptors ER and AR through crocin I, thereby regulating the expression of renal uric acid transport protein, which is beneficial to promoting uric acid excretion and provides a new natural drug for hyperuricemia; 2. The crocin I of the present invention can downregulate the expression of uric acid reabsorption-related transport proteins URAT1 and GLUT9, while upregulating the expression of uric acid excretion-related transport proteins OAT1 and ABCG2. It regulates renal uric acid metabolism from two directions: reducing uric acid reabsorption and promoting uric acid excretion, which is beneficial to reducing blood uric acid levels. 3. The crocin I of the present invention is derived from saffron, is of natural origin and has good safety, and is suitable for the prevention and treatment of hyperuricemia and kidney damage induced by hyperuricemia. Attached Figure Description

[0007] Figure 1 The figure shows the effect of crocin I on serum uric acid (SUA), creatinine (CRE), and blood urea nitrogen (BUN) levels in a hyperuricemia model mouse. Figure 2 Figure 1 shows the effect of crocin I on the mRNA expression level of uric acid transporter protein in the duodenum of a mouse model of hyperuricemia. Figure 3 Figure 1 shows the effect of crocin I on the mRNA expression level of uric acid transporter protein in the jejunum of hyperuricemia model mice. Figure 4 Figure 1 shows the effect of crocin I on the mRNA expression level of uric acid transporter protein in the ileum tissue of a mouse model of hyperuricemia. Figure 5Figure 1 shows the effect of crocin I on the expression level of uric acid transporter mRNA in the kidney tissue of a mouse model of hyperuricemia. Figure 6 Western blot results and grayscale analysis of the effects of GLUT9 and OAT1 protein expression in mouse kidney tissue; Figure 7 The figure shows the effect of crocin I on the mRNA expression levels of GLUT9, SLC22A12, and ABCG2 in uric acid-induced HK-2 cells. Figure 8 The figure shows the effect of crocin I on the mRNA expression levels of GLUT9, SLC22A12, and ABCG2 in uric acid-induced HIEC-6 cells. Figure 9 A schematic diagram illustrating the mechanism by which sex hormone receptors regulate the expression of uric acid transport proteins; Figure 10 The Western blot results and grayscale analysis of the effect of crocin I on GLUT9 protein expression via the sex hormone receptor pathway are shown. Detailed Implementation

[0008] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof. Unless otherwise specified, the instruments, reagents, and materials involved in the following embodiments are all conventional instruments, reagents, and materials already existing in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods involved in the following examples are all conventional experimental methods and detection methods already existing in the prior art. In the examples, crocin I was purchased from Chengdu Pusi Biotechnology Co., Ltd. (batch number PS011052, CAS number 42553-65-1), with the chemical formula C. 44 H 64 O 24 It has a molecular weight of 976.97, is a red solid, and is soluble in 50% methanol and 50% ethanol. The purity is not less than 98.0% (area normalization method) as determined by high performance liquid chromatography (HPLC), and the actual purity is 98.32% (440nm). Mass spectrometry and nuclear magnetic resonance detection results are consistent with its chemical structure.

[0009] Example 1: In vivo pharmacodynamic study of crocin I in a mouse model of hyperuricemia SPF-grade KM mice, weighing 20±2g, were acclimatized for 3 days and then randomly divided into a normal control group, a model control group, a positive control group (benzbromarone), and a crocin I treatment group, with 10 mice in each group. Except for the normal control group, the mice in the other groups were administered potassium oxonate 1g / kg combined with hydrochlorothiazide 0.2g / kg by gavage once daily for 7 consecutive days to establish a mouse model of acute hyperuricemia. After successful model establishment, the crocin I treatment group was administered crocin I 50 mg / kg (suspended in 95% ethanol and diluted with physiological saline) daily by gavage, along with continuous gavage administration of potassium oxonate 1 g / kg combined with hydrochlorothiazide 0.2 g / kg. The positive control group was administered benzbromarone 20 mg / kg daily by gavage, along with continuous gavage administration of potassium oxonate 1 g / kg combined with hydrochlorothiazide 0.2 g / kg. The model control group was administered potassium oxonate 1 g / kg combined with hydrochlorothiazide 0.2 g / kg by gavage every two days for 14 consecutive days. The normal control group was administered an equal volume of physiological saline by gavage. During the experiment, mice had free access to water and food, and their general condition (mental state, activity, coat color, food and water intake, and mortality) was observed daily.

[0010] 1. Serum biochemical index detection: After the last administration, mice were fasted for 12 hours but allowed to drink water. Blood was collected from the orbital venous plexus and whole blood was collected into 1.5 mL EP tubes. The blood was allowed to stand at room temperature for 30 min until it was fully coagulated. Then, the blood was centrifuged at 4℃ and 3000 r / min for 15 min. The upper light yellow serum layer was carefully aspirated and transferred to a new EP tube. Hemolysis of serum samples should be avoided, as hemolyzed samples may interfere with the test results.

[0011] Serum uric acid (SUA) was measured using the Nanjing Jiancheng uric acid test kit (C012-1-1), and the procedure was performed according to the kit instructions. Serum creatinine (CRE) was measured (sarcosine oxidase method, microplate method) using the Nanjing Jiancheng creatinine assay kit, and the procedure was performed according to the kit instructions. Serum blood urea nitrogen (BUN) determination (diacetyl oxime colorimetric method) was performed using the Nanjing Jiancheng blood urea nitrogen test kit, following the instructions in the kit's manual. See results Figure 1 As can be seen from the figure, compared with the model control group, the serum uric acid, creatinine and urea nitrogen levels of mice in the crocin I treatment group all showed a decreasing trend, indicating that crocin I has a uric acid-lowering effect and a renal protective effect.

[0012] 2. Total RNA extraction from tissues and real-time quantitative PCR (qPCR) After euthanizing the mice, kidney and intestinal tissues (duodenum, jejunum, and ileum) were quickly harvested. The tissues were rinsed with pre-cooled physiological saline to remove blood contamination, flash-frozen in liquid nitrogen, and stored at -80°C for later use. Total RNA was extracted using the TRIzol method: approximately 50-100 mg of tissue was added to 1 mL of TRIzol reagent and homogenized thoroughly on ice, then incubated at room temperature for 5 min. 200 μL of chloroform was added, and the mixture was vigorously shaken for 15 s, then incubated at room temperature for 3 min. The mixture was centrifuged at 12000 g for 15 min at 4°C, and the supernatant was collected. An equal volume of isopropanol was added, and the mixture was incubated at room temperature for 10 min. The mixture was centrifuged at 12000 g for 10 min at 4°C, and the supernatant was discarded. The precipitate was washed with 75% ethanol, dried, and dissolved in an appropriate amount of DEPC-treated water. Finally, 20 µL of DEPC-treated water was added to the precipitate to dissolve the RNA precipitate, and the RNA concentration and purity were determined in preparation for subsequent experiments.

[0013] The extracted RNA was prepared into a reverse transcription reaction system according to the following system, and the reverse transcription reaction was carried out under the following conditions to synthesize cDNA;

[0014] Reverse transcription reaction conditions

[0015] Real-time fluorescence quantitative assay Add the following components to a 96-well PCR plate in the following order (single reaction volume: 10µL): q-PCR reaction system

[0016] Mix well, centrifuge, and test under the following conditions:

[0017] The primer sequences for mouse-derived GAPDH, ABCG2, GLUT9, and URAT1 are as follows:

[0018] The results are as follows Figure 2 As shown, compared with the model control group, the mRNA expression level of mGLUT9a in the duodenal tissue of mice treated with crocin I was decreased, while the mRNA expression level of OAT1 was increased. This indicates that crocin I downregulates the transcription of reabsorption genes and upregulates the transcription of secretory genes.

[0019] like Figure 3 As shown, compared with the model control group, the mRNA expression level of mGLUT9a in the jejunal tissue of mice treated with crocin I was decreased, while the mRNA expression level of OAT1 was increased. This indicates that crocin I downregulates the transcription of reabsorption genes and upregulates the transcription of secretory genes.

[0020] like Figure 4 As shown, compared with the model control group, the mRNA expression level of mGLUT9a in the ileum tissue of mice treated with crocin I was decreased, while the mRNA expression level of OAT1 was increased. This indicates that crocin I downregulates the transcription of reabsorption genes and upregulates the transcription of secretory genes.

[0021] like Figure 5 As shown, compared with the model control group, the mRNA expression level of mGLUT9a in the kidney tissue of mice treated with crocin I was decreased, while the mRNA expression level of OAT1 was increased. This indicates that crocin I downregulates the transcription of reabsorption genes and upregulates the transcription of secretory genes.

[0022] 3. Western blot analysis of proteins (1) Cell lysis buffer: Prepared according to the ratio of 100 μL RIPA + 1 μL PMSF (100 mM) + 1 μL protease / phosphatase inhibitor mixture (100×), and stored at -20℃ for later use.

[0023] Protein standard solution: The concentration used is 1 mg / mL. Take 30 μL of the protein standard stock solution (30 mg / mL) into a 1.5 mL centrifuge tube, add 870 μL of PBS to make up the difference and mix before use.

[0024] 1× Electrophoresis buffer: 3.03g Tris + 14.4g glycine + 1g SDS, diluted with pure water to 1L, ready for use.

[0025] 1× Transfer Buffer: 3.03g Tris + 14.4g glycine + 200mL methanol, diluted with pure water to 1L, ready for use.

[0026] 5% Skim Milk Powder (Blocking Solution): Dissolve 2g of skim milk powder in 40mL of TBST and set aside for later use.

[0027] BCA working solution: Solution A: Solution B = 50:1, prepare and use immediately.

[0028] (2) Sample preparation and protein quantification (BCA method) Transfer approximately 20-50 mg of tissue to 1.5 mL EP (Epidermal Extract), homogenize with liquid nitrogen or use a homogenizer, remove the homogenized animal tissue, and add 70 μL of cell lysis buffer to each well. Incubate on ice for 30 min to lyse. After lysis, centrifuge at 12000 rpm for 20 min at 4 °C. Discard the precipitate to obtain the protein solution.

[0029] Prepare the standard curve for the 96-well plate according to the table below.

[0030] Add 2 μL of the protein sample to each well, followed by 18 μL of PBS. Add 200 μL of prepared LCA working solution to both the sample and standard wells simultaneously, and incubate at 37°C for 30 min. After incubation, measure the absorbance (OD value) of each well at 562 nm using a microplate reader.

[0031] Plot a standard curve using Excel with the concentration of the standard on the x-axis and the corresponding OD value on the y-axis, and obtain the regression equation (R²). 2 (Requires ≥0.99). Calculate the protein concentration of the sample based on the regression equation.

[0032] (3) SDS-PAGE gel preparation and electrophoresis Assemble the glue-forming plate and place it in the glue-forming frame, ensuring a tight connection at the bottom, and check for leaks with pure water.

[0033] To prepare two 10% separating gels, add 5 mL of 30% Acrylamide, 3.8 mL of Tris-HCl (pH=8.8), 5.9 mL of deionized water, 150 μL of 10% SDS, 150 μL of 10% APS, and finally add 6 μL of coagulant. After stirring evenly, quickly pour the prepared separating gel into the gel casting plate, adding 7 mL of separating gel to each plate, and add 1 mL of anhydrous ethanol to remove air bubbles for pressing. Allow to solidify at room temperature for 30 min.

[0034] Discard the anhydrous ethanol used for pressing the gel. Prepare a 5% stacking gel by adding 1 mL of 30% Acrylamide, 0.75 mL of Tris-HCl (pH=6.8), 4.1 mL of deionized water, 60 μL of 10% SDS, 60 μL of 10% APS, and finally 6 μL of coagulant. Stir well and quickly pour the mixture onto the top of the separating gel. Insert a 1.5 mm comb into each of the 15 wells and allow it to solidify at room temperature. Once completely solidified, remove the comb and add 20 μL of sample to each well.

[0035] Connect the electrophoresis apparatus to the power supply and set the initial voltage to 80V (constant voltage). After the bromophenol blue indicator enters the separating gel (about 30 minutes), adjust the voltage to 120V and continue electrophoresis until the bromophenol blue indicator reaches 1 cm from the bottom of the gel. Avoid excessive voltage, which may cause the gel to heat up and affect the protein separation effect.

[0036] The PVDF membrane is pre-activated by soaking in methanol, ensuring it is fully wetted. After electrophoresis, remove the gel, rinse off any foam with water, and cut away the stacking gel and excess separating gel according to the molecular weight of the target protein and the marker location, retaining the gel region containing the target protein. With the black side down in the transfer clamp, place the following layers on the clamp in sequence: a sponge pad, three layers of filter paper, gel, PVDF membrane, three layers of filter paper, and another sponge pad, ensuring each layer is free of air bubbles. Secure the clamp and place it in the transfer tank. Place two ice packs in the transfer tank to ensure a low-temperature transfer environment; alternatively, crushed ice can be placed outside the transfer tank for cooling. Connect the transfer apparatus to the power supply and set the transfer conditions to 200mA for 2 hours and 10 minutes.

[0037] After transfer, the PVDF membrane was immersed back-to-back in 5% skim milk blocking buffer and blocked on a shaker for 2 hours. After blocking, the membrane was washed three times with TBST for 7 minutes each time. The membrane was then cut to the desired target protein size. The target protein was placed in the corresponding primary antibody dilution buffer and incubated overnight at 4°C. The primary antibody dilution buffer was recovered, and the membrane was washed three times with TBST for 7 minutes each time. Then, the membrane was incubated with the secondary antibody corresponding to the primary antibody source for 1 hour and 10 minutes on a shaker at room temperature. Finally, the membrane was washed three times with TBST for 7 minutes each time.

[0038] The developing solution should be prepared fresh for immediate use. Mix developing solution A and solution B in a 1:1 ratio, and evenly drop the mixture onto the membrane surface. Develop using a gel imaging system. Scan the protein immunoblot bands using ImageJ software to obtain grayscale values. Calculate the relative expression level of the target protein by dividing the grayscale value of the target protein by the grayscale value of the internal reference protein, and then perform statistical comparison using Prism9.

[0039] The results are as follows Figure 6 As shown, compared with the model control group, the expression level of GLUT9 protein was decreased and the expression level of OAT1 protein was increased in the kidney tissue of mice treated with crocin I. Similar to the results of the qPCR above, this again demonstrates that crocin I can downregulate reabsorption proteins and upregulate secretory proteins.

[0040] Example 2: In vitro pharmacodynamic study of crocin I on a uric acid-induced HK-2 cell model HK-2 cells were cultured in MEM (containing NEAA) containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at 37°C in a 5% CO2 incubator. When cell confluence reached 70%–80%, the cells were passaged or plated. For plate formation, the culture medium was aspirated from the HK-2 cells to be used in the experiment, and the cells were rinsed with PBS buffer (pH 7.2–7.4). After removing the PBS, the cells were digested with 0.25% trypsin (containing 0.1% EDTA) for approximately 30 seconds. When the cells became loose and sandy, three times the volume of complete culture medium was added to stop the digestion. The cells were then centrifuged at 800 rpm for 3 minutes in a 5 mL centrifuge tube. After centrifugation, the supernatant was discarded, and 1 mL of complete culture medium was added and mixed to obtain a cell suspension. After counting the cells, a 12-well plate was used, and HK-2 cells were seeded into each well at a density of 2.5 × 10⁶ cells / well. 5 Cells were collected at 1.5 mL total culture medium volume per well. When the cell confluence reached 70%–80%, the following groups were set up: blank group (no treatment), model group (2 mmol / L uric acid), positive control group (2 mmol / L uric acid + 50 μmol / L benzbromarone), and treatment group (2 mmol / L uric acid and 20 μmol / L crocin I). The above experimental groups were induced for 24 h and then the samples were collected. The mRNA expression levels of SLC22A12, GLUT9, OAT1, and ABCG2 were detected by real-time quantitative PCR (qPCR).

[0041] The primer sequences for human GAPDH, ABCG2, GLUT9, and URAT1 are as follows:

[0042] like Figure 7 As shown, compared with the model control group, the mRNA expression levels of GLUT9a and SLC22A12 in HK2 cells of the crocin I-treated group were decreased, while the mRNA expression levels of ABCG2 and OAT1 were increased. This indicates that crocin I downregulates the transcription of reabsorption genes and upregulates the transcription of secretory genes.

[0043] like Figure 8 As shown, compared with the model control group, the mRNA expression levels of GLUT9a and SLC22A12 in HIEC-6 cells treated with crocin I were decreased, while the mRNA expression levels of ABCG2 and OAT1 were increased. This indicates that crocin I downregulates the transcription of reabsorption genes and upregulates the transcription of secretory genes.

[0044] Example 4: Study on the mechanism by which crocin I regulates the expression of uric acid transporter protein via the sex hormone receptor pathway After counting the cell suspension, take a 6-well plate and seed HK-2 cells into each well at a density of 2.5 × 10⁶ cells. 5 Cells were collected at 2 mL per well. When the cell confluence reached 70%–80%, the following groups were established: blank group (no treatment), model group (2 mmol / L uric acid), positive control group (2 mmol / L uric acid + 50 μmol / L benzbromarone), treatment group (2 mmol / L uric acid and 20 μmol / L crocin I), estrogen receptor antagonist group (2 mmol / L uric acid and 100 nmol / L PHTPP), and androgen receptor antagonist group (2 mmol / L uric acid and 5 μmol / L Flutamide). The cells were collected after 24 h of induction. Cell lysis buffer was prepared according to the ratio of 100 μL RIPA + 1 μL PMSF (100 mM) + 1 μL protease / phosphatase inhibitor mixture (100×) and stored at -20°C for later use. Remove the cell culture dish (6-well plate) after drug stimulation, discard the culture medium, rinse once with pre-chilled PBS, then add 70 μL of cell lysis buffer to each well and incubate on ice for 30 min. After lysis, transfer the cell-containing lysis buffer to a 1.5 mL EP tube and centrifuge at 12,000 rpm for 20 min at 4 °C. Discard the precipitate to obtain the protein solution.

[0045] This study used Western blotting to detect the effect of crocin I on GLUT9 protein expression under uric acid stimulation.

[0046] The results are as follows Figure 10 As shown, uric acid stimulation can upregulate the expression of GLUT9 protein in HK-2 cells, while crocin I treatment can reduce the uric acid-induced GLUT9 protein expression level. Treatment with the estrogen receptor antagonist PHTPP and the androgen receptor antagonist Flutamide can both affect the uric acid-induced GLUT9 protein expression, suggesting that sex hormone receptors ER and AR are involved in the expression regulation of the uric acid transporter GLUT9.

[0047] Based on the results of Examples 2 to 4, crocin I can treat hyperuricemia by targeting hormones to regulate the expression of uric acid transport proteins, thereby reducing serum uric acid levels (see schematic diagram of the mechanism by which sex hormone receptors regulate the expression of uric acid transport proteins). Figure 9 The above experimental data were obtained based on the cell lines and treatment conditions described in this embodiment and are not extrapolated to other cell types and treatment concentrations.

Claims

1. Application of crocin I in the preparation of drugs for the prevention and / or treatment of hyperuricemia.

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